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Full text of "Proceedings of the Indian Academy of Sciences Plant Science Section B Vol_91"

Proceedings of the Indian Academy of Sciences 

Volume 91, 1982 

CONTENTS (Plant Sciences) 

Thaxteriellopsis lignicola and its Moorella anainorph 

C V Sitbramanian and G Sekar 1 

A contribution to the embryology of Alysicarpus monillfer B.C. 

V Seshavatharam 9 

Noa-inheritance of isomerism in cocoyams T Venkateswarlu 17 

Apomixis in Cenchrus glaucus Mudaliar ct Sundaraj C Shanthamma 25 

Regeneration of plantlets from callus of Elettaria cardamomum Maton 

TV K Srinivasa Rao, S Narayanaswamy, E KChako and R Dore Swamy 37 

Studies in Cyperaceae : XVII Novelties in Finibristylis (L.) Vahl and their 
vegetative anatomy E Govindamjalu 43 

Embryological studies in three species of Cymbopogon Spreng (Poaceae) 

S P Choda, Harsh Mitter and Ravfnder K JBhanwra 55 

Reproductive efficiency of secondary successional herbaceous populations 
subsequent to slash and burn of subtropical humid forests in north eastern 
India K G Saxena and P S Ramakrishnan 61 

Vesicular arbuscular mycorrhiza in subtropical aquatic and marshy plant 
communities R Chaubat, G D Sharma and R R Mishra 69 

Chandrasekharania : A new genus of Poaceae from Kerala, India 

V J Nair, V S Ramachandran and P V Sreekumar 79 

Chromosome relationships of spinous solanums 

P Kirti and G S Rao 83 

Groundnut rust-its survival and carry-over in India 

P Subrahmanyam and D McDonald 93 

Correlated promotion of ray-floret growth in chrysanthemum by potassium 
chloride, gibberellic acid and sucrose 

P Pardha Saradhi and H Y Mohan Mam 101 

Nuclear behaviour during heartwood formation in Acacia auriculiformis 

A. Cann K V Bhat and J D Patel 107 

Identity of Ficus macrocarpa Wt. ex King (= F. amplocarpa nom. nov.) 
and F. guttata (Wt.) King-A reinvestigation with anatomical evidence 

E Gonndarajalu and f Masilamoney 115 

Tke gentts JdcJdefia in Sottth India Ram Udar and Adanh Ktfaidr 131 



ii Contents 

Geocalyx Nees-a rare marsupial genus from India 

Udar, S C Srivastava and Dhirendra Kumar k 



Ontogeny of the paracytic stoma: Variations and modifications 

Parveen Farooqui (nee Kiawai) 14 

Growth response of some thermophilous fungi at different incubation 
temperatures S Singh and D K Sandhu If 

Studies on Beggiatoa : Distribution and growth in aquatic habitats of 
Visakhapatnam M R R Mohan and A Narayana Rao 15 

Photoperiodic control of extension growth, bud dormancy and flowering of 
Nerium indicum Mill, and Thevetia pemviana Schum. 

Kushal Singh, Surinder Kumar and K K Nanda 11 

Interaction of kinetin with B group vitamins on the seedling growth of 
green gram (Phaseolus radiatus L.) 

P Gopala Rao and J Kodandaramaiah 18 

Leaf architecture of apocynaceae / S S Mohan and J A Inamdar 1 

Impact of extension growth and flowering on the cambial 'activity of 
Delonix regia Rafin A K M Ghouse and Shamima Hashmi 2C 

Pharrnacognostic studies on the flower of Mesua ferrea L. 

Usha Shome, Shanta Mehrotra and H P Shanna 2J 

Effect of C0 3 in overcoming self-incompatibility barriers in Brassica 
campestris L. var. toria A S Dhaliwal and C P Malik 21 

Pharmacognosy of the stems of Portulaca quadrifida L. and Portulaca 
oleracea L. / Lai and A M Khan 23 

Structure and function of a sub-tropical humid forest of Meghalaya I. i i 
Vegetation, biomass and its nutrients Jasbir Singh and P S Ramakrishnan 2^ 

Structure and function of a sub- tropical humid forest of Meghalaya II. 
Litter dynamics and nutrient cycling 

Jasbir Singh and P S Ramakrishnan j 2i 

Structure and function of a sub-tropical humid forest of Meghalaya III. 
Nutrient flow through water Jasbir Singh and P S Ramakrishnan 2( 

Anatomy of the seedling of the Leguminosae I 

Umavathi Hegde and V D Tilak 2 

Cork-warts in Eucalyptus species Parveen Farooqui (nee Kidwai) 2 

Pericarpial sclereids in some Mimosaceae 

S Rangaiah, I L Kothari and G L Shah 25 

Viability and infectivity of zoospores of Sclerospora graminicola (Sacc.) 
Schroet in the soil C R Ramesh and K M Safeeulla 3( 

Initiation, development and structure of root nodules in some members 

of the tribe Tnfolieae (Papilionaceae) G L Shah cttld M Gopala Fao 3( 



Contents iii 

Turnera ulmifolia var. elegans x T. ulmifolla var. angustlfolia crosses and 
its bearing on the taxonomy of the species 

K Rajeev, P I Kwiachan and C A Nlium 319 

Airborne pollen grains of Visakhapatnam : A combined field and air samp- 
ling study A Janaki Bai and C Subba Rcddl 329 

The floral anatomy of Kniphofia uvaria Hook. (Liliaceae : Knipholieae) 

N P Vaikos and R M Pal 351 

Transmission of seed-borne inoculum of Macrophomma phaseolina from 

seed to plant Tribhuwan Singh and Dalblr Singh 357 

Effect of water stress on opening and longevity of flowers in Gladiolus 

/ V Ramanuja Rao and H Y Mohan Ram 371 

Petal venation in Trigonella (Papilionaceae) Mohlnl Gupta 379 

Responses of cotton-cultivars to long day conditions 

J G Bhatt and M R K Rao 389 

Seed germination and seedling establishment of two closely related Schiina 
species Ram oojh and P S Ramakrishnan 397 

Anther and pollen development in cotton liaploids and their parent, 

S S Mehetre 409 

Changes in proteins, amino and keto-acids in different seedling parts of 
Cyamopsis tetragonolobus Linn, during' growth in light and darkness 

Prem Gupta and D Mukherjee 417 

Effect of ridge gourd pollen on zoospore germination of Pseudoperonospora 
cubensis and its significance in epidemiology 

Amarnatha Shetty, H S Shetty and K M Safeeulla 427 

Leaf proteinase and nitrate reductase activities in relation to grain protein 
levels and grain yield in four species of grain amaranth 

K Ramamurthy Naidu, Y Seethambaram and VSR Das 433 

Cell division in Staurastrum gracile Ralfs. under the scanning electron 
microscope Vidyavati 443 

Leaf surface studies of some medicinal salvias H P Sharma and Usha Shomc 449 

Morphological and metabolic changes in the egg and zygote of Lagers- 
tromeia speciosa, I. Cell size, vacuole and insoluble polysaccharides 

P Raghavan ami V J Philip -465 

The floral anatomy of Pity a spathacea Mexz- (Promeliacene) with ;p cial 
refereuce to nectaries R A Rulkarni and R M Pai 473 

Cytological studies on certain acanthaceae from Central India 

MIS Saggoo and S S Sir 479 

Heterotrophic bacteria associated with seaweed 

R Lakshmctnapemmalsamy and A Ptirwhothaman 487 



IV 



Contents 



Association of chlorophyll content., phyllotaxy^ photosynthesis and B-group 
vitamins in some 3 and C 4 plants 

P Gopala Rao and J Kodandaramaiah 

Effect of morphactin, AMO-1618 and DPX 1840 on the endogenous levels of 
hormones and its implication on apical domnance in Glydne max. Linn. 

/ S Duai.U K Kohli and K S Chark 

Taxonomic importance of epidermal characters in the Indian Thespesia 
Corr. (Malvaceae) S Raja Shanmukha Rao and N Ramayya 

Embryological studies in Launaea midicaulis Hook. 

P S Chikkannaiah and B S Hiremath 

Quantitative profile structure of certain forests in the Kmnaun Himalaya 

A K Saxena and J S Singh 

Contributions to our knowledge of Indian Algae HI. Euglenineae-Part I. 
The genus Euglena Ehrenber M T Philipose 



fittc. Indian Acai Sci. (Plant Sci.j, Vol. 91, Number 1, February 1982* pp. 1-1 
Printed in India. 



Thaxteriellopm lingicota and its Moorelta ariamorph* 

C V SUBRAMANIAN and G SEKAR 

Centre of Advanced Study in Botany, University of Madras, Madras 600 005, India 

MS received 14 October 1981 ; revised 18 December 1981 

Abstract. A. Moorelht anamorph referable to M. spedosa Rao and Rao, is described 
for Tliaxtertetlopsis ligtiicola Sivaixesan, Panwar and Kaur. The connection is 
established from a s-tudy of single ascosporc cultures of the fungus established 
for several (6) South Indian collections. Both the teleomorph and anamorph are 
described and brief notes are given of characteristics of this fungus in culture. 

Keywords. Tltaxtenellojpsis Itgnicola ; Moorella spedosa ; toleomorph ; anamorph. 



1. Introduction 

During the survey of mierofuagi under the project ' Fungus $lora of South 
India \ several collections of a Loculoascomyeete with setose ascomata were 
made. Single ascospore cultures of the fuaigu* produced aoi anamorph referable 
to the hyphomycctc genus Moorella. The Moorella anamorph was also found in 
association with the Loculoascomyeete on the natural substrate. The teleomorph 
was identified as Thaxlerltllopsti lignlcola Sivanesan, *acnwar and Kaur, and the 
anamorph as MooreJUi spedosa Rao ajid Rao. 

The monotypic genus T/iaxtertellopsis was erected by Sivanesan et at (I9*t6) 
with T. lignicola collected from Mount Abu, India as the type. It is characterized 
by non-ostiolatc, setose, cupulatc ascomata, superficial on a subiculum, with 
bitunicate asci containing hyaline or subhyaline ascospores, which are traaisversely 
multiseptate. Some of the ascosporcs, in addition, develop one longitudinal or 
oblique septum, rarely two in one or two of its cells. 

In all the collections; studied, the fimgus agrees in all details with the type descri- 
ption of T. lignlcola except for the difference in the number of the septa in ascos- 
pores. The transversely multi septate asco.xpores, with one or two longitudinal 
and obliquo septa in sonic., typical of T. lignlcola were seen only in one collection 
from the Silent Valley, Kerala (Herb. FSI 3363). In the remaining collections 
the ascospores were predominantly transversely 5-septate. The present study 
shows that in T. l/gnicola the ascospores are initially 5-septate ; the centrum is 
hyaline to white, as seen in most of the collections. As the ascospores mature, 
additional transverse., longitudinal and oblique septa develop ; the centrum becomes 
light brown, as seen in the single collection from the Silent Valley. The additional 



* Memoir No, 352 from Centre of Advanced Study in Botany, 



2 C V Subramanian and G Sekar 

transverse, longitudinal and oblique scptc. develop in any sequence aond are forme 
within any cell, without any regular pattern, in the originally 5-septate ascospore 

2. Description of the fungus 

Thaxteriellopsis lignlcola Sivanesan, Pan war and Kaur, Kavaka 4 : 39, 197 
Aaamorph : Moorella spedosa Rao and Rao, Mycopath. Mycol. appl. 22 : 5 
1964. (figures 1-19.) 

Colonies on the substrate conspicuous, superficial, widely effused, black, consi 
ting of velvety growth of anamorph, interspersed with scattered to gregarious asc< 
mata of teleomorph. Surface mycelium composed of septate, branched, dai 
brown, creeping hyphae 4-7/j wide, constricted at septa when cells are short an 
swollen, smooth when the cells are long cylindrical. Immersed mycelium consi 
ting of septate, branched, light brown or dark brown hyphae ca. 5p wide. 

Ascomata superficial, connected to the creeping mycelium by septate, bro\\ 
pendant hyphae ca. 5p wide, originating from the lower half of the ascoma, son 
seated on a stroma directly on the substrate, black, cupulate, in water becomir 
spherical to broadly spherical or obconic-spherical, 20(WOO// high, 250-37( 
diam., non-ostiolate, with numerous dark brown, septate, uabraached setae, wil 
rounded ends, mostly on the upper half of the ascoma. Setae up to 250/z Ion 
4-5-6-0/* wide. Wall of the ascomata somewhat fleshy, 35-50^ thick, pseud< 
parenchymatous, composed of 6^9 layers, made up of polygonal cells, 5-20 : 
5^13/j ; cells of 'ostiolar' region comparatively smaller, 5-10 x 3-5/j ; eac 
cell with a large oil droplet, oozing out in teased mounts ; outer layers darkbrowj 
gradually becornbag light brown to hyaline towards the inner layers ; cells of tl 
inner layers flattened. 

Asci in a basal hym^ium, bitunicate, long-cylindrical tocylindric-clavats, shor 
pedicellate, 90->150 x 15-30/f, generally 8-spored ; sometimes fewer-spored. 

P^eudoparaphyses present, attached both to the roof and the basal hymeniun 
septate, hyaline, branched, ca. 1-5^ wide. 

Ascospores irregularly tristichous, 23-50 x 5-5-13 (-^16)^, initially with 
transverse septa. Mature ascospores with additional transverse, longitudin* 
and oblique septa, up to 6-12-transversely septate aoid often with up to 3 long 
tudinal and/or oblique septa, clavate to clavate-fusiform or fusiform \ general] 
the second and thir doells from apex broader, tapering towards the rounded end 
curved to straight, thin-walled, slightly constricted at septa in large spores, faint] 
striate, hyaline to smoky brown in mass. 

Conidiophores erect, straight or slightly bent, arising directly from creepin 
mycelium, septate, smooth, up to 420/z long, blackish brown and 7-11/z wide 2 
base, gradually becoming pale brown and 4^7/j wide at apex, bearing up to si 
whorls of branches at intervals, with one to six branches per whorl ; branchc 
1-4-celled, 4-5-7-5/J wide. Conidiophore often terminating in an apical who: 
of branches or a conidiogenous cell. Terminal cell of each branch conidiogenou; 
Conidiogenous cells hyaline, light brown to brown, flask-shaped to globose o 
cylindrical, often proliferating, polyblastic, denticulate. Conidia helicoid, tight! 
coiled Hi times, borne on short cylindrical denticles ca. I// long, 3-.10 septafc 
generally 6-septate, 1 W5/j diam. ; filaments 4-6/* wide, hyaline, finally becomin 
smoky brown with faint str-j^ttons,.*- <-....,. 



Thaxteriellopsis liguicola and its Moordla 




II 



Figures 1-11. tfiaxteriellopsis lignlcola and its Moordla anamorph. t. Vertical 
soctiaa o,f ascoma (Kerb. FSI 3359) x 200. 2. Predominaatly 5-septatc ascos- 
p^res (Kerb. FSE 3151) X 700. 3. M'Uure ascosporcs showing longitudinal and 
oblique septa (Herb FSI 3363) x 480. 4. Part of a conidiopliorc with ccmidiogenous 
colls and a coiidium initial (Herb. FS[ 3151) x 1200. 5. Conidia from natural 
substrate (Herb. FSI 3363) x 750. 6. Genniii'-Ued ascosporc (Herb. FSI 21 2) 
X 375. 7. A conidiogenous cell with a developing -co ntdium (from culture, Herb. 
FSt 3l5l)"x 1200, 8. A short conidiophore with a solitary terminal conidium (from 
culture, Herb. FSt 3151) x 1065. 9, 10, 11. Mature brown conidia from a. 
6 moatli's old culture (Herb, FSI 3151) X 1600, 



Thaxteriellopsis ligniaola and its Moorella anamorph 




Figures 12-19. Thaxteriellopsis lignicola and its Moorella anamorph. 12. Ascus 
(Herb. FSI 3363). 13. Ascospores (Herb. FSI 3363). 14. Young conidia deve- 
loping directly on, a hypha (from culture, Herb. FSI 1423). 15. A conidium borne 
on a single conidiogenous. cell produced laterally on a hypha (from culture, Herb. 
FSI 1423). 16. A conidiophore with several conidia produced from its apical part 
(from culture, Herb. FSI 1423). 17. Conidia from natural substrate (Herb. FSI 
3151). 18. Conidia (from culture, Herb. FSI 3151). 19. Brown conidia from ^ 
$ month's old culture (Herb. FSI 3151). 



6 C V Subfamanian and G Sekar 

Collections examined : 

All collections by G. Sekar. 
Kamataka State 

Ch indst. wood, Ksmphole Forest, near Sakleshpur, Hassan District, 31 October 
1977, Herb. FST 1423 ; on indet. wood ; Muadwar Pimpley Forest, near Londa 
Belgaum District, 2? November 1980, Herb. FST 3204 ; on indet. bark, Karwar, 
North Kanara District, 18 November 1980, Herb. FSI 3151. 
Kerala State 

On indet. wood, Silent Valley, Palghat District : Kumattanthod, 5 December 
1980, Herb, FSI 3225, 3230 ; Dam site, 6 December 1980, Herb. FSI 3257 ; camp 
site, 7 Dxember 1980, Herb. FSI 3261 ; near Valaiyampara Estate, 8 December 
1980, Hfeib. FSI 3271 ; Mathrithod, 11 December 1980, Herb. FSI 3321 ;. Valajya^ 
parathod, 13 December 1980, Herb. FSI 3356, 3359. On wood and bark of 
Syzygium cumini (Linn.) Skeels, Valaiyaparathod, Silent Valley, Palghat District, 
13 December 1980, Herb. FSI 3363. On indet. wood, Karapara Forest, Palghat 
District, 21 December 1980, Herb. FSI 3413, 3417 ; 24 December 1980, Herb. 
FSI 3461. 
Tamil Nadu State 

Oa wood of Syzygium cumini S:egaltheri, Kalakad, Tirunelveli District, 13 
February 1979, Herb. FSI 2048. On indet. wood, Muadanthurai Forest, Tirune- 
lveli District, 16 February 1979, Herb. FSI 2128. On indet. bark, Muadanthurai 
Forest, Tiruaelveli District, 18 February 1979, Herb. FSI 2166. On wood 
of Mango, Kakkanalla bridge, Mudumalai Forest, Nilgiri District, 12 January 1981, 
Herb. FSI 3480. 

3. Cultural studies 

Ascospores germinate on potato dextrose agar within 8 h at 25-30 C. Germ 
tubes are produced from any one or all the cells of ascospores (figure 6), more 
frequently from the end cells. Single spore isolations on potato dextrose agar 
are slow growing, attaining 1cm. diameter in 10 days. The colonies are olive 
green to olivaceous brown, velvety, with compact margin, restricted in growth 
with aerial mycelium and dark brown submersed mycelium. The conidiogenous 
cells are either intercalary or terminal on hyphae, or on a simple conidiophore 
(figures 8 and 16). Typical dark brown erect conidiophores with whorls of 
branches bearing conidiogenous cells are produced after three months in culture. 
Conidia are produced on narrow, cylindrical denticles, ca. lju long and are loosely 
or tightly coiled or juit bent and curved. Conidium size is comparable to that 
on the natural substrate, but soim may be a little larger. In a long standing 
culture, conidia (figures 9-^11 and 19) become brown, up to 12-septate and measure 
up to 23/j diameter with filaments 9/j wide, constricted at the septa. 

4. Discussion 

Thaxteriellopsis is a member of the Loculoascomycetes and belongs to the Pleos- 
porales ; in having Pleospora type of centrum. It is closely related to Thaxtertella, 
as pointed out by Sivanesan et al (1976), aoid possibly to Tubeufia. The anainorpl} 



ThaxterieUopsis Ugnicola and its Moofella anamofph 7 

of Thaxteriellopsis, as shown here, is Moorella and that of Thaxterlelta and Tubeufia 
are Helicorna and Helicosporiitm respectively. It is interesting to note thet all the 
three teleomorphs mentioned above have somewhat fleshy ascornata, vrith usually 
hyaline ascospores and their anamorphs are dematiaceous hyphomycetes producing 
helicoid conidia on denticles. This is the first time a Moorella aaiamorph is con- 
nected with a teleomorph. 

Barr (1980), notes that the illustration and description of Thcateriellopsis ligni- 
cola are suggestive of Boerlagiomyces Butzin. However, even if this v/ere to be 
confirmed by further study of the types, the generic name ThaxterieUopsis \vill have 
to be retained according to the rules of priority. The name ThaxterieUopsis 
Ugnicola is therefore used here for the teleomorph of our fungus. 

Acknowledgements 

One of the authors (GS) is grateful to University Grants Commission for the award 
of Junior Research Fellowship, under the project * Fungus Flora of South India *. 
They also thank Mr V Kaviyarasan for photographic assistance. 

References 

Barr M E 1980 On the family Tubeufiaeeae (Pleosporales) ; Mycotaxon 12 137-167 
Sivanesan A, Panwar K S and Kaur S J 1976 Thaxterieljopsis Ugnicola gen, et sp. nov., a new 
Locmloascomycete from India ; KqvcikQ 4 39-42 



l?roc. Indian Acad. Sci, (Plant Sci), Vol. 91, Number 1, February 1982, pp. 9-15, 
Printed in India. 



A contribution to the embryology of Alysicarpus monilifer D.C. 

V SESHAVATHARAM 

Department of Botany, Andhra University, Waltair 530003, India 

MS received 25 April 19S1 ; revised 4 February 1952 

Abstract. Embryology of Alysicarpus monilifer D.C. is described. The anther wall 
comprises the epidermis, the fibrous endothecium, two middle layers and uniseriate 
glandular tapetum. Pollen is shed at the 2-celled stage. The ana-campylotropus 
ovule is crassinucellate and bitegmic. The archesporium in the ovule is hypodermal 
and cuts off a parietal cell. Embryo sac development conforms to the Polygonum 
type. Endosperm development is nuclear. A chalazal part of the endosperm 
remains free nuclear and is haustorial in function. Embryogeny conforms to 
Period I Megarchetype IV and Series B a . The embryogenic classification of the tribe 
is discussed in the light of the present observation and earlier reports. 

Keywords. Embryology ; Alysicarpus. 

1. Introduction 

Tne tribe Hedysareae of the Papilionaceae includes 47 genera and 7000 species 
distributed in the tropical and subtropical regions of the world (Rendle 1925). Of 
these only 14 genera are known embryologtcally. The researches of Soueges (1947, 
1953a, 1953b, 1955, 1956), Johansen (1950), Rau (1951, 1953, 1954), Smith (1956), 
Goursat (1961), Kaprokar (1964) and Dsshpande et al (1976) reveal the cmbryogeny 
of 15 species belonging to 11 genera of this tribe. The embryology of Alysicarpus 
monilifer D.C. is described here. 

2. Material and methods 

Buds, flowers and fruits of varying ages were collected from the plants growing 
in the university campus, during the rainy season and were fixed in FAA. Custo- 
mary methods of dehydration and embedding, etc. were followed. Sections cut 
between 8 and 10 microns thick were stained with safranin and fast green. The 
endosperm was observed in whole mounts after making suitable dissections. 

3. Observations 

3-1. Microsporogertesis and the development of pollen 

The archesporium in the anther is hypodermal represented by a single row of 4-^5 
cells. The primary parietal layer by further divisions forim 2-3 parietal layers 
of which the innermost forim the secretory tapetum (figure 1). The tapetal cells 
remain uninu^leate throughout. Some of the cells on the connective side adjoining 



10 



V Seshavatharam 



the tapetum also behave like the tapetal cells in assuming a dense cytoplasm and 
deeply stained nuclei. The hypodermal parietal layer forms the fibrous endo- 
thecium (figure 3). The two middle layers are crushed in the mature anther. 
Division of the microspore mother cell is simultaneous and cytokinesis takes place 
by furrowing (figure 2). The pollen is shed at the 2-celled stage. 




Figures 1-9. 1. T.S. anther lobe showing wall layers and sporogenous tissue ; 
2. Same showing cytokinesis in pollen mother cells ; 3. Same showing fibrous 
endothecium and two celled pollen grains ; 4. L.S. portion of ovule showing a 
linear tetrad of megaspores ; 5. LS ovule showing the integuments endosperm and 
barrier tissue at the globular stage of embryo ; 6. Mature embryo sac ; 7. Nuclear 
endosperm showing chalazal elongation ; 8. LS young seed showing the embryo 
and endosperm ; 9. LS portion of the ovule at the chalazal regions showing the 
incipient integumentary tapetum and thick walled barrier tissue, 



Embryology of Alysicarpus monilifer D.C. 1 1 

3*2. Megasporogenesis and female gametophyte 

The ovule at maturity is campylotropous bitcginic and crassinueellate (figure 5). 
The single celled archesporium in the ovule is hypoderinal and cuts off a 
parietal cell. Tlie mogaspore mother cell undergoes the usual meiotic divisions 
resulting in a linear tetrad of magaspores of which the chalazal is functional 
(figure 4). Tne development of the mature embryo sac conforms to the Polygonum 
type. Tnc mature megagametophyte is 8-nucleate with an egg apparatus, two 
polar nuclei and three antipodal cells (figure 6). The antipodal cells are ephemeral 
and degenerate prior to fertilization. 

3*3. Endosperm 

Tlie primary endosperm nucleus divides much earlier than the zygote and the 
development of the endosperm is of the nuclear type. The endosperm remains 
free nuclear until the embryo reaches the early dicotyledonous stage (figure 8). 
During the course of its development the embryo sac enlarges enormously and 
encroaches on the surrounding nucellar tissue. This is more prominent at the 
micropylar region where it comes in direct contact with the innermost layer of the 
inner integument. At the chalazal region the tubular free nuclear part assumes a 
haustorial role (figure 7). The active growth of the endosperm haustorium at the 
chalazal end is arrested by the development of thick walled barrier tissue (figure 5). 
The cells of the innermost layer of the inner integument have dense contents and 
arc prominent (figure 9). 

3-4. Embryo 

The developmental sequence in the embryogeny is illustrated in figures 10 to 23. 
The zygote divides transversely resulting in a two celled proembryo. The apical 
cell (ca) divides by an obliquely vertical wall resulting in two unequal cells 
(figure 11). Another oblique division in the larger derivative cell'of the tier (cal) 
cuts off an epiphyseal initial (e) (figure 12). The basal cell (cb) undergoes a trans- 
verse division resulting in m and ci. Further divisions in the apical quadrant 
demarcate the tiers / and /'. Periclinal divisions in both the tiers differentiate the 
dermatogen. The epiphyseal initial undergoes a vertical division and by further 
divisions contributes to the stem tip in the mature embryo. 

The cell m divides by a vertical wall and both the cells divide transversely. Their 
upper derivatives contribute to the root tip and the root cap, while the deriva- 
tives of the lower cells together with those of tiers /* and n' contribute to the long 
and massive suspensor. A schematic representation of the zygote derivatives 
and their destinations in the mature embryo is given below : 

Stem tip 
i Cotyledons 

-I Hypocotyl and radicle 
Zygote 

Root tip and root cap 
Suspensor 




12 



V Seshavatharam 




21 



3 mm 



Figures 10-23. Stages in the development of embryo. 

Thus the enibryogeny according to Soueges scheme (Crete 1963) falls under 
Period I, Megarchetype IV and Series B 2 . 



4* Discussion 

The archesporium in the ovule is hypodermal in all the species investigated so far 
in the tribe the sole exception being Desmodium pmiculatum where a subhypo- 
dermal archesporium was reported by Rembert (1969). However, this needs 
verification in view of its rare occurrence in the family Papilionaceae. 

In Alysicarpus morilifer wall formation in the endosperm is initiated after the 
differentiation of the cotyledonary lobes in the embryo and is further restricted 
to the upper half of the embryo sac, while the chalazs 1 part functions as the haus- 
torium. A similar feature has earlier been reported by Rau (1953) in Desmodium 
tfiflomm, D. tortulosum, D. pulchellum, Eleiotis soraria and Aeschynomene indica ; 
by Kapuskar (1964) in Aeschynomene aspera and by Deshpande et al (1976) in 
Zornia diphylla. Stylosanthes mucronata (Rau 1953) is so far the only member 
of the tribe where a cellular endosperm is not organised at all in the developing 
seed. 

The tribe Hedysareae shows considerable variation in the mode of embryo 
development and in the nature and organisation of the smpensor. The variation 
met with in the embryogeny of the tribe is summarized in table 1. 



Embryology of Alystearpus monilifer D.C. 



13 



Table 1. Variation in the embryo development among the members of the tribe 
Hcdysarcac. 



Species investigated 



Author 



Embryo typo, afler Embryo type, after 
Johanscn 1950 Soueges 1948 



Coronilla minima 

Desmodium canescens 
D. canadense 
Hcdysaruni coronarium 
Hippocrepis comosa 
lespcdeza violacca 

Jrnithopus jpcrpusittus 
}ttobrychis sativa 
korpiurus vermicutata 
lornia diphylla 

ieschynoment indica 
i. aspcra 

Ittevigatum 



Soueges 1947 Coroniiia variation Period I, Megarchc- 

of the Oaugrad type VI, series A 
type 

Rau 1954 do. do. 

Ran 1954 do. do. 

Soueges 1956 ' do. do. 

Soueges 1955 - do. do. 

Rau 1954 do. do. 

Soueges 1953a do. do, 

Soueges 1953b do. do. 

Goursat 1961 do. do. 

Dcshpande and do. do. 
Bhasin 1976 

Rau 1951 do. do. 

Kapuskar 1964 do. do. 

Rau 1954 do. Period I, Megarche- 

type VI, Series B 



tlysicarpus monilifer 


Present study 


Alysicarpus varia- 
tions of the 
Onagrad type 


Period I, Megarche- 
type IV, Series B 


Irackis kypogca 


Smith 1956 


Solanad type 


Period I, Megarche- 
type V, Series C 


fedystrum nutats 


Johansen 1950 


Caryophyllad type 


Period II, Megarche- 
type IV, Series A 



The cmbryogeny in a majority of the species follows a more or less uniform 
pattern agreeing with that origii\3lly described by Soueges (1947) in Coronilla 
minima and belongs to the first embryogenic group under Period I, Megarchetype 
n and Series A. According to the system of Johaoiseji (1950) the embryogeny 
a all these species conforms to the Onagrad type, but does not fit into any, of 
he variations proposed by him. Hence it is now proposed to erect a new varia- 
ion designated as * Coronilla variation * under the Onagrad type to accommodate 
11 these genera of the tribe Hedysareae, belonging to Period I, Megarchetype VI 
nd Series A, where cb = s. 



14 V Seshavatharam . . 

Rau (1954) observed that Desmodium laevigatnm differs in its embryogeny from 
the rest of the members of the tribe which necessitates further investigations to 
evaluate the significance of this variation. 

Alysicarpus monilifer shows further deviation from the fundamental type, where 
the embryogeny falls under the second group along with Desmodium laevigatum 
but belongs to the fourth megarchetype in period I where cb = iec + co + s. In 
this respect Alysicarpus differs from the rest of the Hedysareae and resembles 
members of Phaseoleae and Galegeae. 

According to Johansen's system, the embryogeny in Alysicarpus rnonilifer 
conforms to the Onagrad type but docs not fit into any of the variations proposed 
under that type. The erntryogeny shows resemblence to the Trifolium variation 
of the Onagrad type in the possession of the epiphyseal initial, but differs in the 
destination of the derivatives of the basal cell cb. In Trifolium variation, based 
on the embryogeny in Trifolium minus (Soueges 1927) the entire basal cell cb 
contributes to the suspensor (cb = s) whereas in Alysicarpus the derivatives of 
the basal cell contribute to the root tip and root cap in addition to suspensor 
(cb = iec -f co 4- s). 

Hence, a new variation designated as Alysicarpus variation is proposed to accom- 
modate the type, having an epiphyseal initial and where the derivatives of the tier 
cb contribute to the root tip and root cap in addition to the suspensor. Alysi- 
carpus is so far the only genus in this tribe where an epiphyseal initial is demar- 
cated in the tier ca and cb = iec + co + s. In this respect it resembles Rhynchosia 
suaveolens of Phaseoleae, and Tephrosia strlgosa of Galegeae (Seshavatharam 1969) 
suggesting a relationship between these tribes and Hedysareae embryogenicaliy. 



Acknowledgements 

The author is grateful to late Professor J Venkateswarlu, for suggesting the 
problem and for his encouragement during the progress of this work. 

References 

Crete P 1963 Embryo. In tycent advances in Embryology of angiosperms (ed.) . Maheswari 
Intl. Soc. Plant Morphologists, New Delhi pp. 171-222 

Deshpande P K and Bhasin R K 1976 A contribution to the life history of Zornia diphylla Pers; 
/. Indian JBot. Soc. 55 115-124 

Goursat M J 1961 Etnbryogenie des Legumineuses. Developpement de Pembryon chezl e Scor- 
piurus vermiculata L. ; C.R. Acad. Sci. Paris 253 307-309 

Johausen D A 1950 Plant Embryology., Waltham Mass . \ 

Kapuskar A T 1964 Studies in the embryology of Legumtnosae II. Embryo development in ' 
Aesckyrtomene aspera Linn. ; Proc. Indian Acad. Sci. B60 87-94 

Rati MA 1951 Development of embryo in Aeschynomene indica Linn. ; New Phytol 50 124-126 

Rau M A 1953 Some observations on the endosperm in Papilionaceae ; Phytomorphology 3 
209-222 

Rau M A 1954 The development of embryo of Cyamopsis, Desmodium and Lespedeza, with 
a discussion on the position of Papilionaceae in the system of embryogenic classification; 
Phytomorphology 4 418-430 ' . 



Embryology of Atysicarpus monilifer D.C. 15 

Rembert D H (Jr) 1969 Comparative megasporogenesis in Papilionaceae ; Am. J. Bot. 56 

584-591 

Rendle A B 1925 Tfie classflcation of Flowering plants, Cambridge University Press 2 

Seshavatharam V 1969 A contribution to the embryology of Papilionoideae and Caesalpinioideae ; 
Ph.D. Thesis, Andhra University. 

Smith B N 1956 Arachis hypozaea. Embryogeny and the effect of peg elongation upon embryo 
and endosperm growth ; Am. J. Bot. 43 233-240 

Soueges R 1927 Embryogenie des Legumineuses. Developprncnt du proembryon Chez le Tri- 
folium minus Rehl ; C.R. Acad. Sci. Parts 184 1018-1019 

Sou6ges R 1947 Embryogenie des Papilionacces. Developpement de 1'embryon Chez le Coronllla 
minima L. ; C.R. Acad. Sci. Pans 224 1254-1256 

Sou6ges R 1948 Embryogenie ct classification 3 fasciculac Paris 

Sou6ges R 1953a Embryogenie des Papilionacecs. Developpement de T cmbryon Chez T Orni* 
tropus perpusillm L. ; C.R. Acad. Sci. Paris 237 1199-1201 

Soueges R 1953b Embryogenie des Papilionacees. Developpement de 1'embryon Chez 1' Onobry* 
chis sativa Lam. C.R. Acad. Sci. Paris 237 450-452 

Soueges R 1955 Embryogenie des Papilionacees. Developpment de 1'embryon Chez T Hippo- 
crepis comosa L. ; C.R. Acad. Set. Paris 24 2100-2103 

Sou6ges R 1956 Embryogenies des Papilionacees. Developpement dc 1'cmbryon Chez 1* Hedysa* 
rum coronarium L. ; C.R. Acad. Sci. Paris 242 704-707 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 1, February 1982, pp. 17-23. 
Printed in India. 



Non-inheritance of isomerism in cocoyams 

T VENKATESWARLU 

Central Tuber Crops Research Institute, Trivandrum 695017, India 

MS received 16 July 1981 

Abstract. The present communication describes the non-inheritance of isomerism 
in prefoliation and contortion of spathes in cocoyams. Importance of isomerism 
from the point of taxonomy and agronomy has been discussed. It is expected that 
a study on the orientation of leaf promordia at shoot apex and their spiral 
descendance would help in unravelling the hidden laws in shoot morphogenesis 
of cocoyams. 

Keywords. Isomerism ; cocoyams ; dextro ; levo ; contortion ; prefoliation ; 
taro ; taunia. 



1. Introduction 

Isomerism in biological materials was described by many workers under different 
names such' as asymmetry (Davis 1962) bioisomerism (Meyen 1973 ; Venkates- 
warlu and Hrishi 1977) enantiomorphism (Davis 1974), isomerism (Bahadur and 
Venkateswarlu 1976 a,b), chinlity (Venkateswarlu 1978) and radtel symmetry 
(Davis 1978). 

Isomerism in prefoliation and contortion of spathes in cocoyams was described 
in detail (Venkateswarlu and Hrishi 1977, Venkateswarlu 1978). In some crops 
this phenomenon is correlated with yield (Davis 1972). The present study is taken 
up to see if the corm yield is associated with prefoliation and if so whether this 
trait is transmitted through cormels and true seeds. Kasinov (1969) described 
that this character is transmitted through fronds in Lemna gibba L. The present 
paper describes the investigations carried out on the pattern of prefoliation in tuber 
and seedling progenies in taro and only tuber progenies in taunia. 

2. Materials and methods 

All the specimens used were taken from the collection of germplasm of edible 
aroids maintained at the Central Tuber Crops Research Institute, Trivandrum. 
The method of scoring a plant into levo and dextro was the same as that reported 
earlier (Venkateswarlu and Hrishi 1977 ; Venkateswarlu 1978). Observations on 
transmission of prefoliation through cormels in taro and tannia were recorded 
in 1978. The seed tubers were selected from labelled plants and sown. Observa- 
tions on prefoliation were recorded $t weekly intervals 1 . Based o the prefoliation 

17 



18 T Venkateswarlu 

pattern, plants were classified as levo, dextro and bichin 1. Observations were 
also made on prefoliaticm pattern on the main pseudostem and on the suckers 
developing from the main clump. 

For inheritance studies in taro seedlings, the nature of contortion in spathe \vas 
recorded at the time of effecting crosses and labelled as C95 L x C 271 D etc. 
as the case may be. Seeds obtained from the cross were sown and observations 
were recorded from the spathes; produced in the seedlings, Observations were 
also made on prefoliation in. taro seedlings, 



3. Results and discussion 

3*1. Observations on Frefoliatfon 

The phyllotaxy in cocoyaim is alternate. Unfurling of leaves is either clockwise 
(levo) or anticlockwise (dextro) (figures 1-6). Plants were labelled as levo, dextro 
and bichiral based on their morphology during the growth period. The corm 
and cormels of labelled plants were sown during 1978 to see whether the nature 
of prefoliation is transmitted vegetatively. The prefoliation pattern of seed setts 
and the resulting new pseudostems in both taro and tannia are presented in table 1 . 

Out of 60 levo tubers planted in taro, 28 grew into levo, 28 as dextro and 4 
bichiral types i.e., levo and dextro in 1:1 ratio. Total dextro tubers planted 
in taro were 21, out of which 9 developed as levo, 12 as dextro. Interestingly, 
no bichiral plants were produced when dextro tubers were planted. 

In tannia, bichiral plants were produced from both levo and dextro tubers. In 
all, 38 levo tubers were planted, from which 11 levo, 21 dextro and 6 bichiral 
plants were produced. From 42 dextro tubers sown, 17 behaved as levo, 21 as 
dextro and 4 as bichiral. There seems to be no association between the seed sett 
and the preudostems in cocoyams similar to that of Costus speciosus (Davis 1978). 

Observations were also made to know whether the new suckers developing from 
the seed sett differ in the nature of isomerism in prefoliation in taro. The data 
are presented in table 2. From the data presented in tables 1 and 2, it is obvious 
that the isomerism in 'prefoliation of cocoyams is not transmissible vegetatively 
which is contradictory to the results of Kasinov (1969) in Lemna gibba. further 
the whole phenomenon is complicated by an irregular pattern the suckers show. 

Similar observations were noticed in the seedlings of taro also. Levo and dextro 
seedlings were produced almost in a 1 : 1 ntio indicating the non-genetic nature 
of the character. This is similar to the findings of Davis (1962) in coconut. 

3-2. Contortion of spathes in taro seedlings 

An attempt was also made to study the inheritance pattern of spathe contortion 
in taro seedlings. Crosses were made in all possible combinations (L x L, 
L x D, D x D and D x i). At the time of flowering, the nature of contortion 
in spathes was recorded. Seedlings of all the crosses flowered except of D >< D. 
The data on the asymmetry of spathe are presented in table 3. It is clear from 
the data that both levo and dextro spathes were produced in almost ecjuai numbers 
the same plant. Davis(1962) also reported similar 1 ; 1 ratio of left 



Non-inheritance of isomerism in cocoyams 



19 




Figures 1-6. 1. Levo prefoliation in taro, 2. Dextro prefoliation in taro, 3. Levo 
contortion of spathe in taro, 4. Dextro contortion of spath in taro, 5. Levo 
prefoliation in tannia, 6. Dextro prefoliation in tannia, 



20 



T Venkateswarlit 




Figures 7-8. 7. Levo clextro prefoliation in the same clump of taro, 8. Lcvo 
and doxtro prefoliation in the same clump of tannia. 



Non-inheritance of isorncrlsm in coco yams 
Table 1. Isomerism in prefoliation of seed setts and new pseiidostems. 



21 



Name of the Nature of No. of 

crop seed sett seed setts 

planted 



Isomerism in aew pseuclostems 



Levo 



Dexlro 



Bichiral 



Total 



Taro 


Levo 


60 


28 


28 


4 


60 




Dextro 


21 


9 


12 


... 


21 




Total 


81 


37 


40 


4 


31 


Tannia 


Levo 


n 


11 


21 


6 


38 




Dextro 


42 


17 


21 


4 


42 




Total 


80 


28 


42 


10 


80 



right spiralled plants in coconut in all the four combinations indicating that this 
character is not genetically controlled. 

Furthermore, the fact that both levo and dextro rotatory leaves and spathes are 
produced on the same plant suggests that this phenomenon is non-genetic 
(figures 7,8). At the same time, it is interesting to see some plants behaving 
either as levo or dextro throughout their growth period with respect to both 
prefoliation and contortion (Venkateswarlu 1978). Such, plants were termed 
unichiral. 

Isomerism, a fairly common mechanism in biological materials has attracted 
the attention of several workers. This is exhibited in different characters like 
prefoliation in leaves, contortion in floral parts, twining in stems and coiling of 
shells in molluscs. 

This phenomenon was widely used over the years by taxonomists, phylogene- 
ticists and agronomists . Hutchinson(1964) used twisting of keel petals and styles 
for taxonornic purposes. Smartt (1976) pointed out that the Asiatic species of 
Phaseolus show clock-wise contortion of style and the American species show 
counter clock-wise contortion. More recently, Jos and Venkateswarlu (1978) 
used twining nature in Asian yams to describe their distribution. It was suggested 
that the phylogeny of dextral types might be basically different from that of 
sinistral types even among the Asian yams. 

In palms (coconut, arecanut and toddy palm) the nature of leaf spiral determines 
the yield. Right spiralled plants were found to be higher than their counterparts 
(Davis 1972). Bahadur et al (1978) pro-bed into the causes resulting in higher 
yield in right spiralled coconut. Their study revealed that the right spiralled 
coconuts were physiologically superior to the left spiralled ones and therefore the 
higher fruit production in right handers was correlated to their efficient metabolism. 

In Dioscorea etsculenta, the levo plants were found to give more yield than the 
dextro plants under artificially controlled conditions. But the vericais were better 
than lefts Ijy 42-63% and the rights by 55*57% (Davis 1972). 



22 T Vertkateswarlu 

Table 2. Isomerism in prefoliation of main stem and the suckers in taro. 



Nature of 


No. of 


Prefoliation in suckers 








main stem 


tubers 


Levo 


Dextro 


Total 




planted 








Levo rotation 


8 


4 


15 


19 


Dextro rotation 


3 


3 


3 


6 


Total 


11 


7 


18 


25 



Table 3. Isomerism in spathes of seedling families in taro. 



SI. Cross particulars Levo Dextro Total 

No. 



1. 95LX 


271 L 


1 


2 


3 


2. 271 L x 


251 L 


1 


1 


2 


3. 271 L X 


135 1, 


4 


3 


7 


4. 271 L X 


268 D 


3 


3 


6 


5. 271 L X 


211 D 


1 


2 


3 


6. 271 D X 


Kovvur L 


2 


1 


3 




Total 


12 


12 


24 



Similar studies conducted in cocoyams did not show any convincing results. 
Moreover, since this character is, not even transmitted vegetatively, the results will 
not have any bearing on its utility as selection criterion. 

At the moment, we are not in a position to understand the factors responsible 
for the expression of levo, dextro, and bichiral nature in the prefoliation and con- 
tortion of spathes in cocoyams. Recent study by Davis (1978) on Costus spetiosns 9 
suggests the involvement of one or more important laws governing plant growth 
and. he hopes that a study on the orientation of leaf primordia at shoot apex and 
their spiral descendance may help in unravelling the hidden law in shoot morpho- 
genesis. 

.'Similar study in cocoyams may also reveal some interesting facts responsible 
for the mechanism as the new leaves and spadices arise from shoot apex. 



Non-inheritance of isomerism in cocoyams 23 

Acknowledgements 

The author is highly grateful to Prof. T A Davis, FAO, Coconut Expert to 
Indonesia and Dr J S Jos for the constructive comments on the manuscript. The 
author is also thankful to Shri P K Thomas, Director of CTCRJ for providing 
facilities and encouragement. 

References 

Bahadur B and Venkateswarlu T 197(>a Isomerism in flowers of four species of Jaiwp\\a\ /. 

Indian Hot. Soc. 55 30-37 

Bahadur B and Venkateswarlu T 1976b /. Indian Bot. Soc. 55 $9-94 

Bahadur B, Madusudhana Rao M, Chandraiah M and Lokendar Rao K 197$ The physio- 
logical basis of handedness in relation to yield in Cocos nucifera, L A proposal ; Incomp. 

Newslett. 9 108-111 

Davis T A 1962 Non-inheritance of asymmetry in Cocos nucifera ; /. Genet. 58 42-50 
Davis T A 1972 Effect of foliar arrangement on fruit production in some tropical crop plants ; 

Tropical Ecology with an emphasis on organic production Athens (U.S.A.) pp. 147-164 
Davis T A 1974 Enantiomorphic structures in plants ; Proc. Indian Nat. Set. Acad. B40 424-429 
Davis T A 1978 Radial symmetry in Costus speciosus ; Phytomorphology 28 373-378 
Hutchinson J 1964 The Genera of Flowering plants (Angiospermae) Dicotyledons (London : 

Oxford University Press) 
Jos J S and Venkateswarlu T 1978 Twining in relation to distribution among Asian yams; 

J. Root Crops 4 63-64 
Kasinov V S 1969 Inheritance of left and right handedness in Lemnaceae and other organisms ; 

Genetika 5 22-29 

Meyen S V 1973 Plant morphology in its nomethetical aspects ; Bot, Rev. 39 205-260 
Smartt J 1976 Tropical pulses (London : Longmans) 

Venkateswarlu T 197S Unichirality in Edible Aroids ; /. Root Crops 4 15-18 
Venkateswarlu T and Hrishi N 1977 Bioisomerism in Edible Aroids ; /. Roof Crops 3 29-32 



Proc. Indian Acad. Sci. (Plant Sci.), VoL 91, Number 1, February . 1988; -pp--' 25^35. 
Printed in India, 



Apomixis in Ceitchms glaucus Mudaliar et Suudaraj 



C SHANTHAMMA 

Department of Post-Graduate Studies and Research in Botany, University of 
Mysore, Manasagangatri, Mysore 570006, India 

MS received 18 July 1980 ; revved 29 December 1JMI1 

Abstract. A detailed cytoembryological investigation in Cenchrus gfaucus revealed, 
that it is ail obligate apomict producing only aposporous embryosacs. Microsparo-. 
genesis disclose chromosomal irregularities and mcgasporogeiiesis occasionally occur 
and the development of sexual embryosac is completely absent. Otv the other hand, 
aposporous initials develop into 4-nuclcatc cmbryosacs. Autonomous development 
of tlie embryo is of common occurrence. 

Keywords. Cenchms glaucus ; obligate ; apospory. 



1. Introduction 

Cenchrus glaucus Mudaliar et Sundaraj, a pentaploid perennial tropical species 
belongs to the tribe Paniccac of Panicoideae. The occurrence of apomixts in this 
genus was first investigated by Fisher et al (1954) in Pennisetum clitoris and Cenr 
chrus setigerus. Later Snyder et al (1955) outlined a detailed study on the forma- 
tion of aposporous embryosacs in C. ciliaris, a polymorphic facultative apornict. 
Prelimiixary investigation has disclosed that another species of Cemchrus, C. glaucus 
an aneuploid with 2/i = 45, where in microsporogeaesis is characterized by 
abnormalities such as uaivalents, muJtivalents and lagging chromosomes, repro- 
duces by gametophytic apomixis. The present study reports the results of cytoent. 
bryological studies revealing the formation of only aposporous embryosac (obligate 
apomict) and the complete absence of sexual embryosac. 

2. Material and methods 

Clones were collected from Agricultural College, Coimbatore and were grown in 
the Departmental Botanical Garden, Manasagangotri, "University of Mysore, 
Mysore. Identification of the material was confirmed by the Botanical Survey 
of India, Coimbatore. A voucher specimen of the material is deposited in the 
Herbarium, Department of Botany, University of Mysore, Mysore, India. 

Florets at appropriate stages of development "were collected between 10 a.m. 
and i p.m. and fixed in a mixture of 3 : 1 absolute alcohol.aavd acetic acid, later 
on stored in 70% alcohol. Pollen mother cells were smeared in 2% acetocannine 
for the study of meiosis. For mitotk studies, root tips squashes were Cdfule follow- 

25 



2f C Shartthamma 

ing Tijo and Levan's (1950) technique. Embryological studies were carried out 
following conventional methods of dehydration, infiltration and embedding. 
Sections were cut at 10-44 microns in thickness and stained in Heidenhain's iron 
alum haematoxylin. 

3. Results 

3*1. Mttrosporogenesis 

During diakincsis and metaphase I, tetravalents, univalents in addition to biva- 
lents are formed (figures I, 5). Chromosome number was confirmed in root-tip 
(figure 11). Occasionally, a bridge and a fragment configuration indicating struc- 
tural changes were seen at anaphase I (figure 2). Anaphase distribution is marked 
by irregularities such as irregular distribution (figure 6), laggards (figure 7) and 
micronuclei formation at dyad stage (figure 8). Second division irregularities 
arc also pronounced with precocious movement of chromosomes at anaphase 
(figure 9). Figure 3 reveals a dyad in division stages with 2 micronuclei in one 




Figures 1-4. Microsporogcnesis. 1. diakinesis stage showing tetravalents, tri 
valents, urtivaleats and bivalents, 2. anaphase I showing bridge fragment configu- 
ration, 3. dyad in division showing single micronucleus at each pole in one of the 
dyad cells, while in the other, one ratcroouicleus, 4. tetrad with chromosomal 
material being left behind. 



Apomlxis in Certchrus glaucus Mudaliar et Sundaraj 



27 




Figures 5-11. Microsporogenesis. 5. metaphase I with multivalents, bivalents 
and umvaleiits, 6. anaphase with irregular distribution of chromosomes, 7. late 
ariaphase with lagging chromosomes, 8. dyad showing micronuclei, 9. dyad in 
division with micronucleus in one of the cells and also showing precocious movement 
of chromosomes, 10. polyad formation, 11. somatic cell with 45 chromosomes 
(magnification x 750). 



Apontfxis in Certchrus glauaus Mudaliar et Sundaraj 



29 




(For figure captions sec page 35) 



of the dyad cells, \vhile in the other a single micronucleus at one pole. On the 
other hand, figure 4 reveals chromosomal material being left behind in a tetrad. 
All these above irregularities lead to polyad formation (figure 10). Pollen sterility 
is about 51%. 



30 C Shanthamma 

3*2. Megasporogeitesis and megagametogenesis 

Megasporogcncsis begins with the differentiation of an hypodermal arehesporial 
cell \\hich Junctions as the megasporo mother cell (figure 27) and proceeds through 
the us ua I meiotic divisions. Figures 16 and 17 reveal degenerating dyad and tetrads. 
In addition, several ovular sections showed the inception of the mciotic divisions 
(figures 13, 14, 15), The presence of either sexual embryosacs or the stages leading 
to their formation wcie not observed in any ovule. 

3 3 . Aposporons embryosac 

Aposporous initials numbering from 1 to 8 in most of the ovules differentiated 
in the vicinity of rnegaspore mother cell (figures 12, 13, 14) or its meiotic products 
(figures 15, 16, 17) within the nuccllus. In the early stages of development 
aposporous initial which consists of a single nucleus located at one end of the 
cell with a prominent vacuole developing below it (figures 18, 29) undergoes the 
first mltotic division resulting in 2 nuclei and both the nuclei are located at the 
same pole (figures 19, 28). The next division would result in a 4-nucleate embryo- 
sac and all the 4-nuclei remaining at the same pole (figures 20, 30). Later on, 
organization of the four nuclei takes place, 3 nuclei contribute to form an egg 
apparatus of 2 synergids and an egg and a solitary polar (figures 21, 31). 
However, synergid nuclei degenerate very early, leaving an embryosac with an 
egg and a polar nucleus (figures 22, 32). Occasionally, the 4-nuclei without 
organization undergo further divisions resulting in the formation of 6 to 8 nuclei 
all grouped together (figure 25). The development of aposporous embryosac 
may be simultaneous (figures 19, 22, 29) or may be non-simultaneous (iigures 20, 
21, 31). 

In addition to the aposporous cells 2 or 3 nuccllar epidermal, cells at the micro- 
pylar end become conspicuous with dense cytoplasm and enlarged nuclei (figure 30). 
In extreme cases aposporous smbryosacs are found at the attachment of the ovule 
to the ovary wall (figure 32). The average value of embryosacs per ovule is indi- 
cated in table 1. 

3-4. Embryo and endosperm 

The association of embryo and endosperm varies in different embryosacs of the 
same ovule. Embryosacs containing well developed embryo and multinucieate 
endosperm occur along with embryosacs containing * n imdivjdcd egg cell and a 



Table 1. Average value of embryosacs per ovule. 



No. of embryosacs per ovule 



1234567 



No. of of ovules examined 15 16 30 15 IS 16 10 10 



Apomixis in Cenchrus glmuus Miulaliar cf Sumlar 



31 



'i-Wft.M. 




**ev^..*ifSS 






-Y 



>*. 



S 



}'*'*{*, 

m ''ASi. 



29?ft-.;75. so V;. "& & y$JKS: : * 







' 



^'P ^' 81 ^' aposponms cmbryosac development and embryo 
endosperm reIat,o n ,Wp, 27. MMC, 28. 2-binuclca.e aposporoas cmbryosac, 
l o'rZr 6 ap Sp0rOUS einb '-y sacs ' 30 " 4-iwIcate aposporous cmbryosac 
^ , , aposporou$ "*ryosac, 32. aposporous cmbryosao developing at 

n P ti0n f th VU 



X 150) 



" y0> 



th 

many Ce " C<l cmbr y w;th 
Sh wing disturbed P laril y (magnification 



Apomms in Cenchrus glauctts Mudaliar et Sundaraj 33 

single polar nucleus in the same ovule (figure 23). Embryosacs containing embryo 
with a single polar nucleus coexist with cmbryosaes containing undivided egg cell 
with endosperm in the same ovule (figure 24). Sometimes embryos lack normal 
polarity (figures 26, 34) and the polar nucleus is found lateral to the embryo. 
Precocious development of the embryo is frequently encountered as in figure 33 
and a polar nucleus is found towards one side. Although, the number of embryos 
varies from 3 to 4, twin seedlings arc only 4%. Those seedlings ako reproduced 
apomictically and triple seedlings were absent. 



4. Discussion 

Cettetmts gluucus is an obligate pcntaploid apomict with chromosome number 
2n = 45. Microsporogenesis proceeds with chromosomal irregularities resembling 
those found in Ccnclirus ciliaris (Fisher et at 1954) resulting in about 51% sterile 
pollen. The presence of high percentage pollen viatility in C. glaucus may iridiocte 
pseudogamous seed formation. The chromosomal abnormalities in this taxon 
are similar to Pemiisetum setaceum, P. villosum (Narayan 1951) which arc also 
obligate apomicts. According to Weimarck (1972) such aberrations in Hierochloe 
species seem to be associated with the occurrence of apomixis. 

Mcgasporogencsis sometimes occurs and embryosac formation is. rarely cnoouru 
tered. Jn aposporous apomicts irregularity of meiosis at the time of megasporc 
formation may affect the ability of the cmbryosac formation. Degeneration of 
all the megaspores sometimes observed in C. glaucus may be due to irregular 
meiosis as reported in Hieracium auraittiacum (Skalinska 1971) and in Poa praten* 
sis (Qrazi et al 1961). However, 4-nucleate unreduced embryosac is of common 
occurrence. On the other hand, the megaspore mother cell or its products degene- 
rate autonomoulsy even before the origin of aposporous initials. Such autonomous 
degeneration even before the origin of aposporous initials is reported in Fertilise turn 
villosum, P. setaceum (Narayan 1951), Paspalum secans (Snyder 1957), Po a grant- 
tica (Skalinska 1959), Heteropogon coittortus (Emery and Brown 1958), Sotfoiochlofl 
schaemum (Brown and Emery 1957), Fenitisetum ciliare (Snyder et al 1955), 
Puniciim maximum (Warmkc 1954) and in Boutdouva curtipertdula (Mohamed 
and Gould 1966). 

The presence of 4-nucleate aposporous embryo^ac in this genus was reported 
first in Ceitchrus clliaris (Snyder et al 1955). The 4-nucleate embryosac develop* 
ment has been described in detail in nearly 14 species in Poaccae. Brown aa\d 
Emery (1958) have reported the presence of 4-nucleate aposporous cmbryosacs 
in 43 of 153 species. 

A striking feature of this species, however, is apart from embryosacs present 
in the nucellar portion, embryosacs are ako found in such extra-nucellar places, 
the broad base of the ovule where it is attached to the pericarp wall. Such nucellar 
embryosacs have been reported in Pertnisetum villosum and P. sctaceum (Nafayan 
1951), P. meziajtifm (Shaaithamma 1974j, Themeda iriandra (Brown and Emery 
1957), Hierochloe odorata (Weimarck 1967), Hieracium wrantiacum (Skalinska 
1971) and Hieracium pratense (Skalinska and Kubien 1972). 

Embryos in C. glaucus arise exclusively from unreduced aposporous embryo- 
sacs, Poiyembryony as studied by Norstog (1957) is high in Poaceae. Armstrong 

P.CB)-3 



34 C Shanthamma 

(1W7) reported 42% polyembryony in a particular strain of Poa. Snyder et al (1955) 
reported 20% of polyembryony in Peimlsetum etliare, but in Cemhrus glaucus only 
4% of the seeds produced twin seedlings. (All the seedlings rcporduced apomicti- 
cally. Triple seedlings were totally absent although in oviilar sections apparently 
2 to 3 embryos per ovule were commonly observed). The low frequency of twin 
seedling produced in C. glaucus may be attributed to the lack of endosperm for- 
mation in cinbryosacs of a single ovule. 



Acknowledgement 

The author is deeply indebted to Dr K N Narayan for his guidance and encoura- 
gement throughout the investigation. 



References 

Armstrong J M 1937 A cytological study of the genus Poa L. ; Can. f. Res. (Bot. Sci.) 15 

281-297 

Bar N L 1960 The grasses of Burma, Ceylon, India and Pakistan (London : Pcrgamun Press) 
Brown W V and Emery W H P 1957 Apamixis in. the Gramincac, Tribe Andrapogoneuc ; 

Themeda triandra and Botkr'wchloa ischaemiun ; Bot. Gaz. 118 246-253 
Brown W V and Emery W H P 195$ Apamixis in the Gramineac, Panicoidcuc ; Am. J. Bot. 45 

253-263 
Emery W H P and Brown W V 195& Apomixis in the Gramincac , Tribe Andropogoneue : 

Heteropogoii contonus ; Madrono 14 238-246 
Fisher W D, Bashaw E C and Holt E C 1954 Evidence for apomixis in Pennisctum ciliare and 

Cenchrus setigems ; Agrori. J. 46 401-404 
Gildfitthuys P J and Brix K 1959 Apomixis in Pennisetiun dubiuni ; S. Afr. J. Ayrte. Sci. 2 

231-245 
Grazi F, Umaerus M and Akerber^ E 1961 Observations on the mode of reproduction and 

embryology of Poa pm tens is ; Hereditas 47 4iJ9~541 
Moharaed A H and Gould F H 1966 Biosystematic studies in the Boittdoua ctirtipeitdula complex 

V., Megasporogenesis and embryosac development ; Am. J. Bot. 53 166-169 
Mudaliar et Sundaraj 195& Cenchrus glaucus ; /. Boftibay Nat. Hist. Soc. 54 926 
Narayan K N 1951 Cytogeitetic studies in apomictic Pennisetum species ; Ph.D. Thesis, University 

California', Berkeley. 

Norstog K J 1957 Polyembryony in Hierochloe odomta (L.) Beauv ; Ohio J. Sci. 57 315-320 
Shanthamma C 1974 Studies in Poaceae Unpublished thesis 
Skalinska M 1959 Embryological studies in Poa granitica Br. B1. 9 an apomictie species of the 

Carpathian range ; Acta Biol. Crac. Ser. Bot. 2 91-112 
Skaiinska M 1971 Experimental and embryological studies in Hieradum auranliacum Z. ; Acta 

Biol. Crac. Ser. Bot. 14 139-155 
Skalinska M and Kubien 1972 Cytological and embryological studies in Hieracium pratcme 

Tausch ; Acta Biol. Crac. Ser. Bot. 15 39-55 
Snyder L A, Hernandez H E and Warmke H E 1955 The mechanism of apomixis in 

Pennisetum ciliare ; Bot. Gaz. 116 209-221 

Snyder L A 1957 Apomixis in Pasjpalum secans ; Am. J. Bot. 44 318-324 
Tjio J H and Levan A 1950 The use of oxyquinoline in chromosome analysis ; Am. Estec. 

Exp. Anta. Dei. 2 21-64 
Warmke H E 1954 Apomixis in Panicum maximum ; Am. J. Bot. 41 5-11 



Apomixis in Certchrus gluitcus Mudaliar ct Similar aj 35 

Weimarck G 1967 Apomixis and sexuality in Hierochloe austral? s and in Swedish Hierochloe 

odomta on different polyploid levels ; Bot. Not. 120 209-235 
Weimarck G 1972 Male meiosis in some Ainphimictic and Apomiclic Hierochloe (Gramineae) ; 

Bot. Not. 126 7-36 



Figures 12-26. Mcga^porogcncsis, aposporous embryo-sac development, and embryo 
and endosperm relationship. 12. LS of nuccllus showing megaspore mother cell 
and 3 conspicuous nucellar aposponnis. cells (x 3000), 13. MMC at diakinesis 
and 2 aposporous cells, ( x 3000), 14. metaphasc I of MMC with a single apos- 
sporom coll initial (x 3000), 15. dyad and 3 prominent nucellar cells (x 3000), 
16, 17- degenerated dyad and tetrad with nuccllar cells (adjoining) prominent 
(x 2000), 18. a group of 4 aposporous ( mi nucleate) crnbryosacs (x 2000), 19. a 
group of 3 aposporous cmbryosacs., 2 cmbryosacs at binuclcate stage and the other 
one is at mctaphase, 20. 3 aposporous. cmbryosac?, one at 4-nucleate stage while 
the third one at anaphase (x 2000), 21. a group of 3 aposporaus embryosacs. at 
dilTercat stages of development and one of the embryosacs is at 4-nucleate stage 
(x 1200), 22. 3 apo.sporo.us. embryosacs showing 2 degenerated synergids, an egg 
and a polar nucleus ( X 2000), 23. two apo&porous embryosacs, one showing many 
colled embryo and free nuclear endosperm and the other embryosac showing 
an egg and a polar nucleus, (x 1200), 24. ovule showing 2 embryosacs, one with 
many celled embryo and a single polar nucleus while the other embryosac with 
a single egg cell and 4 endosperm nuclei (x 1200), 25. a group of 4-aposparous 
embryosacs, 3 embryosacs arc binuclcate and the remaining one showing unorga- 
nized 6-nucieate condition (x 1 200), 26. an aposporous embryosac with disturbed 
polarity of embryo (x 1200). 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Ho. 1, February 1982, pp. 37-41. 
(0) Printed in India. 



Regeneration of plantlets from callus of Elettaria cardamonwm Mat on 

N K SR1NI.VASA RAO, S NARAYANASWAMY*, E K CHACKO 

and R DORE SWAMY 

Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural 

Research. (ICAR), Bangalore 5(50080, India 

*Emeritus Scientist, Council of Scientific and Industrial Research, New Delhi 

MS received .3 November 1980 ; revised 1 October 1981 

Abstract. Embryo callus and callus of rootstocks of in w'/ro-raigod seedlings of 
Elettaria canlamomtwi were grown on MS medium supplemented with C\V -1-2, 
4-D -1- BAP. Differentiation of Shoot buds, roots and leaves leading to tho develop- 
ment of plantlets could be induced irt callus by withdrawing 2, 4-D or substituting 
it by IAA or NAA in low concentrations. 

Keywords. Callus culture ; cardamom ; regeneration. 



1. Introduction 

Reports on induction of shoot buds and whole plants from tissue cultures of both 
monocotyledonous and dicotyledon or s plants have been numerous in ree en t years 
as evident from the spate of publications or the subject (see reviews by Murashige 
1974 ; Narayanas\vamy 1977). Clonal propagation through tissue culture has 
been successful with many spice and condiment plants such as Foenicttlum vulgcrc 
(Maheshwari and Gupta 1965), Anerhum graveolens (Ratnambs and Chopra 1974), 
Carwn corvi (Ammirato 1974) and Capsicum annuum (Ounay and Rao 1978), 
Spectacular rate of multiplication of turmeric (Curcuma longa) plants have been 
reported in cultures of young vegetative buds isolated from the root stock (Nada- 
guada et al 1978). This prompted us to investigate the potential for organogenesis 
in tissue cultures of the cardamom (Elettaria car damomurn Maton of Zingiberaceae) 
widely used as a condiment. This paper reports the successful regeneration of 
shootbuds and plantlets from seedling callus of the herbaceous perennial species, 

2. Methodology and results 
2 1 . Seed germination 

Dry seeds of cardamom were surface sterilised by 0-lJj mercuric chloride solution 
to which a few drops of the detergent Teepol had been added. After washing 



Contribution number 944 of Indian Institute of Horticultural Research. 

37 



38 N K Srinivasa Rao et al 

thoroughly in sterilized water, the seeds were sown on White's (1963) nutri 
agar. Slender seedlings -were obtained in three weeks on incubation at 26 C 
Whole seedlings bearing tke first sheathing leaf and plumule were transferred 
Murashige and Skoog's (1962) medium (MS) to which auxins, cytokinins t 
coconut water (CW) as specified (table 1) and sucrose (2%) had been added. 
EDTA was used as the iron source. Each treatment comprised 12 replicai 
Embryo callus was also obtained directly from seeds sown on the medium (8gur< 
containing an auxin such as 2, 4-D (2,4-dichlorophyenoxy acetic acid). 

2-2. Callus induction 

Five-week-old 3 cm long seedlings bearing the first sheathing leaf and plum 
were transferred to MS medium to which CW (18% v/v) H- 2, 4-D(2mg/l) 
indole-3 -butyric acid (IB A 2mg/l) or naphthalene acetic acid (NAA 2mg/l) a 
benzylaminopurine (BAP 2mg/l) had been added in combinations as need 
Proliferation of cells from the rootstock was observed in 75% of the cultures res 
ting in the formation of exuberant callus in 6 weeks after incubation (figinc 
MS supplemented with CW (18% v/v) 4- 2, 4-D (2mg/l) + BAP (0-5mg/0 \ 
conducive for callus initiation and growth which couJd be augmented by the ad 
tion of casein hydrolysate (CH, lgm/1.) in the medium. Neither yeast cxtr 
250mg/l nor malt extract (250mg/l) proved favourable for callus growth; 2, 4 
could, however, be replaced by IBA for callusing. Propicmocarmine squasl 
of the proliferating callus showed cells of diverse sizes and shapes (figure 
Trachcidal differentiation of cells was marked. 

2-3. Regeneration of shoot buds 

Callus growing on. MS + CW + 2, 4-D \vas subcultured on medium devoid 
2, 4-D but containing 1AA. (2mg/l) or NAA (lmg/1). Three weeks after trans 
green nodular structures developed in the callus indicating the initiation of organic 
growth centres. CalJus grown on medium with higher concentrations of the an? 
became friable and was not conducive for shoot bud induction. But induct! 



Table 1. Responso of cardamom calli to growth regulators in vilro oit soqucn 
transfer. 



SI. Media composition Nature of 

No. (Hormone concentrations in mg/1) response 



1 . MS -I- CW (18% v/v) 4- 2, 4-D (2) Callusing good 

2. MS -h CW(18% v/v) -f 2, 4-D (2) -1- CH(1g/l) -h BAP CaHusiag exuberant 
(0-5) 

3. MS -f CW(18% v/v) -f- lAAor IBA(l) -hNA\(2) Callus grew as vascular nodul 

4. MS-hCW(18%v/v)4-lAA(l)^BAP(2) Shoot bud initiation in call 

(80%) 

5. MS 4-CW(tO% v/v) 4-BAP(2~5) -f-UA (1) 4-6 jjhoot buds per subcultu 



Regeneration in Elettarla aardarnoinum 



39 




Figures 1-5. Callus induction and shoot bud regeneration in cardamom. 
1. Calhtiiitg of seedlings in vitro on MS ~f GW (1 8 %v/v) 4- 2,4-D (2 mg/1) -{-BAP 
(ft P 5 mg/1) 6 weeks after incubation, X 1 *5. 2. Nodular callus derived from root 
stock of seedling transferred to MS medium -h 1. AA (t mg/l) ~h NAA (2 mg/l) 
x 1-5. 3. Propionocatmine squash preparation from root-stock callus piece 
showing free cells and cell aggregates x 800. 4. Regeneration of a shoot bud 
in primary callus grown on MS -f BAP (2 mg/l) + 1 AA (mg/l), x 1 5. 5. Cluster 
of shoot buds regenerated from callus Subcultures ort MS 4- CW (10 %v/v) -I- BAP 
(2mg/l)-HAA (I mg/l) x 1-5. 



Regeneration m Elettaria cardamomum 41 

scurrcd if MS medium was supplemented with CW (10% v/v) 4- BAP 
1-5 mg/1) with or without the addition or 1AA and incubated for 6 weeks under 
00 lux (figures 4,5). Four to six shoot buds could be obtained from each callus 
ibeulture of uniform size, Rooting occurred at the base of individual shoot 
uds on prolonged incubation in the same medium (aged cultures) or vrhen indivi- 
ual shoottets were isolated and grown on White's medium to which NAA (2mg/l) 
nd sucrose (1%) had been added. 



Discussion 

.egeneration of plantlets in callus culture is an alternate means of propagation 
i cardamom. Callus subcultures could develop 4-6 rcgcncnmts, each of which 
as capable of rooting, when isolated and grown, forming a whole plant. Plant- 
is propagated thus might not conform to parent genotype, .having been obtained 
om seedling calii. Nevertheless, tissue culture provides a method by which a large 
umber of strains could be obtained for selection of desirable variants. Also, it 
tTers a method of rapid multiplication of elite varieties through multiple shoot 
duction. Preliminary studies have shown that test tiibc plants could be success- 
illy transferred to soil. 



cknowledgements 

he authors thank J>r G S Randhawa, the then Director, Indian Institute of 
horticultural Research, Bangalore, for his keen interest and for providing faci- 
:ics for tissue culture work. Thanks are also due to Dr I> Oundu Rao, Senior 
ant Pathologist, for the supply of cardamom seeds. 



ifcmicos 

.mnirato I* V 1974 The affects of ab&igio ucid ott the development of Somatic embryos: from 
colls of caraway (Canwt carvi L.) ; Bat. Gaz. 135 328-337 

iitay A L and Rao P S 1978 In vitro regeneration from liypocotyl and cotyledon. ox?lanttf of 
rod pepper (Capsicum annum) ; Plant Sci. Lett. 11 3(55-372 

ahoshwari S C and Oupta R. P G 1965 Production of adventitious cmbryo'idtf in vitro from 

stem callus of Foenicutuni vttlgare ; Planta 67 384-386 

urasliigo T 1974 Plant propagation through tissue culture ; Ann. Rev, Plant Physio 1 . 25 
135-166 

urasbuge T and Skoog'F 1962 A revised medium for rapid growth and bioassays with tobacco" 
tissue cultures ; Physhl. Plant. 15 473-497 

idaguada R S, Mascarcnhas A F, Hendrc R R and Jaganathan V 1978 Rapid multipli- 
cation of turmeric (Curcuma longa Iiirtit.), plants by tissue culture ; Indian /. Exp. Bio I. 16 
120-122 

urayanaswamy S 1977 Regei\cratioit of plants from tissue cultures; In Fundamental and Applied 
Aspects of Plant Cell, Tissue ami Organ Culture (ed$.) J Reinort and Y P S Bajaj (Berlin : 
Springer-Verlag) pp. 179-250 

>tnamba S P and Chopra R N 1974 In vitro induction of embryoids from liypocotyl and 
cotyledons of Anelhwn graveotens seedlings; Z. Pflanzenphysiol. 73 452-455 

ute P R 1963 The Cultivation of Animal and Plant Cells (New York : Ronald Press) 



Proc. Indian AcadL Set. (Plant Sci.) Vol. 91, Number 1, February i982, pp. 43-53. 
Printed in India. 



Studies in Cyperaceae : XVII Novelties in Fimbristytis (L.) Vahl and 
their vegetative anatomy 



EGOVINDARAJALU 

Department of 'Botany, Presidency College, Madras 600005, India 

MS' received 27 March 1 980 

Abstract. T\vo novelties of Fimbristylis collected from Tamil Nadu (Madras Stale) 
arc described and illustrated. One of them belong* to tli section Fuscac and the 
other one to Cyinosae. The vegetative anatomical characters of these two novelties 
not only fall within 1 the ambit of the range of anatomical variability of the genus 
as a whole but their respective anatomical features in combination appear to be 
characteristic and different from those of the species already known anatomically . 

Keywords. Novelties \n Fimbristylis ; vegetative anatomy. 

Fimbristylis scabnsquama Govind $/>. nov. Sect Fuscac Ohwi (figure 1) 

Peremis. Rhizoma btevisshna, Lignosa. Cu/mj 1-2, pentaquetri, valdo costati, 
sulcati, glabri, rigidi, erecti, straminei-atrobrunnei, scabridi in dimidio superiore, 
tecta ovato-lanceolatis sq.i\amis et denique fibro&is sq.uarnis, 15-25 cm x 0-6- 
0-8 mm. Folia multa, spiraliter disposita, canaliculata cum marginc incurva 
rccurva, brunnea-atrobrunnea, demxini evadentia nigra, eligiilata, aci\mir.aia 
(acuta), incrassata et in margine dimidio superiore scabrida, culmo quam angus- 
tiora, 10-15 cm x J-2 mm ; vaginae omnes lammiferae, glabrae, oblique trim- 
catae, nitideac vel pallidae, briunae aliquando evadcn& nigtae, pi\\smimisve cor- 
ncac. Inflorescentia simplex, contracta, capitata, consistens (2-) 3-8 (-10) spiculis, 
l-2-l-8cm longa et hta. Bracteae anguste ovatae, aristatae, plusminusve 
scabridae ia margine aristae, glabrae, inflorescentia multo breviores, 0- 5-1 -Ocm 
longae. Radii primari O, raro cum-unus radius adest, triquetroi^s, laeves, usque 
1cm longus, Spiculae elliptico ovatae, acutae, castaneo^bmnnae, plerumquc 
fasicutatae, aliquando soiitariae, sessiles, subteretes, 10-16 florae, 6-7 x 2-3 mm^ 
Glumae late ovatae, acutae-stibacutae, distichae in dimidio inferiore vel plus 
minusve per totum proprie oblique et lineariter scabridae in nudatis partibus, 
anguste scariosae ad marginem, pleramque ciliolatae in dimidio superiore margine 
cum lateribus inervis, cymbiformes, charatceae, nitidae, adpressae, mi\cronatae, 
4-0-4'2 (incluso mucrone) x 3-0-3 ; 2ram ; carina aliquando valida 3 (-5) nervia 
ncrvis excurrentibus in mucronem ; mucro plerumque recurvata, 0*5 mm longa ; 
nervis lateralis levior. Rhachilla pannose alata, excavata. Stamina 3 ; filainenta 
longe flexuosa, tenues, tortuosa, 2-5-2'8min longa ; anthera brun^ea, apwiculata 

43 
P.(B)-4 



44 



E Govindarajalu 




Figure la-g. Fimbmtylis scabrisquama Govind. sp. uov. a. habit x \\ b. style 
and stigma x 11; c. spikelet x 5; d. glume, lateral view x .10; c. stamen 
x 8; f. nut x 23; g. glume, spread out x 10; (from Govindarajalu, 
12,110, type). 

cum plus minusve apice setacea, Uneares, distincte calcarata adbasim, 1-9-2*0 mm 
pnga. Stylus triqueter, vix dilatata ad basim, distincte fimbriata in superiore 
tertia parte vel dimidia, 24-2-5 mm longus ; stigmata 3, brevis ciliata per totum 
stylo breviora, 1-7-1' 8 mm longa, Nux angusta oblongo ovata, triquetra, tricos- 
tulata, alba stramiaea cum leviter lateralis convexa> stipitata, non vimbonulata 
cum triangulf riter apice plana, aliquantulum aspera ob minutas transverse elon- 
gates tuberculas in dimidio superiore, 0-6-0 -8 xO'3-0-4mm; cellulae epicar- 
picae in dimidio superiore circulares (plus minusve hexagonales), minutae, reticu- 
latae ; stipes 0-12^0- 15 mm longae. 

Perennial. Rkizome short, woody. Claims 1-2, pentaquetrous, strongly ribbed, 
sulcate, glabrous, rigid, erect, stramineous-dark brown, scabrid in upper half, 



Novelties in Fimbristylis (L.) Vahl 45 

covered by ovate-lanceolate scales and ultimately by the fibrous remains of the 
scales, 15-25 cm x 0-6-0 -8 mm. Leaves many, spirally arranged, canaliculate 
with incurved margin, recurved, brown-dark brown finally tending to become 
black, cligulate, acuminate (acute), thickened and scabrid in upper half margin, 
shorter than culms, 10-< 15 cm x 1-2 mm ; sheaths all laminiferous, glabrous, 
obliquely truncate, shining or dull, brown sometimes becoming black, more or 
less horny. Inflorescence simple, contracted, capitate consisting of (2-^) 3-8 (-10) 
spikelets, 1-2^1 -&cm long and broad. Bracts narrowly ovate, aristatc, more or 
less scabrid in the aristatc margin, glabrous, much shorter than inflorescence, 
0-5-4'Ocm long. Primary rays O, rarely when 1 present triquetrous, smooth 
up to 1 cm long. Spikelets elliptic ovate, acute, castaneous brown, usually clus- 
tered, sometimes solitary, sessile, sub terete, 10-16 flowered, 6-7 x 2-3 mm. 
Glumes broadly ovate, acutc-subacutc, distichous in lower half or more or less 
throughout, characteristically obliquely and linearly scabrid in uncovered parts, 
narrowlys carious at margin, ususlly ciliolate in upper half margin with nerve- 
less sides, cymbiform, chartaceous, shining, adpressed, mucronate, 4 -0-4 -2 (inch 
mucro) x 3 -0-3 -2 mm ; keel rather strong, 3 (-5) nerved, nerves excurrcnt into 
mucro; mucro usually recurved, 0-5 mm long; lateral nerves rather faint. 
Rhachllla raggedly winged, excavated. Stamens 3 ; filaments long, flexuous, 
slender, tortuous, 2-5-2-8 mm long ; anther brown, apiculatc 'with somewhat 
setaceous apex, linear, distinctly spurred at base, 1-9-2- Omm long. Style tri- 
quetrous, hardly dilated at base, distinctly fimb riately hairy in upper 1/3 or 1/2, 
2-4-2-5 mm long ; stigma 3, shortly hairy throughout, shorter than style, 1-7- 
1-8 mm long. Nut narrowly oblong ovate, triquetrous, tricostulatc, whitish 
stramineous with slightly convex sides, stipitate, non umbonulate with triangularly 
flat apex, somewhat rough due to minute transversely elongated tubercles in upper 
half, 0-6-0-8 x 0-3-0*4 mm; epicarpic cells in upper half circular (somewhat 
hexagonal), minute, reticulate ; stipe 0*12-0- 15 mm long. 

Govindarajalu 12,110, Venniyar toVaraiyattumottai, Highwayys Mts., Maciurai 
Dt.,Tamilnadu occurring in open grassy slopes (type : PCM) ; Isotypcs: 12,1 10 A 
(CAL) ; 12,110 B (MH) ; 12,110 C (PCM). 

Related to F. eragrostis (Nees) Hance but differs in having culms covered 
at the base by ovate lanceolate scales and ultimately by their fibrous remains, 
much narrower canaliculate acuminate (acute) recurved leaves, simple contracted 
capitate short inflorescence consisting of lesser number of spikelets, primary rays 
when present triquetrous, clustered smaller spikelets, obliquely and linearly scabrid 
glumes, longer recurved mucro, slender tortuous very long 'Maminal filaments, 
anthers with somewhat setulose apex, distinctly fimbriately hairy style, stigmas 
shorter than style, narrowly oblong ovate triquetrous tricostulate non umbonu- 
late smaller nuts with triangular flat apex and rough surface marked by somewhat 
transversely elongated tubercles in upper half and reticulately arranged circular 
epicarpic cells. 

Note ,; Easily observable field and herbarium characters of this novelty are the 
presence of ovate lanceolf te scales at the base of culms which are ultimately becom- 
ing fibrous, involute canaliculate leaves recurving towards the soil with a tendency 



46 



E Govindarajalu 



to become ultimately black, contracted capitate inflorescence and scabrid sub- 
distichous-distichous glumes. Not common in the said locality. This is named 
after its characteristic obliquely and linearly present scabrid condition of the 
glumes. 

Fimbristylis tortifolia Govind. sp. nov. Sect Cymosae Ohwi {figure 2) 

Perennis. Rliizoma crassa, lignosa, horizontaliter vel oblique rcptans (interdum 
verticaliter crescens) sine ullis obviis internodis. Culmi aliquantum tcnues, pcnta- 
goai, incrassati et foliati ad basim, plerumque fbxuosi (erecti), solitari, lacvc*., 
glabri, costati, sulcati, dense tectis multis basibus foliatis et demum fibrosis fila- 
mentis, plerumque curvati ad basim (15-) 20-35 cm x 0-75-1-0 mm. Folia 




Figure 2a-h. Fimbristylis tortifolia Govind. sp. nov. a. habit x ; j>. s tyjc 
and Stigma x 11 ; c. spikelet x 5; d. glume, lateral view x 6;'e. stamen 
x 10; f. glume, spread out x 6; g. outer epicarpic cells, diagrammatic' 
h. nut x 15; (from . Govindarajalu 12,000, type). * 



Novelties in Fimbristylis (L.) Vahl 47 

multa, ftexuosa, omnes recurva vel varie tortuosa vel saltern infima folia recurva, 
glabra, canaliculata, involuta, longinque et aliquantulum scabrida, laevis ad in- 
fimum dimidium marginem eligulata, leviter lata, acuta, culmo breviora, multi- 
nervia, non catinata, 8-15 cm x 1-0-1 -5 mm. Vaginae brunnae, valde nerviae, 
glabrae, omnes laminiferae, infimae denique evadentes filamenta fibrosa, non 
corneae cum lateribus angustis rnembranaceis stramineis, oblique truncatae. 
Irtflorescentia simplex, capitzta (raro cum uno radio addita), consister.s 3-8 (-10) 
spiculis, 8-10 cm longa et lata. Bracteae late ovato lanceolatae, atrobrunneae, 
late ad basim cum arista aliquantulum scabrida, rigidae, erectae-oblique erectae, 
aequilongae vel leviter longiores, in (foreseen tia 8-15 mm longae. Radii O. Spiculae 
ovato lanceolatae, acutae, teretes, castaneobrunaae, multiftorae, sessiles, 6-9 x 
2-5-^3 'Omm. Olumae late ovatae, acutae, glabrae et laeves iid apicem, patens 
cum distincte margins hyalina et cum lateribus uno nervis in dimidio una quoque, 
eglandulosae, non nitidae, distincte mucronatae, 4-5-5-5 (iticluso mucrone) x 
4-0 mm ; carina distincta, 5 nervia, excurrentes in mucronem ; mucro recurvata 
vel erecta, 0-6-0-7 mm longa. Rhachilla pannose alata, excavata. Stamita3; 
anthera Unearo oblonga, lutea ad apicem rotundata, non setacea, ad basim cal- 
carata (lobata), 0-75-0 -8 mm longa. Stylus triqueter cum leviter dilatata pyra- 
midalibasi, glaber vel leviter fimbriatus post trifurcationem, 1-75-2-0 mm longus ; 
stigmata 3, papilhta, stylo longiora (aequilonga), 2-25-2 -5 mm longa. Nux 
late obovata triquetra, tricostulata cum lateribus convexis, atrobrunnea demum 
evadens nigra, distincte stipitata, umbdnulata, distincte tuberculata in dimidio 
superiore 1- 1-1-25 (inclusis stipitibus) x 0-75-0-8 mm ; stipes 0-1-0-2 mm 
longus ; cellulae epicarpicae extimae in dimidio superiore transverse elongatae, 
hexagonales, tessellatae, occurrentcs in 10-^12 regularibus verticalibus seriebus 
in una quoqae facie. 

Perennial. Rhizome thick, woody, horizontally or obliquely creeping (sometimes 
vertically growing) without any obvious internodes. Culms rather slender, penta- 
gonous, thickened and leafy at base, usually flexuous (erect), solitary, smooth, 
glabrous, ribbed, sulcate, densely covered by many leaf bases and ultimately by 
their fibrous strands, usually curved at base (15-*) 20-3 5 cm x 0-75-4 -Omm. 
Leaves many, flexuous, all recurved or variously tortuous or at least the lowermost 
leaves recurved, glabrous, canaliculate, involute, distinctly ard somewhat scabrid 
in the margins, smooth in lower half margins, eligulate, slightly broad, acute, shor- 
ter than culms, many nerved, non keeled, 8-15 cm x 1-0-1 -5 mm; Sheaths brown, 
strongly nerved, glabrous, all laminiferous, lowermost ultimately becoming fibrous 
strands, non horny with narrow membranous stramineous sides, obliquely trun- 
cate. Inflorescence simple, capitate (rarely 1 ray added), consisting of 3-^8 (-40) 
spikelets, 8-10 mm long and broad. Bracts broadly ovate lanceolate, dark brown, 
broad at base with somewhat scabrid arista, stiff, erect-obliqu,ely erect, as long as 
or slightly longer than inflorescence, 8-15 mm long. Rays O. Spikelets ovate 
lanceolate, acute, terete, castaneous brown, many flowered, sessile, 6-9 x 2*5- 
3-0 mm. Glumes broadly ovate, acute, glabrous and smooth at apex, patent with 
distinct hyaline margin and 1 lateral nerves in each half, eglandular, not shining, 
distinctly mucronate, 4- 5-5- 5 (incl. mucro) x 4-0 ram; keel distinct, 5 nerved, 
excurrent into mucro j mucro recurved or erect, 0'6-0*7 mm Itfng. Rhachilla 



48 E Govindarajalu 

raggedly winged, excavated. Stamens 3 ; anther linear, oblong, yellow, rounded 
at apex without bristles, spurred at base (lobed), O'75-O' 8 mm long. Style trique- 
trow; with slightly dilated pyramidal base, glabrous or slightly hairy behind tri- 
furcation, 1*75-2- Omm long ;. stigma 3, papillose, (as long as) longer than style, 
2 -25-2 -5 mm long. Nut broadly obovoid, triquetrous, tricostulate with convex 
sides, dark brown ultimately tending to become black, distinctly stipitate, unibonu- 
late, distinctly tubercled in upper half, 1-1-1 -25 (incl. stipe) x 0-75-0-8 mm ; 
stipe 0-1-^0 -2 mm long ; outer epicarpic cells in upper half transversely elonga- 
ted hexagonal, tessellated, occurring in UM2 regular vertical rows in each face. 

Govindarajalu 12,000, Kulikad, Highwavys Mts., Madurai Dt., Tamilnadu, 
commonly occurring in wet open grassy rocks (type : PCM) ; Isotypes : 12,000 A 
(CAL) ; 12,000 B (MH) ; 12,000 C (BLAT) ;. 12,000 D (BSI- ; 12,000 E-V (PCM). 

Related to F. rigidiuscula Govind. (sect. Cymosae Ohwi) but differs in having 
usually flexuous culms usually covered at base ultimately by fibrous strands of 
leaf bases, usually all or at least the lowermost leaves flexuous recurved or variou- 
sly becoming tortuous, less scabrid or more or less smoooth margined leaves, 
slightly broader non keeled many nerved acute leaves, non horny brown sheaths 
with stramineous sides, ovate lanceolate spikelets, broadly ovate patent acute dis- 
tinctly mucronate and distinctly hyaline margined glumes with 1 lateral nerve in 
each half, long recurved or erect nxucro, shorter anthers with non bristly rounded 
apex, shorter style, stigma usually longer than style, broadly obovoid triquetrous 
tricostulate distinctly stipitate smaller nuts with distinct tubercles in Tipper half 
and transversely elongated hexagonal epicarpic cells occurring in lesser number of 
rows in each face. 

Note : The following are the outstanding characters of this novelty that can 
easily be recognized in the field as well as in the herbarium. The flexuous culms 
are curving at the base and covered by fibrous strands of several leaf bases ; hori- 
zontally or obliquely creeping rhizomes ; flexuous or variously tortuous non 
keeled leaves with more or less smooth margins ; simple capitate inflorescence 
consisting of 3-<6 ovate lanceolate spikelets. The name is based on the tortuous 
condition of the leaves. 

Vegetative anatomy 

Materials and methods 

The materials for the anatomical investigation were taken from their respective 
isotypes. The methods that were followed in earlier works are adopted here 
(Govindarajalu 1966, 1968 a,b, 1975 et seq). Most of the descriptive terms used 
here are those that have been recommended by Metcalfe and Gregory (1964). The 
typological terminologies proposed by Chaedle and Uhl (1948 a,b) for designa- 
ting the types of vascular bundles and metaphloem are followed. 

Fimbristylfs scabrisquama Govind 

Lwf-^Abaxial surface : Intercostal cells moderately long, axially elongated ; cell 
walls tbin, smooth ; end walls straight. Stomata (L. 45-49-5 /on ; W. 27*0- 



Novelties in Fimbristylis (L.) Vahl 49 

i-.S //m), elliptic oblong, thick-walled ; subsidiary cells low dome-shaped ; inter- 
omatal. cells long with concave ends. Silica cells short, broad, thin-walled 
rcumng in 3 continuous rows, each one of them containing 2-3 cone-shaped 
[ica bodies surrounded by satellites. 

Adaxial surface : Cells isodiametric, somewhat hexagonal ; cell walls thin, 
nooth. Stomata wanting. Other details as in al axial surface. 

T.S. lamina : Lamina examined 2 -2 mm wide. Outline *W* shaped, assymm- 
ric I (figure 3,d). Keel inconspicuous. Margins dissimilar, one margin acute 
id the other one somewhat rounded (figure 3,d). Cuticle uniformly thick, 
alliform cells not differentiated. Adaxial epidermal cells uniform in size and 
tape and larger than those of the abaxial. Sclerenchyma strands : adaxial 3 
It. 72-90 jum ; W. 45-^54 /*m), pentagonal ; abaxial associated with each vl 
It. and W. 54-90 /(m), rounded (puiviniform) ; submarginal strands (Ht. 3 6 /on; 
r . 157-5 /*in), pulviniform. Adaxial hypodermis consisting of 2-3 layers of in- 
ited radially elongated or rounded colorless perenchyma cells. Chlorenchyma 
diating restricted to a axial one half of lamina. Vascular bundles c. 20 in 
imber comprising large, medium and small vb's and not showing any regular 
ternation with each other ; large vb's (type III A) oval in outline while medium 
id small vb's rounded (type I) ; metaxylem vessel elements (D. 27 /n in dia- 
eter) ; metaphloem of 'regular type*. Bundle sheaths 3 layered ; M.S. in 
I vb/s fibrous, complete ; in large vb's O.S. and I.S. parenchyinatous, the former 
mplete \vhile the latter incomplete and in the case of small vb's both complete, 
icumvasdular sclerenchyma absent. Tannin idioblasts common. 

Ciilm--Epidermis 9 surface view : Cells axially elongated with thin somewhat 
tucms walls, Stomata (L. 31-5-36-0 /on ; W. 27-0-31-5 jum, more or less 
borbicular, thick-called ; subsidiary cells dome-shaped ; in terstomatal cells long 
th concave ends. Silica cells, short, rather narrow, thin-walled, occurring in 
more or less continuous rows and each cell containing usurlly 2-3 cone-shaped 
ica bodies surrounded by satellites. Silica particles very commonly and charac- 
ristically present in cells in between the ribs and interstomatal cells ; likewise 
unded silica bodies characteristically present in subsidiary cells (figure 3,f). 

T.S. Culm; Long diameter of cv 1m examined 0-8 mm. -Outline subcircular 
th ribs and furrows (figure 3,e). Cuticle uniformly thick (thickness 13 '5/jm) 
roughout. Epidermal cells thin-walled, similar in size and shape except at the 
PS. Stomata in line with epidermis ; guard cells with outer ledges only ; sub- 
>matal chamber very narrow and small. Hypodermjs consisting of 3-4 (-5) 
fers of radiating chlorenchyma cells, the continuity of which interrupted by jeri^ 
,eral ring of vb's (figure 3,e). Sclerenchyma strands (Ht. 45-0-67-5 /on j W/ 
0-45*0 /a), variable, pulviniform, rounded, triangular. Ground tissue made 
> of Large parenchymatous cells having. intercellular spaces, vascular bundles 
28 in number comprising I oth large and small vb's, arranged in two peripheral 
igs, the former forming; the inner ring and the latter outer ring (figure 3 t e) ; 
:ge vb's 6 in number, oval shaped (type HI B), the remainder rounded (type I) j, 



50 E Govindarajatu 

large vb's containing protoxylem lacunae ; metaxylem vessel elements (D.c. 
22*5 //min diameter); metaphloern of Regular type'. Bundle sheaths, see 
lamina. Circumvascular-selerenchyma little or rot developed. Tannin idioblasts 
abundant i n hypodermis. 

Rhizome. Transverse section: Diameter of the rhizome examined 3 -2 mm. 
Epidermal cells thick-walled, isodianntric. Cortex very broad, organized by 
thick-walled parenchyma cells ; cells compactly arranged without intercellular 
spaces and containing abundant starch. Subepidermal fibrous strands (Ht. 174-0- 
290-0 /on ; W. 92-8-174-0 /on), triangular. Endodermoid layer made up of 
single layer of cells with *U' shaped thickenings and forming a wavy limiting layer. 
Stele central containing many vb's more or less rounded in outline, amphivasal 
(type V); metaxylem elements (D. c. 9-0 urn in diameter). Central ground 
tissue sim'.iar to that of cortex. Tannin idioblasts large, very common throughout 
ground tissue. 

Root. Transverse section : (figure 3,c). Diameter of the root examined 0-7 mm. 
Exodermis single layered consisting of cells similar in size and shape with suberized 
cell walls. Cortex : recognizable into 2 zones ; outer zone narrow consisting 
of 4-5 layers of compactly arranged parenchyma cells ; inner broad, lacunose 
due to concentrically arranged air-cavities ; air-cavities separated by radiating 
row^ of parenchyma cells. Endodermis single layered consisting of cells with 
*U' shaped thickenings and broad lumina. Pericycle of single layer of thick-walled 
cells. Metaxylem vessel central, solitary, rounded in outline (D. c. 45-0 /on 
in diameter). Protoxylem units 7-8 in number. Metaphloem not easily disting- 
uishable. Central ground tissue parenchymatous. 

Fimbristylis tortifolia Govind. 

Lezf-Abaxial surface : Intercostal cells axially elongated, thin-walled, smooth 'j 
end walls straight. Stomata (L. 45-54 /mi; W. 18-0-22-5 //m), narrowly 
oblong ;. subsidiary cells low dome-shaped ; interstomatal cells short with concave 
ends. Silica cells rather broad, short, moderately thick-walled, present in 2-3 
continuous rows ; each cell possessing (1-) 2-3 cone-shaped silica bodies with 
satellites. 

Adaxial surface : Cells broad, isodiametric ; cellwalls thin, smooth, end walls 
straight Stomata absent. Silica cells occurring in a single more or less dis- 
ontinuous row ; other details see abaxialc surface. 

T.S. lamina : Lamina examined 1-6 mm wide. Outline flatly triangular (fig- 
ure 3, a), asymmetrical with abaxial ribs and furrows. Keel slightly developed, 
somewhat rounded or bilobed (figure 3, a). Margins dissimilar; one margin 
sloping towards abaxial side, the other more or less truncate with depression 
(figure 3,a). Cuticle thick on either surface, Adaxial epidermal cells slightly 
larger than those of the abaxial. Bulliform cells not distinctly differentiated, (fig- 
ure 3,a). Sclerer.chyma strands : adaxial 5 in number (Ht. 40-5-45-0 //m; 
W, 54-0-67*5 //in), pentangular ; abaxial strands .(Ht. and W. 36-0-49-5 /<m),. 



Novelties In Fimbrisiylis (L.) Vahl 



51 



more or less rounded or pulviniform and associated with vb's (figure 3, a). Adaxial 
hypodermis consisting of 4-5 layers of compactly arranged inflated translucent 
polygonal parenchyma celts. Chlorenchyma restricted to abaxial one third of 
lamina. Air-cavities absent except at substomatal cavities. Vascular bundles 
18-20 in number of two different sizes out of which 3 large and the rest small ; 
both belonging to type III A and arranged without any regular alternation ; 
median vb. adaxially capped by radiating rows of large translucent parenchyma 
cells (figure 3, a) ; metaxylem vessel elements (O. 18 /on in diameter) ; meta- 
phloem of 'regular type '. Bundle sheaths in all vb's 3 layered ; O.S. parenchy- 
ittatous, complete ; M.S. fibrous, complete ; I.S. parenchymatous., incomplete 
in all vb's . Tannin idioblasts not observed. 



T.C. 




CU. 



Figure 3 a f. a, b. Flmbristylis torti folia Oovind. a. transcction of lamina 
x 44; b. transection of culm x 33 ; <?-/. F. scabrtequama Oovind. c. Transtection: 
of root x 44; d, translection of lamina x 33; e. traitscction of culm 
x 44; f. surface view of epidermal cells (note pressewce of silica bodies in 
subsidiary cells and silica particles in epidermal and interstom^tal cells) x 226. 
(All pasted on their respective isotypes). 



52 E Govindarajalu 

Culm. Epidermis, surface view : Cells axlally elongated ; cell walls thin, smooth, 
Stoma.ta (L. 38-0 /mi ; W. 27-0-28-8 /(in), oblong elliptic ; subsidiary cells low, 
dome-shaped ; interstomatal cells long with concave ends. Silica cells present 
in a single discontinuous row ; cells long, and each one of them containing 5-6 
cone-shaped silica bodies without satellites. 

T.S. Culm : Long diameter of culm examined 1-2 mm. Outine polygonal or 
somewhat subhexagonal with ribs and furrows (figure 3,b). Cuticle uniformly 
thiclc (thickness c. 9 jj,m) throughout. Epidermal cells thin-v^alled, . similar , 
in.size-a,nd shape. Hypodermis consisting of 6-8 layers of non radiating chloren- :j 
chyma cells interrupted by fibrous strands and vb's (figure 3,b). Air-cavities repre- ;j 
sented by substomatal spaces. Sclerenchyma strands (Ht. 54-0-112-5 //m ; 
W. 99*0-135-0 /zm),. usually pulviniform (triangular ovate). Ground tissue con- 
sisting of large parenchyma cells arranged \vith intercellular spaces. Vascular 
bundles c. 43 in number comprising both large (type III A) and small vVs (type I) 
large vb's 7 in number somewhat oval or subcircular in outline containing pro- 
toxylem lacunae ; small vb's circular in outline and arranged peripherally in the 
form of undulating ring following the contour of the culm while large vb's 
forming the inner ring (figure 3,b) j metaxylem vessel elements (D. 18-0-22-5/zm 
in diameter); metaphloem of 'regular type'. Bundle sheaths in all vb's 3 
layered ; O.S. and I.S. parenchymatous, the former complete and the latter in- 
complete ; M.S. fibrous, complete in all vb's. ;;/ 

Root. Transverse section : Diameter of the root examined 0-6 him; Exodermis : 
single layered consisting of cells uniform in size and shape ; cell' walls suberized, 
Cortex : recognizable into two zones, the outer zone narrow consisting of Slayers 
of compactly arranged thick-walled parenchyma cells ; inner cortex rather broad, 
lacunose ; air-cavities concentrically arranged and separated by radiating rows 
of parenchyma. Endodermis of single layer made up of slightly tangentially 
elongated cells with *U' shaped thickenings. Pericycle consisting of a single 
layer of thick-walled cells. Metaxylem vessel solitary, central, circular in outline, 
thick-walled (D. 67-5 /jm in diameter) ; protoxylem units 8 in number alternating 
with as many metaphloem units ; each unit of the mstaphloem consisting of a 
group of 4 cells of which 2 being large sieve tube elements and 2 companion 
cells. Ground tissue made up of thick-walled parenchyma. 



References 

Cheadle V I and Uhl N W 1948a Types of .vascular bundles in the Monocotyledoneae and 
their relations to the late metaxylem conducting elements ; Am. J. Bat. 35 486-496 

Cheadle V I and Uhl N W 1948b The relation of metaphloem to the types of vascular bundles 
in the Monocotyledons; Am. J. Bot. 35 578-583 

Govindarajalu E 1966 Systematic anatomy of South Indian Cyperaceae : Bulbostylis Kunth 
/. Linn, Soc. (Bot.) 59 289-304 

Govindarajalu E 1968a Systematic anatomy of south Indian Cyperaceae: Fuirena Rottb.; /, 
Linn. Soc. (Bot.) 62 27-40 



Novelties in Fimbristylis (L.) Vahl 



53 



Govindarajalu E 1968b Systematic anatomy of south Indian Cyperaceae: Cyperus L. subgen. 
Kyllinga (Rottb.) Suringar; /. Linn. Soc. (Bot.) 62 41-58 

Govindarajalu E 1975 The systematic anatomy of South Indian Cyperacene: Eleocharis R.Br., 
Rhynchospora Vahl and Selena Berg.; Adansonia 14 581-632 

Metcalfe C R and Gregory M 1964 Some new descriptive terms for Cyperaceae with a dis- 
cussion 1 of variations in leaf form noted in the family; Notes Jodrell Lab. I 1-11 



Key to figure lettering and text abbreviations 

A.B.E. abaxial epidermis: 

A.C. air-cavity 

AD.E. adaxial epidermis 

CH. chlorenchyrna 

CU. cuticle 

E. epidermis 
B.C. epidermal cell 
EN. encloderrris 
EX. exodermis 

F. fibres 

G.T. ground tissue 

I.C. interstomatal cell 

1,CO. inner cortex 

M.V. metaxylem vessel 

O.C. outer cortex 

PH. metaphloem 

P.I*. protoxylem lacuna 

P,X. protoxylem 

R.P. radiating parenchyma 

S. sclerenchyma 



S.B. silica body 

S,P. silica particle 

S.S. sclcrenchyma strand 

ST. sttoma 

T.C. translucent cell 

T.i. tannin idioblast 

V.B. vascular bundle 

XY. metaxylem 

Text abbreviations : 

c. circa 

D, diameter 

Ht. height 

l.S. inner sheath 

1'. length 

M.S. middle sheath 

O,S. outer sheath 

vb*s vascular bundles (sing, vb.) 

W. width 



Procf. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number I, February 1982, pp. 55-60. 
Printed in India. 



Embryological studies in three species of Cymbopogon Spreng 
(Poaceae) 

S P CHODA, HARSH MITTER and RAVINDER K BHANWRA 

Department of Botany, Panjab University, Chandigarh 160 014, India 

MS received 17 September 1980 ; revised 24 September 1981 

Abstract. The embryology of Cymbopogon nardua var. confetti floras, C. martinii 
var. Motia and C. par kef i has been studied. C. /utrdus and C. martinii have been 
observed to be seed-sterile owing to failure of fertilization while in C. parkeri, the 
seed-setting is only about 8-00%. 

Keywords. Seed-sterility ; Cymbopogon ; Andropogoneac ; Poaccac. 



1. Introduction 

The genus Cymbopogon belongs to the tribe Andropogoneae of the subfamily 
Panicoideac and it is represented by 24 species in the Indian sub-continent (Bor 
1960). A number of species of Cymbopogon yield essential oils and are used in 
perfumery. The embryology of C. mart mil and C. nervatus has been worked out 
by Brown and Emery (1958) although of a preliminary nature. The present paper 
deals with the embryology of Cymbopogon nardus (L.) Rendle var. Confertiflorus 
(Steud.) Stapf ex Bor, C. martinii (Roxb.) Wats var. Motia and C. parkeri Stapf, 
to find the nature of seed sterility observed in different species. 



2. Materials and methods 

The material of C. nardus and C. martinii was collected from the Botanical Gardens 
of the Panjab University while that of C. parkeri from Shiwalik hills in the months 
August to November. Conventional methods of dehydration and embedding 
were used. The sections were cut at 5-10 /an and stained with safranin and fast 
green. For studying the growth of the pollen tube in the style, the method given 
by Khoshoo and Vij (1963) has been followed. For this purpose the gynoecia were 
fixed in 1 : 3 acetic alcohol, after 3, 6, 9 and 24 hrs of pollination. After half an 
hour the styles were transferred . to 30% ethyl alcohol for preservation. The 
ovaries were transferred to 1 % solution of acid fuchsin for- about 20 min. Subse- 
quently, the styles were cleared in lactic acid at 60 C and the whole mounts 
were made in pure lactic acid. 

55 



56 S P Choda, Harsh Mitter and Ravftider K Shanwra 

3. Observations and discussion 

3.1. Microspofogenesis and male gametophyte 

Anthers are tetrasporangiate (figure 1) and the anther wall consists of an epidermis, 
followed on the inner side by the endothecial layer, a single middle layer and the 
tapetum (figures 2, 3). The epidermal cells in mature anthers show deposition of 
oil droplets in C. nardus and endothecial cells develop fibrous thickenings 
(figure 10). The middle layer is ephemeral. The tapetum is of the glandular 
type and its cells become binucleate in C. nardus while they remain uninucleatc 
in C. martinii and C. parked. As seen in transections the MMC are disposed in 
4 or 5 rows (figures 2, 3). Meiosis in MMC is normal and it is of the successive 
type leading to the formation of isobilateral microspore tetrads (figures 4-7). The 
development of the male gametophyte occurs as described in other members of 
the family and the pollen grains are shed at the 3-celled stage (figures 8, 9). The 
pollen grains are monocolpate with a thick smooth exine and slightly thinner 
intine. The pollen fertility is about 90% in C. nardus and C. parked and about 
70% in C. martinii. 

3.2. Ovary and ovule 

The ovary contains a sessile, bitegmic, pseudocfassinucellar hemianatropous ovule 
(figures 16, 17). The inner integument is composed of cells 2-3 layers thick but 
it usually fails to grow over the nucellus so that a definite micropyle is lacking 
(figure 16). In Pennisetwn typhoideum (Narayanaswami 1953) and Capillipediutn 
huegelii (Choda and Bhanwra 1980) the inner integument does not grow over the 
nucellus but in most other species of the family the micropyle is formed by the 
inner integument (Narayanaswami 1954, 1955, 1956). In Cymbopogon parkeri 
where some seed-setting is observed, the inner integument is found to degenerate 
after fertilization unlike that in Saccharum offidnarum (Artschwager et al 1929) 
and Sorghum vulgar e (Artschwagei? and McGuire 1949), belonging to Andropogo- 
neae where they persist in mature caryopsis. 

The outer integument on the upper side of the ovule is 2-3 cell layered but it 
covers only about a third of the ovule (figure 17), a feature characteristic of the 
subfamily Panicoideae (Chandra 1963 ; Venkateswarlu and Devi 1964). The 
outer integument on the lower side of the 6vule shows about the same growth as 
the inner integument and is 2-5 cell layered (figures 16-18). 

The nucellar epidermis undergoes 2-3 periclinal divisions in the region of the 
micropyle (figures 16, 18) by the time the megaspore tetrads are formed. In 
C. nardus, the nucellar cells in this region become conspicuous owing to their large 
size, dense contents and prominent nuclei (figure 16). The formation of a parietal 
tissue due to periclinal divisions in the nucellar epidermis near the micropyle has 
been reported in many species belonging to the subfamily Panicoideae (Chandra 
1963 ; Venkateswarlu and Devi 1964). The nucellar tissue is absorbed by the 
developing embryo and endosperm and is hardly traceable in the mature caryopsis 
of C. parkeri (figures 26, 27). 



Embryology of Cymbdpogon Spreng. (Paaceae) 



'51 




19 



Figures 1-19. Microsporangium, microsporogenesis, male gametophyte, megasporo- 
genesis and female gametophyte. 1, 3, 11, 16-19. Cymbopogon ncrdus. 2, 12, 
14, 15. C. maninii. 4-10, 13. C. parkeri. 1. TS of the anther, 2. TS anther 
lobe showing periclinal division in the inner secondary parietal layer, 3. TS anther 
lobe showing wall layers and sporogenous cells, 4-7. Stages in microsporogenesis, 
8, 9. 2- and 3 -celled pollen grains respectively, 10. Portion of wall layers showing 
epidermis and fibrous thickenings of the endotheciura, 11. LS ovule primordium 
at archesporial cells stage, 12. LS ovule at megaspore mother cell stage, 13, 14. 
Megasporogenesis, 15, 16. 2- and 4-nucleate embryo sac stages respectively, 
17. VS of the ovary and ovule, 18. Embryo sac showing egg cell, two synergids, 
two polar nuclei and antipodal complex of several cells, 19. Embryo sac showing 
egg apparatus, polar nuclei and degenerating antipodal cells. (See explanation of 
abbreviation in p. 58). 



58 



S P Choda, Harsh Uitier aitd Ravinder K Bhaitwra 




Figures 20-27. Post-pollination development. 20, 22. C. tfd/Y/ws ; 21, 23-27- 

C. parkcri. 20-22. Degenerating embryo sac and shrivelled ovule ; 23-27. 
Some stages in caryopsis development. 

ANT antipodal ; CO co4eoptilc ; COZ coleorhiza ", E egg ; ELembryomc 

leaf ; END endodermis ; EPI epidermis ; IP inner parietal layer ; MES- 

mesocotylc ; PN polar nucleus ; R radicle ; RC root cap ; S -sporogcnous 
tissue ; SO-scutelluni ; SYN synergid. 



3.3. Embryo sac development 

The single hypodermal archesporial cell is differentiated in the nucellus, which 
increases in size and functions as the megaspore mother cell (figures 11, 12), It 
divides meiotically so as to form a linear tetrad of megaspores (figure 13). In 
C martinii^ however, the upper dyad cell degenerates without undergoing division 
(figure 14). The chalazal megaspore functions and develops into the polygonum 
type of embryo sac having aft egg cell, two synergids, a central cell with its two 



Embryology of Cymbopogon Spreng. (Poaceae) 5J> 

polar nuclei and three antipodal cells. The latter proliferate further and form 
9-26 cells in C. nardus, 12-15 cells in C maninll and 9-16 cells in C. parkerl The 
multiplication of the 3 antipodal cells is commonly reported in grasses 
(Venkateswarlu and Devi 1964 ; Maze and Bohm 1973). 

In C. nardus and C. martini!, the pollen grains germinate on the stigrnatic hairs 
but the pollen tubes fail to reach the embryo sac due to some unknown factor. 
The embryo sac and the ovule eventually shrivel and undergo disintegration 
(figures 20-22). Occasionally the ovules become enlarged and they contain endo- 
sperm nuclei formed probably due to autonomous divisions of the secondary 
nucleus, but there is no embryo formation. In C. parkeri, however, about 8% 
seed-set has been noticed. The primary endosperm nucleus starts dividing earlier 
than the zygote. The endosperm is of the nuclear type (figure 23) as is reported 
in other grasses. The endosperm becomes completely cellular at globular stage 
of the proembryo. Figures 23-27 show some of the stages in the development 
of embryo in the species. The sequence of early development of the embryo could 
not be traced but the structure of the mature embryo is similar to that described 
by Reeder (1957) in other members of the tribe Andropogoneae. Seed-sterility 
in family Poaceae has been previously reported in Helaria belangeri and H. mutica 
by Brown and Coe (1951) and in Digitaria decumbens by Sheth et al (1956). 

In H. belangeri and H. mutica, the degeneration of the female gametophyte may 
occur any time after megaspore formation and this has been suggested to be the 
cause of seed sterility. In the present species, however, the degeneration of the 
female gametophyte occurs only after it has attained maturity. 

Poor seed-set as in C. parkeri and complete sterility as exhibited by C. nardus 
and C. martinii appear to be compensated by the predominance of vegetative 
propagation as a means of survival ; consequent on perturbation of sexuality. 



References 

Artschwager E, Brandos E W and Starrett R C 1929 Development of flower and seed of some 

varieties of sugarcane ; /. Agric. Res. 39 1-30 
Artschwager E and McGuire RC 1949 Cytology of reproduction in Sorghum vulgare ; J. Agric. 

Res. 78 659-673 
Bor N L 1960 Grasses of Burma, Ceylon, India and Pakistan (Excluding Bambuseae) ; (London, 

New York, Paris : Pergamon Press) 
Brown W V and Coe G E 1951 A study of sterility in Helaria belangeri (Stcud.) Nash and 

H. mutica (Buckl.) Benth ; Am. J. Bat. 38 823-830 
Brown W V and Emery W H P .1958 Apomixis in the Gramineae : Panicoideae ; Am. J. Bot. 

45 253-263 

Chandra N 1963 Som-3 ovule characters in the systeraatics of Gramineae ; Curr. Sci. 32 271-279 
Choda S P and Bhanwra R K 1980 Apomixis in Capillipedium huegelll (Hack.) Stapf 

(Gramineae) ; Proc. Indian Natl. Sci. Acad. 46 572-578 
Khoshoo T N and Vij S P 1963 Biosystematics of Citmttus vulgaris var fislulosus ; Caryologia 

16 541-552 
Maze J and Bohrn L R 1973 Comparative embryology of Stijpa elmeri (Gramineae) ; CanJ. 

Bot. 51 235-247 
Narayanaswami S 1953 The structure and development of the caryopsis in some Indian millets. 

1. Pennisetum typhoidewn ; Phytomorphology 3 98-112 



60 S P Choda, Harsh Mitter and Ravinder K Bhanwra 

Narayana-swami S 1954 The structure and development of the caryopsis in some Indian millets. 

2. Plaspalum scrobiculatum ; Bull. Torrey Bot. Club. 81 288-299 
Narayanaswami S 1955 The structure and development of the caryopsis in some Indian millets. 

4. Echinochloa frumentaceae ; Phytomorphology 5 161-171 
Narayanaswami S 1956 The structure and development of the caryopsis in some Indian millets. 

6. Setaria itdica\ Bot. Gaz. 118 112-122 

Reeder J R 1957 The grass embryo in systematics ; Am. J. Bot. 44 756-768 
Sheth A A, Yu L and Edwardson J 1956 Sterility in pangola grass Digitaria decumbens ; Agron. 

J. 48 505-507 
Venkateswarlu J and Devi P I 1964 Embryology of some Indian grasses ; Curr. Sci. 33 104-106 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 1, February 1982, pp. 61-tfg. 
Printed in India, 



Reproductive efficiency of secondary successional herbaceous 
populations subsequent to slash and burn of sub-tropical humid 
forests in north-eastern India 



K G SAXENA and P S RAMAKRISHNAN 

Department of Botany, School of Life Sciences, North Eastern Hill University, 
Shi Hong 793014, India 

MS received 10 January 19&1 ; revised 5 December 1981 

Abstract. Three categories of secondary successional herbaceous communities 
subsequent, to slash and burn, viz., early success-ional non-sprouting, early succes- 
sional sprouting and late suceessional populations were investigated for tlieir 
reproductive efficiency considering leaf component since it is the chief organ of 
photosynthesis. Early successional non-sprouting populations were found to be 
rcproductively the most efficient whereas the early successional sprouting popu- 
lations allocated more to vegetatively reproducing organs. While the high reproduc- 
tive potential of early successional non-sprouting species was. associated with vigour 
and production efficiency of the species, this relationship was stronger with the 
latter characteristic. On the other hand, early successional sprouting populations 
showed inverse relationship between vegetative and sexual reproductive effort. The 
strategy of late successional species seems to be to maximize vegetative growth in a 
closed habitat. The significance of these strategies is discussed in the paper. 

Keywords. Growth strategies ; leaf area ratio ; reproductive effort ; 
communities ; adaptation. 



1, Introduction 

Slash, and barn agriculture, locally known as s Jhum ', is the most prevalent form of 
cropping in the hill regions of north-eastern India. The early successional herba- 
ceous communities constitute an important phase in the fallow development during 
secondary succession subsequent to .cropping. This community, which holds the 
ground for about 5-6 years, often gets arrested at this stage due to the shortening 
of the Jhum cycle, the intervening period before the cropping is done on the same 
site (Ramakrishnan et al 1981). 

Optimization of reproductive output in plants is attained through a favourable 
partitioning of the available resources for various life-activities such as mainte- 
nance, growth and reproduction (Abrahamson and Gadgil 1973). Much is known 
about the resource allocation pattern in relation to the reproductive strategy of 
different plant species in an attempt to explain the ecological success of a species 
in a given environment (Harper and Ogden 1970; Ogden 1974; Abrahamson 
1975, 1979 ; Newell and Tramer 1978). While such an approach has yielded 

61 



62 



K G Saxena and P S Rantakrishnart 



valuable information, little effort has been made to relate the reproductive gfowff i 
strategy with the leaf growth (McNaughtoa 1975; Bazzaz and Harper 1977 ; 
Primack 1979). This approach for evaluating the reproductive strategy of plants 
is more relevant because, leaf as an organ is the chief region of photosynthetic 
activity. In the present paper, early successional non-sprouting and sprouting, 
and late successional herbaceous populations have been compared for their growth 
and reproductive characteristics considering leaf as the sole organ responsible for 
energy capture and its overall distribution. The non-sprouting species, obviously, 
are all established through seeds alone. Sprouting species, though they may also 
come through seeds, are those that are established through sprouts alone. 



2. Study area and climate 

The study was earned out in Burnihat (26 N latitude and 91-5E longitude) in 
the Khasi Hills about 90 km north of Shillong, on precambrian rocks which are 
represented by gneiss, schists and granites. The soil is red sandy loam and of 
laterite origin. The pH ranges from 5 to 7. The angle of slope generally ranges 
from 20 to 40. 

Climatically the year can be divided into three distinct seasons ; the dry summer 
runs from mid-February to May and the rainy season extends from May to 
September with an annual rainfall of 2200 mm. The latter is a warm period with 
high humidity. The mild winter which is practically rainless except for a few 
showers, extends from November to February. The annual maximum and 
miaimum temperatures are 33 C and 7 C respectively (figure 1). 

3. Methods of study 

Four fallows which were slashed in January 1978 and freshly burnt in Mar&h 
1978 and two 40 year old forested fallows were selected for this study. While 



maximum temperature 
* minimum temperature 

A rainfall 




500 

400 

300 

200 

100 





2 



Figure 



JFMAMJJASONO 

Time period (months ) 
1. Ombrothermic diagram of the study area (1978). 



Sncaesslortal herbaceous communities 63 

mixed cropping is done normally at least for one year (Ramakrishnan et al 1981), 
for the purpose of the present study, the site was left directly as a fallow after 
the burn instead of being cultivated. The fire was a high intensity burn, since 
the slash burnt was derived from a 20 year regtowth. For each herbaceous 
species, phonological observations were made throughout the year using three 
permanent quadrats (50 cm x 50 cm) which were harvested at the fruiting stage. 
Those species which did not flower, were harvested at the end of the growing 
season. The samples were taken from uniformly monospecific patches of each 
species in order to minimize the variation due to micro-environment. Sample 
size ranged from 7 to 25 individuals. Below ground parts were carefully 
washed and leaf blade and seed components were detached. In situations where 
senescence started at flowering or fruiting stage, the fallen leaves and fruits or 
seeds were also included. Different components were dried at 80 5C in a 
hot air oven, for 48 hours and then weighed. 

Leaf area (by planimeter) and leaf dry weight were estimated using three repli- 
cates ith 20 leaves per replicate. Total leaf area per plant was obtained by 
dividing total leaf biomass by dry weight per unit leaf area. Leaf area ratio was 
calculated as leaf area (cm 2 ) per unit (g) biomass. 

Density values for different species were estimated by using 50 randomly placed 
1m 2 quadrats in each fallow (Misra 1968). Twenty quadrats at random were 
harvested for estimating the average biomass per plant. This alongwith the 
density values were used for calculating biomass per m 2 . Each shoot was consi- 
dered as a separate individual, in the case of the rhizomatous species. Community 
analysis was done at the end of the growing season. 



4. Results 

Table 1 shows the density and biomass values of different species in the early and 
late successional communities, Amongst the early successional annuals, Engeron 
linifolius was numerically the most dominant followed by Panicum maximum and 
Cassia tora. However, C. tora having the lowest density, contributed maximum 
to the herbaceous biomass. Six other annuals were present in a small proportion 
and therefore are considered together. Amongst the early successional non- 
sprouting perennials, Eupatorium odoratum was the most dominant. About 64% 
of the herbaceous biomass was contributed by the sprouting species in the early 
successionai communities. Thysanolaena maxima, though having higher relative 
density than Saccharum arundinaceum, contributed lesser in terms of biomass 
compared to the latter. In the late successional herbaceous communities, 
Oplisrnenus compositus was the most dominant component. 

'Species like Engeron linifolius, Eupatorium odoratum, Saccharum amndinaceum 
and Thysanolaena maxima which were the most dominant component in the early 
successional communities, had lower leaf area ratio compared to the less frequent 
s pecies like Euphbrbia hirta, Borreria articularis, Digitaria adscendens and Mimosa 
pudica. Leaf area ratio of the late successional species was, generally much 
higher than the early successional species. Even . odoratum and JP. maximum*, 
which are common in the early and late successional stages, exhibited higher leaf 
r&io in the l^e successiowal communities. Reproductive effort which 



64 KG Saxena and P S Ramakrishnan 

Table 1. Density and biomass of different species in tlie early and late successional 
herbaceous communities. 



Density Relative Biomass Biomass 

, (individuals/ density (%) (g/m 2 ) contribution 
m 2 ) (JQ 



Early successional non-sprouting 
populations : 

Annuals : 

Erigeron linifoliua 3-45 10-11 6-38 0-19 

Panicum maximum 1-96 5-75 13-52 0-40 

Cassia tora 1-10 3-22 16-61 0-49 

Others (TJ = 6) 2-40 7-05 7-05 0-50 

Perennials : 

Eupatorium odaratum 3-75 10-99 1087-35 32-03 

Panicum khasianum 2-45 7-18 15-44 0-45 

Others (n 4) 1-60 5-57 6-35 0-.19 

Early successional sprouting 
populations 

Thysanolaena maxima 9-05 26-53 728-53 21-46 

Saccharum arundinaceum 6-20 18-18 1305-10 38-45 

Imperata cylindrica 150 4-40 87 -38 2-58 

Others (/* = 2) 0-35 1-02 111-61 3-26 

Late successional populations 



Annuals : 










Panicum maximum 


5-62 


16-11 


3-37 


3-59 


Oryza granulata 


1-85 


5-30 


12-21 


13-02 


Perennials : 










Oplismenus compositus 


21-46 


61-53 


27-84' 


28-82 


Centotheca lappacea 


2-35 


6-74 


11-16 


11-90 


Others (# = 7) 


3-60 


10-32 


40-07 


42-67 



n is the number of species, 

worked out in relation to leaf growth (seed (mg)/10 cm 2 leaf) indicates much higher 
values for species like E. odoratum and E. linifolius in early successional commu- 
nities. Amongrt the early successional sprouting perennials, Imperata cylindrica 
and Grewia elastica did not flower in the first post fire year and M. pudica had 
much higher reproductive effort than the others, The late successional species, 
on the other hand, had comparatively very low values for reproductive effort ; 
in Hedychium coccineurn and Curculigo recwvata flowering was not observed 
during the year of study (figure 2). 



Succession^ herbaceous communities 



65 



200 



100 



20 




O 

? 

? 40 

o 

UJ 

ft GO 

Figure 2. Leaf area ratio and reproductive effort (seed mg/10 cm 2 leaf) of secon- 
dary successional herbaceous populations. Non-sprouting early successional 
populations, (a) Annuals, (b) perennials, (c) sprouting early succes&ional perennials, 
late successional populations, (d) annuals, (e) perennials. From left to right the 
different columns are : Erigeron limifolius Willd., Rottboelia goalparensis Bor., 
Cassia torn L., Crossocephalum crepidioides (Benth.) S., Brachiaris distachya (L.) 
Stapf., Panicum maximum Facq. , Euphorbia hirta L.,Borreira articular is (L.f.) Wild., 
Mollugc stricta L., Eupatorium cdoratum L,, Setaria palmifolia (Koen.) Stapf.. 
Paspatidium putictatum (Burm.) A. Camus., Panicum khasianum Munro., Manisuria 
granularis L.f., Digitaria adscenderts (H,B,K.) Henr., Saccharum arundinaceum 
Hook f., Thysanolaena tnaxima (Roxb.) O. Ketze., Grewia elastica Royle 
Irnperata cylindrica Beauv,, Mimosa pudica L., Oryza granufata Nees et Arn. 
Panicum maximum Facq., Rumex itepatensis Spreng., Eupatorium odoratum L. 
Hedychium cocclneum Ham. Carex cruciata Wahl., Oplismenus compositus Beauv. 
Centotheca tappacea Deov., Curculigo recurvata Dryand., Cypems globosus Allioni. 
Chlorophytum arundinaceum Baker. 



These population characteristics were compared to assess their ecological 
importance. Pairwise comparison was made by Mann-Whitney's two sample 
rank test. Early succes&ional non-sprouting category had significantly (I 5 < 0-05) 
lower leaf area ratio but higher reproductive effort than the late successional 
category. Early successional sprouting species showed significantly (P < 0-05) 
lower leaf area ratio than those of the early successional non-sprouting and late 
successional types. However, its reproductive effort was not significantly 
different (P >0-05) from both the categories. 

Regression analysis showed that while, reproductive effort was negatively 
correlated with leaf area ratio in the early successional non-sprouting category 
(r = 0-72, P < O'Ol), positive correlation existed in the early successional 
sprouting category (r =0-86, P < 0-05). Further, ir the early successional non- 
sprouting category only, reproductive effort was positively correlated with leaf 
area per plant (figure 3). In the late successional category, no significant relation- 
ship could be detected between leaf characteristics and reproductive effort. 



K G Saxenci and P S Ramakrishnan 



Annuals 
O Perennials 



<-> 



Q i 
UJ ' 




*" 2 ^ 6 6 10 

LOG LEAF AREA (cm 2 ) 

Figure 3. Relationship between leaf area (cm 2 )/plant and reproductive effort (seed 
mg/10cm 2 leaf) in the non-sprouting early success-ional category. 



Early successional non-sprouting populations when considered separately as 
annuals and perennials did not differ significantly between themselves for their 
leaf area ratio or reproductive effort. However, the negative correlation obtained 
for these categories, between leaf area ratio and reproductive effort, was more 
significant for the perennials (r = 0-93, P < 0-01) compared to the annuals 
(r =0-65, P < 0-05). Reproductive effort was found to he positively correlated 
with absolute leaf area in the perennials only (r = 0-80, P < 0-05) and not in 
the annuals (figure 3). 



5. Discussion 

Leaf area ratio is an important structural concept as it expresses the proportion 
of as^imilatory surface to respiratory mass (Evans 1972). Though the different 
species exhibit a range of variation, late successional populations which occupy 
the habitat of a low light regime, have higher leaf area ratio than the early succes- 
sional ones as the adaptation in the former is to synthesize and maintain the 
maximum light interception surface whereas the latter occupying a productive and 
open environment divert their resource budget to other life purposes as growth 
and reproduction. Higher leaf area ratio in shaded environments compared to 
that in the open was also reported by Myerscaugh and Whitehead (1977) and 
Bazzaz and Harper (1977). Lower leaf area ratio of early successional sprouting 
species compared to that of the early successional non-sprouting types may be 
accounted as due to the preferential allocation of photosyntbates to the under- 
ground organs of the farmer. The somewhat lower reproductive effort of the. early 
successional sprouting populations compared to the non-sprouting ones, though 
not statistically significant (P > 0-05), might have evolved due to the failure of 
their regeneration through seedlings (Wilson 1971), 



Successioftat herbaceous, communities 67 

Absolute leaf area of a plant gives an idea about its capacity of light inter- 
ception and vigour while Leaf area ratio, the ratio of light interception surface and 
total biomass (cm 8 leaf area/g biornass) indicates the efficiency of dry matter 
production on leaf area basis. Significantly positive correlation of leaf area and 
negative correlation of leaf area ratio with reproductive effort in the early succes- 
sionai non-sprouting category show that high reproductive potential is associated 
with the vigour and also the production efficiency of the species. Comparatively 
stronger correlation of leaf area ratio with reproductive effort tlau that of the 
absolute leaf area with the reproductive effort in the early successional non-sprout- 
ing species indicates that reproductive success here depends more upon the pro- 
duction efficiency rather than the overall vigour of the plant. In contrast, early 
successional sprouting populations exhibited positive correlation of leaf area ratio 
with sexual reproductive eifort. Thus a species like Mimosa pudlca which has 
a high leaf area ratio allocates more for sexual reproductive effort. Also it so 
happens that this species is less vigorous in its vegetative regeneration compared 
to others like Sac char wn arundinaceum and Thysanolaena maxima., and thus 
compensates more through sexual reproduction. Complete paucity of flowering 
in the first post-fire year, as in Imperata cylindrica and Grewia elastica has been 
shown for a number of shrub species (Gill 1975). This aspect of the problem is 
receiving our attention. Within the late successional group no signitican 
(jP>0-05) relationship between leaf characteristics and reproductive effort wa 
found, suggesting that sexual reproduction is not related with production effici- 
ency or vigour of the plant ; the strategy seems to be to maximize vegetative- 
growth in a closed habitat. 

Considering the annuals and perennials of the early successional non-sprouting 
category separately, certain differences are apparent. While reproductive effort 
seems to be dependent upon production efficiency in both the cases, it was positively 
correlated with absolute leaf area in the case of perennials alone indicating that 
the vigour of the plant is less critical for the annuals having a single possibility 
of flowering during their life-span. 

MacArthur and Wilson (1967) pointed out that organisms in open environ- 
ments are selected for greater reproductive capacity (r-strategy) while those in 
closed environments are selected for greater ability to compete for resources, though 
at the cost of lower reproductive potential (it-strategy). Grime (1974, 1977) has 
extended this argument by describing three primary strategies in plants which. 
are related to their ability to withstand competition, stress and disturbance. Here, 
ruderal and stress tolerant strategies correspond to the extreme of r- and jf-selec- 
tion while highly competitive species of productive environments occupy an inter- 
mediate position. The findings of the present study clearly indicate that early 
successional non-sprouting populations are of ruderal type as they are equipped 
with the strategy to maximize seed production in order to colonize a disturbed 
habitat whereas early successional sprouting and late successional populations 
direct their synthetic capacity for competition and stress tolerance respectively by 
economizing on the reproductive growth. Vegetative reproduction has been looked 
merely as a growth in a horizontal plane (Harpeu 1977) and, therefore, is not 
considered in the present study. 



6S J? G Saxena anct P S Rcmakrishnm 

Acknowledgement 

This research was supported by the Department of EBvirenmcjit, Government of 
India, under the ' Man and Biosphere ' programme. 

References 

Abrahamson W G 1975 Reproductive strategics in dewberries ; Ecology 56 721-726 
Abrahamson W G 1979 Patterns of resource allocation in wildflower populations of fields and 

woods ; Am. J. Bot. 66 71-79 
Abrahamson W G and Gadgil M 1973 Growth and reproductive effort in golden rods (Solidago, 

Compositae) ; Am. Nat. 107 651-661 
Bazzaz F A and Harper J L 1977 Demographic analysis of the growth of Linwn itsitatissimwn ; 

New Phytol. 78 193-208 
Evans G C 1972 The quantitative analysis of plant growth (Oxford : Blackwell Scientific 

Publications) 

Gill A 3Vf 1975 Fire and the Australian flora : a review ; Aust. For. 38 4-25 
Grime J P 1974 Vegetation classification by reference to strategies ; Nature (London) 250 

26-31 
Grime J P 1977 Evidence for the existence of three primary strategies in plants and its relevance 

to ecological and evolutionary theory ; Am. Nat. Ill 1169-1194 
Harper J L 1977 The population biology of plants (San Fransisco : Academic Press) 
Harper J L and Ogden J 1970 The reproductive strategies of higher plants I. The concept of 

strategy with special reference to Scnecio vnlgans L. ; /. Ecol 58 681-698 
MacArthur R H and Wilson E O 1967 The theory of island biogeography (Princeton NJ | 

Princeton University Press) 

Misra R 1968 Ecology workbook (New Delhi : Oxford and IBM Publication) 
McNaugaton S J 1975 r- and ^-selection in Typfa ; Am. Nat. 109 251-261 
Myerscough P J and Whitehead F H 1967 Comparative biology of Tussilago far far a L,, 
Chaiwerteriott angustifoliuni (L.) Scop. , Epilobium montanum L. and Epilobium adenocaulon 
Hauskon. II. Growth and ecology ; New Phytol. 66 785-823 
Newell S J and Tramcr E J 197S Reproductive strategies in herbaceous plant communities during 

succession ; Ecology 59 228-234 
Ogden J 1974 The reproductive strategy of higher plants. II, The reproductive strategy of 

Tussilago farfara L. ; /. Ecol 61 291-324 
Primack R B 1979 Reproductive effort in anuual and perennial species of Plantago (Plantagi- 

naceae) ; Am. Nat. 114 51-62 

Ramakrishnan P S, Toky O P, Mishra B K and Saxena K G 19SI Slash and burn 
agriculture in north-eastern India ; In Fire regimes and ecosystem properties (eds.) H A 
Mooney, J M Bonnicksen, N L Christensen, 3 E Lo-tan and W A Reincrs. USDA For. 
Serv. Gen. Tech. Rep. pp. 560-584 

Wilson M F 1971 Life history consequences of death rates ; Biologist (Phil, Sigma Sac,) 53 
49-56 



Proe. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 1, February 1982, pp. 69-77. 
(g) Printed in India. 



Vesicular arbusculai mycorrhiza in subtropical aquatic and marshy 
plant communities 



R CHAUBAL, G D SHARMA and R R MJSHRA 

Department of Botany, North-Eastern Hill University, Shillang 793 014 9 India 

MS received 20 November 1980 ; revised 4 December 1981 

Abstract. Occurrence of vesicular arbu&cular mycorrhiza in live subtropical ponds, 
i.e. , cutrophic (jPl, P2 and P3), running water (T4), oligoiropluc lake (PS) and marshy 
plant community (M) was studied. It was. observed that the plants growing in P\ , P$ 
and M habitats exhibited the vesicular arbuscular mycorrhizal association, whereas 
the fungal association was. lacking in plants of jP2, P3 and P4 ponds. The cndo- 
gonaceous spore population was estimated from water and sediments of the different 
ponds and it was. found that cnxlophytcs in sediments arc less in terrestrial 
habitats and completely absent from water samples. The bioassay studies revealed 
that plants without mycorrhizal association grew poorly and all the cndophytcs 
isolated could establish vesicular arbu&cular mycorrhizal associations in pot cultures. 

Keywords. Vesicular arbuscular mycorrhiza ; subtropical aquatic community ; 
endophytes ; bioassay. 



L Introduction 

Vesicular arbusciilar mycorrbiza (VAM) is universal in occurrence (NicoJson 1967; 
Mosse 1973) and is useful to the host plant in various ^ays ; in enhancing the 
uptake of nutrients (Kayman 1975) and water (Safir etal 1972), in resisting against 
pathogen (Marx 1975) and in increasing the effective absorption surface of roots 
(H&yman and Mosse 1971). Most of the rushes and sedges, however, are reported 
to be non-mycorrhizal (Powell 1974 ; Khan 1974 ; Harley 1969 ; Gerdemann 
1975). Recently, a few temperate (Soaderg&ard and Laeg&ard 1977) and tropical 
(Bagyaraj et al 1979) aquatic species were reported to be mycorrhizal. In the 
present study five aquatic and one marshy sub-tropical plant community were 
examined for occurrence of VAM and endophytic fungi. To test the ability and 
efficiency of these endophytes a bioassay \*as also developed to test its potential 
use in propagation of VAM in suecessiona communities of aquatic systems. 

2. Materials and methods 
2*1. Site selection 

Five fresh water bodies and one marshy habitat in ShiUong (altitude 1450 m, 
latitude 25-34N and longitude 91*5i6 E) \vere selected for the present study. 

69 

P.CJB-7 



70 R Chaitbal, Q D Sharma and R R Mishra 

The ponds, are designated as PI, P2, P3, P4, P5 and M (marshy habitat). Pl # 
P2and P3 are eutrophic poads, P4 is a running stream and P5 is an oligotrophic 
lake. PI remains dry in summer and receives water during the rainy season. 

2-2. Collection of root samples and assessment of VAM 

Intact plants with roots from different localities were collected in containers. The 
roots were washed with tap water and cut into segments of approximately 1 cm in 
length (100 segments from five plants). Further, the root segments were processed 
and stained for VAM infection by the Phillips and Hayman (1970) technique. 
Percentage of root infection was calculated in the presence of either vesicle, 
arbuscules or both by counting the infected segments by the slide method (Mishra 
et at 1981). 

2-3. Estimation of endogonaceous spores 

Fifty ml of water and 30 g of sediment were collected by water sampler from each 
site in five replicates. The sediment was wet sieved and decanted (Gcrdemann 
and Nicolson 1963) and water was filtered through. Whatman No. 1. filter paper, 
tne spores retained on the sieves and filter paper were examined under a binocular 
microscope. 

24. Sioassay study for infection efficiency of endogonaceous spores In pot cultures 

Sterilized maize seeds were germinated in sterilized moist chambers. Five seed- 
lings (2 cm radicle stage) were transplanted to pots (11 x 10") containing steri- 
lized soil (soil 4- sand in equal amount w/w). The plants were inoculated with 
endophytes isolated from the sediments ; uninoculated pots received soil with 
microflora but were devoid of mycorrhizal propagules. All the pots were regularly 
watered. Plants were harvested 15, 30 and 45 days after transplanting. Root 
infection, shoot height, dry weight and leaf production were recorded at each 
harvesting. 

2.5. PkysiGQchemical analysis of water and soil 

pH of soil and water was measured by electronic digital pH meter. Organic 
carbon (Walkey method), nitrate (phenol disulphonic method) and phosphorus 
(Bray's method) were estimated as outlined by Jackson (1967). 

3. Results 

Five subtropical aquatic species, viz. Rotala rotwdifolia, Paspalum dilatatumt 
Polygonum hydropiper, Nymphaea alba and Hydrilla verticellata have been observed 
for the first time as mycorrhizal (table 1, figures 1-4). Vesicular arbuscular my- 
corrhiza (VAM) was observed in JP1, P5 and M plant communities. Plants 
growing in F2, P3 and P4 ponds \vere devoid of VAM infection. Percentage 
infection was highest in PS plants followed by PI and was least in plants from 
the marshy (M) habitat (table 2). Vesicles and hyphae were regularly observed. 
The population of endogonaceous spores in general was low in all the sediments 
from different sites and the number did not differ significantly (table 2). No 



Vesicular arbusculaf mycorrhiza 



71 



Table 1. Percentage occurrence of vesicular arbuscular mycorrhiza in root* of 
different plant species of sub-tropical aquatic and marshy communities. 



Aquatic Marshy 
commu- Plant species VAM (%) commu- Plant species 
nity nity 


VAM % 
(root) 


PI Rotala rotundifolia 


10-00 


Impatiens chinensis 


100-00 


Paspalum dilatatum 


60-00 


Drosera sp. 


64-00 


Polygonum hydropiper 


56-00 


Utricularia sp. 


73-00 


Cyperus distans 


0-00 


Soncfius sp. 


52-00 






Polygonum cajpitatum 


51-00 


PZ Cardamine hirsuta 


0-00 


Drymaria cordata 


33-00 


C. macrophylla 


0-00 


Plantago major 


19-00 


Scirpus articulate 


0-00 


Nasturtium indica 


0-00 


S. juncoides 


0-00 


Anemone rivalaris 


0-00 


Eleocharis congesta 


0-00 


Steudnera colocasioides 


0-00 


P 3 Rotala rotundifolia 


0-00 


Oen&thera javinaca 


0-00 


Cyperus distans 


0-00 


Brassica juncea 


12-00 


Eleocharis congesta 


0-00 


Rumex nepalensis 


28-00 


Spargonium ramoswn 


0-00 


Galium rotundifolium 


16-00 






Panicum brevifolium 


46-00 


P 4 Hydrilla vertidllata 


0-00 






Alternanthera philoxeroides 


0-00 






Monochoria hastata 


0-00 






Hydrocotyle sibthorpioides 


0-00 






Lasia spinosa 


0-00 






Pa Rotala rotundifolia 


3-00 






Hydrilla vertidllata 


16-00 






Nymphaea alba 


12-00 







Table 2. Eridogonaceous spore population and frequency of YAM jmycorrbizal 
plants in different localities. 





Mean spore 


population/30 g/rnl 








soil/water 




Sites 






- Frequency (%) of 








Water 


Soil 


mycorrhiza 


(per 30 ml) (per 30 g) 


PI 





27 


75 


P* 





33 


X) 


P. 





6 





P* 





7 





A 





11 


100 


M r 


a 


34 


71-3 



72 R Chanhal, G D Sharma and R R Mishra 

endogoiiaceous spores were found in \vater. The pot culture studies on infection 
efficiency of endogonaceous spores from different sites revealed that they may 
infect and establish in roots growing in soil and that their efficiency may differ 
(table 3). TMnoculated seedlings did not grow well, whereas the seedlings with 
mycorrhizal association gre^ better. 

Soil and water from all the sites were acidic in nature. Organic carbon was 
very lew in water samples and highest in soil of marshy land. Nitrate and phos- 
phate were highest in P2 and Fl and lowest in P5 and M sites (table 4), 



4. Discussion 

The study reveals that VAM occur rarely in aquatic subtropical plant communities, 
but they are not completely absent. The results are, therefore, contrary to the 
vieus of Harley (1969), Gerdemann (1975), Powell (1974) and Khan (1974), but 
support the findings of Sondergaard and Laegaard (1977) and Bagyaraj et at 
(1979). Apart from environmental factors like light, temperature and aeration, 
the occurrence of VAM and its intensity may be regulated by the nutrient status 
of the aquatic system. Gerdemann (1968) observed that mycorrhiza may tie low 
in rich soil. The amount of infection in a fe\v species, i.e., Rotala rotundifolia 
and Hydrilla verticellata differed insignificantly in different \vater systems depending 
on the nutrient status and other physical factors of these systems. In general, 
jpl, P5 and M sites favoured VAM establishment but P2, P3 and P4 ponds 
did not possess any mycorrhizal association. It seems that temporary drying of 
PI and M habitats in summer may initiate the VAM establishment. A similar 
trend was also observed in other environmental conditions (Read et al 1976). 
The high percentage occurrence of VAM in, PS may be attributed to its eligotro- 
phic nature (Sonderga?rd and Laegard 1977), as Hydrilla veniddlata was my- 
corrhizal in P5 community but not in P4. Besides the root system, the shoot 
also helps the aquatic plants in absorbing the nutrients from water and this may 
be one of the reasons for the absence or less frequent mycorrhizal association in 
certain plants. Sutcliffe (1962) suggested that in aquatic plants, the roots primarily 
act as anchors ; on the other hand, I>enny (1972) concluded that the nutrients 
may enter through roots and shoots. Therefore, it seems that phosphorus uptake 
may depend oa the efficiency of the root system, i.e. 9 some root systems may be 
adaptive enough to draw the phosphate at low level even in the absence of VAM 
association (Powell 1975). VAM may not be of much importance in plants grow- 
ing in rich medium (P2 9 P3 and P4 system) but it may help the hosts in absorp- 
tion of the nutrients growing in low level nutrient systems (PS). Therefore, two 
conditions, i.e., temporary drying of the aquatic system and the oligotrophic nature 
seem to be more favourable for VAM development. The presence of endophyte 
spores in all aquatic, systems suggests that these spores probably enter into the 
water system through run off from terrestrial ecosystem, their subsequent develop- 
ment, however, is governed by water regime, light, aeration and other factors 
(Mosse 1973). The presence of endophyte spore in P2 community may further 
indicate that either these species are not capable of causing infection or plants may 
not be susceptible in such systems (Reeves et al 1979 ; Miller 1979). The bio- 



Vesicular arbuscular mycorrhiza 




Figures 1-4. 1. Spore, Glom:is sp. infecting foot tissuo of Panicitm brevlfolium 
(x 1,000), 2. Vesicles and hyplue m root tissue of l*np atiens balsamia (x 100), 

3. Obovato vesicles of Ghtnus sp. in tho root tissue of Rotala rotundifolia (X 400)^ 

4. Round vogiclos with oil globule and degenerating stage of laypha in root tissue 
of Nyrnphaea alba (x 400), 



Vesicular arbuxcular myaarrhiza 



75 



3 


+ + 4- + 


4- + I 


It 3 


9 ? o o 
8 3 <* i 

rH eS s S 


O O V* 

<N >A O 

2 8 *- 




$ 8 S'c' 


"** "^ VQ OO 


* ^ cm 


J S I 


O t"* r- <NI 
CS C4 J~n c^ 


* <x> * 

C-4 C4 A 


5 6 


VO OO V> V0 


t^. ,00 -^ 


i 


+ > , , 


1 4- l 


?. *! 


p p o o 

O O O C5 
CN O CO 00 

T-H IN T 1 


O CN O 
<Q 

^ s ^ 


O 4-> 

jr/5 " sH* 


ON vi >n oo 


fNjj ON Q> 

\43 ^ C4 


(M * 

s 


n r^. ^ vjs 


n ^ ' .* 


a 


" : : 


* I 


- I 1s 

TQ ^ 


9 ? o o 
JCj IS g 


? 

& $ 


2 ill 

tn r ^ 


E S S 


<n ^ V 


s* 


^ ^: * ^ 


^r ^ m 


i ^^ 




"OB 


il^ 




< | 



76 R Ckauhal, G D Sharrna and R R Mishm 

Table 4. Physico-chemical characters of soil and water in different water bodies . 



Site 



pH 



Org. Carbon (%) Nitrate (ppm) Phosphate (ppm) 



water 



soil 



water 



Soil 



Soil 



water water 



A 


6-5 


8-3 


0-05 


4'9 


2-2 


0-20 


3-6 


6-62 


p* 


6-4 


6-3 


Q'07 


7-6 


1-6 


0-17 


3*8 


7-8 


Pa 


6-0 


6-3 


0-05 


5-3 


1-6 


0-07 


1-07 


3-18 


Pi 


6-6 


6-5 


0*06 


5-6 


1-8 


0-09 


0-1 


2-01 


p* 


6-1 


6-3 


0-04 


3-9 


1-4 


0-05 


o-oi 


1-96 


M 




6-2 


0*00 


8-0 


0-1 




- 


1*42 



assay studies using maize (Zea mays) as test plant suggested that these endo- 
phytes may develop VAM and enhance the growth of plants, when placed into 
sterilized soil. It is of great ecological importance to study the establishment, 
germinatioa and entrance of these endophytes into the hosts under diverse 
environmental conditions, in understanding of the developmental pattern of aquatic 
and terrestrial communities. Baylis (1959) suggested that VAM play a significant 
role in the evolution of plant communities. Therefore, studies in controlled 
conditions in aquatic habitats on the esta lishment of VAM will provide infor- 
mation towards further understanding of succession in aquatic systems and <would 
aid management of aquacuitural systems. 



References 

Bagyaraj D J, Manjunath A and Patil R B 1979 Occurrence of vesicular arbuscular mycorrhiza 
in some tropical aquatic plants ; Trans. Br. Mycol Soc. 72 164-165 

Baylis GTS 1959 Effect of VAM on growth of Gnselinia littoralis (Cornaceae) ; New Phytol. 

58 274-280 

Denny P 1972 Sites of nutrient absorption in aquatic macrophytes ; /. Ecol. 60 819-329 
Gerdemann J W 1975 Vesicular arbuscular mycorrhiza. In The development and function of roots 

(eds.) J G Torrey and Clarkson (London : Academic Press) 
Gerdemann J W 1968 Vesicular arbuscular mycorrhiza and plant growth; Ann. Rev. Phyto-^ 

pathol 6 397-410 

Gerdemann J W and Nicolson T H 1963 Spores of mycorrhizal Endogone species extracted from 
soil by wet sieving and decanting ; Trans. Br. Mycol. Soc. 46 235-243 

Harley J L 1969 The biology of mycorrhiza (London : Leonard Hill) 

Hayman D A 1975 Phosphorus cycling by soil microorganisms and plant roots. In soil Micro- 
biology (ed.) N Walker (London : Butterworth) 

Hayman D A and Mosse B 1971 Plant growth responses to VAM I. Growth of Endogone 
inoculated plants in phosphate deficient soils ; Neb Phytol. 70 19-27 



Vesicular arbuscutar mycorrhizd 1i 

kson M L 1967 Soil chemical analysis (New Delhi : Prentice Hall) 

.an A G 1974 The occurrence of mycarrhizas la Halophyfcos, hydrophytes and xerophytes and 

of Endogow spores in the adjacent soils ; /. Gen. Microbiol. 81 7-14 
irx D H 1975 Mycorrliizae of exotic trees in tlxe Peruvian Andes and synthesis of ectomyoor- 

rhizae on niexican pines ; For. Sci. 21 353-35$ 
Her RM 1979 Some occurrence of vesicular arbuscular mycorrhizae in natural and disturbed 

ecosystems of Red desert ; Can. J. Bat. 57 619-623 
shra R R, Sharma G D and Kharsyntiew IB 1981 Response of inoculum density in maize ; 

Experientia 37 568-569 
:>sse B 1973 Advances in the study of vesicular arbuscular mycorrhiza ; Ann. Rev. Phytopathol. 

11 171-195 
colson T H 1967 Vesicular arbuscular mycorrhiza a universal plant symbiosis ; Sci. Prog. 

(Oxford) 55 551-581 

illips J M and Hayman D A 1970 Improved procedures for clearing roots and staining para- 
sitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection ; Traits. 

Br. Mycol Sac. 55 158-161 
wel C L 1974 Effect of P-fertilizer on root morphology and P-uptake of Carex coriacea ; 

Plant Soil 41 651-667 
wel C L 1975 Plant growth responses to VAM VIII Uptake of P by Onion and clover 

infected with different Endogorte spore types in 32 P-labelled soils ; jNew PhytoL 75 563-566 
?ad D J, Kovchiki H K and Hodgson J 1976 Vesicular arbuscular mycorrhiza in natural 

vegetation systems ; New PhytoL 77 641-653 
;cves B F, Wagner D, Moorman T and Kiel J 1979 The role of endomycorrhiza in rcvcgc- 

tation practices in semi arid west. L A comparison of incidence of mycorrhizae in severe y 

disturbed W. natural environments ; Am. J. Bot. 66 613 
tir G R, Boyer J A and Gerdemann J W 1972- Nutrient status and mycorrhizal enhancement 

of water transport in Soybeans ; Plant Physiol. 49 700-803 
ndcrgdard M and Laegaad S 1977 Vesicular arbuscular mycorrhiza in sonic aquatic vascular 

plants ; Nature 268 232-233 
tcliffc J F 1962 Mineral salts absorption in plants (Oxford : Perfiamon) 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number i, April 1982, pp. 79-82. 
Printed in India. 



Chandmsekhamnia : A new genus of Poaceae from Kerala, India 

V J NAIR, V S RAMACHANDRAN and P V SREEKUMAR 

Botanical Survey of India, Caimbatore 641003, India 

MS received 26 June 1981 ; revised 30 March 1982 

Abstract. A new genus, Chandrasekharania and a new species Chandrasekharania 
keralensis under it are being described from Cannauore District, Kerala. 

Keywords. Chandrasekharania keralensis ', Poaceae ; new genus; new species. 



1. Introduction 

Intensive explorations are being conducted in various parts of Kerala to clearly 
understand the flora of this botanically rich area. During these tours a number 
of very interesting plants were collected. One of these belonging to the family 
Poaceae., on critical study with reference to available literature (see Bor 1960 ; 
Ved Prakash et al 1978) and the specimens available in the Herbarium of Southern 
Circle, Botanical Survey of India, Coimbatore (MH) and the Central National 
Herbarium, Calcutta (CAL), turned out to be quite distinct. So a specimen along 
with a detaile^l description and analytical sketches was sent to Dr Thomas A 
Cope, Herbarium, Royal Botanic Gardens, Kew, for expert opinion. He confirmed 
that our collection belongs to a new genus. This along with its type species is 
described here. 



2. Latin description 

Chandrasekharania V J Nair, V S Ramachandran et P V Sreekumar gen. nov. 
Gramen annuum. Culmi erecti vel decumbentes. Folia lanceolata vel ovato- 
lanceolata, basibus cordatis. Ligulae obscurae. Inflorescentia contracta, racemi- 
flora, spiculae omnibus similaribus, lateraliter compressis, floribus duobus, pedi- 
cellatis, pedicellis numquam articulatis. Flosculi similes, hennaphroditi. Glumae 
inaequales, coriaceae, ovatae vel ovatolanceolatae, 7-nervatae, aristatae, aristis 
curtis et rectis. Glumae infernae breviores in dorsi parte superiore pilis basi 
tuberculatis. Lemmata 5-nervatae, coriacea, elliptico-lanceolata, apicibus 
breviter emarginatis, aristatis ad sinus. Aristis curtis et rectis. Paleae ovato- 
ellipticae, hyalinae, binervatae, bicarinatae, carinis ciliatis, apicibus bilobatis. 
Ovarium glabrum. Styli 2, distincti. Stigmata plumos.a. Stamina 3, filamentis 
brevissimis. Lodiculae 2. Grana ellipsoidea. 

79 



80 V J If air 9 V S Ramachandran and P V Sreekumar 

Species typica sequens : 

Chandrasekliarania keraknsis V J Nair, V S Ramachandran et P V Sreekumar 
sp. nov. 

Gramen annuum usque 40 cm altum. Culmi graciles, striati leaves et glabri. 

Nodis inferioribus radicantes. Folia 2-4 -5 cm longa,, 5-8 mm lata, marginibus 

et paginis ambabus pilis densis vel sparsis, basi tuberculatis. Vaginae striatae, 

glabrae, internodiis breviores. Inflorescentia, contracta, ovoidea vel oblonga, 

spiciformis; racemiflora, 1 5-2- 5 cm longa, 1-1 5 cm Uta. Spiculae 5-6 mm longae. 

Pedicelli 0-5-1 -5 mm longi. Glumae infernae 7-nervatae, nervi duo alternati et 

mediani prominentes, nervi cateri inconspicui, ovatae, 3-5-4 mm longae, ari&tatae, 

arista ca. 3 mm longa, costa et arista scabridus, inargines ciliolati, dimidia supera 

paginarum dorsalia dense tecta pilis, basibus tubercularibus. Glumae superae 

5 5-6 mm longae, ovato-lanceolatae acuminatae, aristatae, 7-nervatae, inargines 

ciliolati nervi scaberuli, arista scabrida, ca. 3mm longa. Lemmata elliptico- 

lanceolata, 4-5 mm longa,, apicibus breviter emarginatis, aristatis ad sinus, coriacea, 

glabra praeter scaberula costas, arista scabrida, 1-5-2 mm longa. Paleae ca. 

4 mm longae, subtiles hyalinae, binervatae, dorsiis dimidiis infernis pilis longiore, 

cariniis ciliatis, apicibus bilobatis, lobi acuti. Gynoecium ca. 2 mm longum. 

Ovarium 0-5 mm longum, oblongum. Antherae ca. 1-25 mm longae. Fila ca. 

0-25 mm longa. Lodiculae cuneatae, apicibus emarginatis. Grana ellipsoidea, 

ca. 1 mm longa, fusca et extremis distalibus maculae ateris. 

Holotypus : Kerala, Cannanore District, Kannoth, 175 m, 18-2-1978, 
V S Ramachandran 54064 (CAL). Isotypi in K et MH. 

3. Description 

Chandrasekharania V J Nair, V S Ramachandran et P V Sreekumar gen. nov. 
Annual grass. Culms erect or decumbent. Leaves flat, lanceolate or ovate- 
lanceolate, base cordate. Ligules obscure. Inflorescence terminal, solitary, 
contracted, racemose. Spikelets ail alike, laterally compressed, two flowered, 
pedicelled, pedicels never jointed. Florets similar, bisexual. Glumes unequal, 
coriaceous, ovate or ovate-lanceolate, 7-nerved, awned, awns short and straight. 
Lower glume smaller with the dorsal surface with tubercle based hairs in the upper 
half. Lemma 5-nerved, coriaceous, elliptic-lanceolate, minutely notched at the 
apex with an awn in the sinus. Awns short and straight. Palea ovate-elliptic, 
hyaline, 2-nerved, 2-keeled, keels ciliate, apex two lobed. Ovary glabrous. 
Styles 2, distinct. Stigmas feathery. Stamens 3. Filaments very short. Lodi- 
cules 2. Grain ellipsoid. 

Type species followsi : 

Chandrasekharania keralensis V J Nair, V S Ramachandran et P V Sreekumar 
sp nov. 

Annual grass up to 40 cm tall. Culms rooting at the lower nodes, slender, 
striate, smooth, glabrous. Leaves 2-4, 5 cm long, 5-8 mm broad, both surfaces 
and margins with dense or sparse tubercle based hairs. Sheaths shorter than the 



Chandrasekharania, new genus of Poaceae 



81 




12mm 



10 \JJ 



Figures 1-13. Chandrasekharania kemlemis gzn. et sjp. nov. 1. Habit. 2. Spikclet. 
3. Lower glutne. 4. Upper glutxxe. 5. Lower lemma. 6. Lower palea dorsal view. 
7. Same ventral view, 8. Upper lemma 9. Upper palea dorsal view, 10. Same 
ventral view. 11. Lodicules and stamens. 12. Gyaoecium, 13. Grain. 



internodes, striate, glabrous, Inflorescence a contracted ovoid or oblong spikate 
raceme, 1-5-2 -5 cm long, 1-1 -5 cm broad. Spdkelets 5-6 mm long. Pedicels 
0-5-1 -5 mm long, scabrid. Lower glume 3 -5-4 mm long, ovate, awned, awn 
ca. 3 mm long, densely hairy on the upper half of the dorsal surface with tubercle 
based hairs, the median and the alternating two nerves prominent, the midrib 
and the awn scabrid, margins ciliolate. Upper glume 5 -5-6 mm long, ovate- 
lanceolate, acuminate, the tip with an awn ca. 3 mm long, scaberulous on the 
nerves, scabrid on the arista, margins ciliolate. Lemma elliptic-lanceolate, 
4-5 mm long, shortly notched at apex with an awn in the sinus, coriaceous, glabrous 
except for the scaberulous midrib, awn scabrid, 1 5-2 mm long. Palea ca. 4 mm 



82 V J Nair, V S Ramachandran and P V Sreekumar 

long, delicate, hyaline, long-ciliate on the lower half of the outer surface, keels 
ciliate, apex bilobed, lobes acute. Gynoecium ca. 2 mm in length ; ovary ca. 
0-5 mm long, oblong. Anthers ca. 1-25 mm long, filaments ca. 0-25 mm long. 
Lodicules cuneate and shallowly notched at apex. Grains ellipsoid, ca. I mm 
long, brown coloured with a black spot at the distal end. 

Holotype : Kerala, Cannanore District, Kannoth, 175 m, 18-2-1978, 
V S Ramachandran 54064 (CAL). Isotypes in K and MH. 



4. Affinities 

Affinities of this new genus are not very clear. According to Dr T A Cope, Kew 
Herbarium, anatomical studies are required to clearly understand its affinities 
(personal communication). 



5. Etymology 

The genus is named after Dr N Chandrasckharaa Nair, the first author's teacher 
and present Joint Director, Botanical Survey of India, Coimbatore, in recognition 
of his outstanding contributions to Indian botany. The specific epithet is after 
'Kerala' the state from where the plant has been collected 



Acknowledgements 

Authors are thankful to Dr Thomas A Cope, The Herbarium, Royal Botanic 
Gardens, Kew, for examining the specimen and giving his valuable opinion and 
R Suudararaghavan, Regional Botanist, (Kew) for help. Rev. Fr. Zachaeus, 
CMI, Principal, Lisieux Higher Secondary School, Coimbatore, has kindly corrected 
the Latin description. 



References 

Bor N L 1960 The Grasses of Burma, Ceylon, India and Pakistan (Oxford, London, New York, Paris : 
Pergamon Press), p. 1-767 : Repr.ed. 1973 (Otto. Koeltz Antiquariat, Koeningstein, BRD) 

Ved Prakash, Shukla U, Pal D C and Jain S K 1978 Additions to the Indian grass flora in 
last two decades ; Bull. Bot. Surv. India 20 143-147 



Prac. Indian Acad. Sci. (Plant Sci.), Vol, 91, Number 2, April 1982, pp. 83-91, 
Printed in India. 



Chromosome relationships of spinous solanums 



P B KIRTI* and B G S RAO 

Department of Botany, Andhra University, Waltair 530003, India 

* Present address : JARl-Regional Station, Jlajendranagar, Hyderabad 500030, 

India 

MS recdved 25 April 19S1 ; revised 29 March 1982 

Abstract. Chramasome pairing wa.s studied in reciprocal hybrids of S. integnfolium 
and S. indlaim and the FI S. integnfolium X 5. surattense. Pairing was generally 
close and meiosis regular with higher chromosome associations. AH hybrids were 
highly sterile. Such sterility could be due to the formation of unbalanced gametes 
following pairing and exchange between partially homeologo-us chromosomes, 
S. integrifolium, S. irtdictitn, S. surattense along with S. melongena and its wild forms 
form a closely related group of taxa. 

Keywords. Solarium indicum ; 5. surattense ; S. integnfolium ; Fj hybrids ; sterility ; 
chromosome relationships. 



1. Introduction 

Chromosomal studies on species hybrids are of considerable importance in 
elucidating species relationships and evolutionary trends. Such studies on 
spinous Solanums, which are important medicinally as well as vegetables, were 
meagre because of the difficulty in producing interspecific hybrids. Magoon 
et al (1962) cited the earliest attempt^ of species hybridization in solaminis 
including those of Sarvayya (1936), Hagiwara and lida (1939) and Tatebe (1939). 
Bhadhuri (1951) first discussed the interrelationships of spinous solanums 
including the origin of S. melongena. Further attempts in this direction were 
made by Zutshi (1967), Rajasekaran (1969, 1970a, b, 1971), Rajasekaran and 
Sivasubramanian (1971), Rangaswamy and Kadambavanasundaram (1974), 
Veerabhadra Rao (1977), Veerabhadra Rao and Rao (1977b, c), Kirti and 
Rao (1978, 1981a, b, c). Most of the cultivars of the brinjal are susceptible to 
various diseases and pests (Krishnaiah and Vijay 1975). Wild species suet as 
S. integnfolium and S. indicum are resistant to some of the diseases and pests. 
However, these species cannot be utilized in egg plant improvement unless the 
interrelationships of the whole group are unravelled. First attempts of crossing 
S. integnfolium and S. melongena were made by Hagiwara and lida (1939), Tatebe 
(1939), Khan et al (1978). Kirti and Rao (1981c) discussed at length the chromo- 
some relationships between these two species as well as S. integrifoUum and 



84 ? 5 Kirti and 3 G S Rao 

S. indicum var. multiflora. The chromosome relationships S. mtegrifolwm, 
S. indicum, S. surattense and other species are discussed in some detail here. 



2. Materials and methods 

Seeds of S. indicum L. and S. surattense Burm. F. (=S. xanthocarpum Schrad 
and Wendi) were collected from plants occurring wild while seeds of S. integrifolium 
were kindly provided by Prof P V Bhiravamurthy of the Andhra University, who 
obtained them from Denmark. 

Method of crossing was the one employed by Veerabhadra Rao and Rao 
(1977a). 

Standard propionic carmine schedule was used for PMC smears. 



3. Results and discussion 

3-1. Crossability 

An on-the field screening for functional pistillate flowers was done since stylar 
heteromorphism is prevalent in spinous solanums. Only flowers with long and 
exserted styles can be used as pistillate flowers in crossing experiments. Rao 
and Veerabhadra Rao (1976) have studied this phenomenon in some detail in 
S. surattense. Shamim Baksh etal (1978) described this in S. integrifolium. In 
S. indicum, the inflorescence is a 3-4 flowered cyme and only the basal most flower 
having the long an.d exserted style sets fruit. Others are generally with shorter 
and inserted styles and do not set fruit. So this phenomenon should be taken 
as an important criterion in the consideration of crossability. 

The ease with which any two species are crossed reflects to some extent the 
nearness of the species concerned with the absence of prezygotic incompatibility 
"barriers. In the present study, S. integrifolium and S. indicum could be crossed 
in reciprocal directions and the percentage of hybrid seed obtained was very 
high (table 1). S. integrifolium could be crossed with S. surattense in only one 
direction and only a solitary hybrid could "be realised. Thus it seems S. integri- 
folium is more closely related to S. indicum on the basis of this observation than 
to S. surattense even though other factors are to be taken into consideration. 

3-2. Morphology of parental species and hybrids 

S. indicum and S. surattense conformed to the classical descriptions of Gamble 
(1957). S. integrifolium is an erect herb with small white flowers, and scarlet 
red coloured and lobed berries. 

Reciprocal hybrids of S. integrifolium and S. indicum resembled each other and 
were intermediate between the parents in some features and resembling either 
of the parents in some respects. Similarly the FI S. integrifolium x S. surattense 
was intermediate. 



Chromosome relalioriships of spinous xoiainmis 




Figures 1-4. Cytology of hybrids ; Diakirwsia-Metaphase-I in S, indicum X 
S. integrifoUutn 1. 10 n -h 1 J? . 2. 12 n . 3. 10 n -fl ir 



Chromosome relationships of spinous s olanums 



87 



Table 1. Results? on crossabihty relationships of S. integrifolium, S. indicum and 
S. Surattense. 





S. integri folium 


S. indicum 


5. integrijolium 




X 


X 


X 




S. indicum 


S. integrifolinni 


S. surattense 


umber of pollinations made 


15 


70 


105 


umber of fruits set 


1 


1 


1 


umber of healthy Seeds per fruit 


165 


174 


1 


Tcentagc of seed g[errmnation 


97-6 


94-2 


100 


>rcentage of hybrid seed obtained in 








relation to the number of * target ' 








ovules* per ovary 


78-9 


56-5 


0-48 



Approximated from the average number of seeds per fruit in parental species, 209 in 
i'rttegrifoliurn and 308 in 5. indicum. 



Table 2. Frequencies of nuclei with different chromosome associations observed 
in hybrids of S. integrifolium, S. indicum and S. surattense. 



integrifoliujn S. integrifolium X Kinds of chromosome 5. indicum x 
S. surattense S. indicmi associations per nucleus S. integrifolium 
requency of Frequency of Frequency of nuclei 


nuclei 


nuclei 


Makinesis 


Diakinesis 


Metaphase-I IV III 


II 


I Diakiriesis 


Motaphase-I 


9 


27 


15 1 


10 


8 


28 


(40-9) 


(44-3) 


(50-0) 




(63-0) 


(73-7) 


3 


7 


1 


9 


2 5 




(13-6) 


(11-5) 






(4-6) 


*> 


t 


.. 


1 


8 


4 2 


> 










(1-8) 




. . 


4 


2 .. 1 


10 


1 4 


1 




(6-6) 


(6'7) 




(3-7) 


(2-6) 


2 


_ 


1 


9 


3 1 




(9-1) 








(0-9) 




4 


IS 


12 


12 


23 


S 


(19-2) 


(29-5) 


(40-0) 




(21-3) 


(2M) 


4 


5 


1 


11 


2 5 


1 


(18-2) 


(8-2) 


(3-3) 




(4'6) 


(2-6) 


22 


61 


30 Total number 


of nuclei 


analysed 108 


38 



gures in parentheses indicate percentage frequency), 
ytetapha$e-I data not available, 



88 P B Klrtl and B G S Rao 

3-3. Cytological observations 

Meiosis proceeds regularly in the parents with the formation of twelve bivalents 
(2n = 24) and regular chromosome separation at anaphase I and II. 

Various types of chromosome associations (figures 1-3) and their frequencies 
observed in the hybrids are summarised in table 2. Higher chromosome associations 
in hybrids indicate chromosomal structural repatterning in the divergence of the 
concerned taxa. 

Mean frequencies of chiasmata in parents and hybrids are summarized in table 3. 
Mean values in hybrids were significantly lower than in parents indicating some- 
what reduced chromosome homeologies. 

Later stages of meiosis followed normally with regular chromosome segregation 
at anaphases I and II. Laggards, bridges without fragments and micronuclei were 
rarely encountered. Despite regular meiotic divisions, all the hybrids were over 
95% pollen sterile. Stebbins (1950) advocated that cryptic chromosomal structural 
differences are responsible for sterility in hybrids with regular meiotic events. 
It is also possible that segregational events, following pairing and exchange of 
segments between partially homeologous chromosomes of the two genomes leading 
to the formation of unbalanced gametes, eventually lead to lethality (Kirti 1978). 




Hybrid obtaine-d 

- Fruits set, seed sel variable from many 

to none -a II shrivelled 
--- Cross unsuccessful 



Figure 4. Crossability relationships of some spinous 



Chromosome relationships of spinous solanums 




Reproductive behaviour of hybrids 

_ Fertile 

_ Partially fertile 
Sterile 

Figure 5. Fertility relationships of some spinous solanums. 



Table 3. Average chiasma frequencies observed in 5. surattense, S. integrifolium, 
S. indicum and their hybrids. 



S.Surat- S.integri- S.indicum S.integri- S.indicurnx S.inte- 

tense folium folium x S.integri" grifolium 

S. indicum folium X S. surat- 
tense 

DiaJc* Diak Meta-I Diak Meta-I Diak Meta-I Diak Meta-I Biak* 



Slumber of PMCst 
analysed 30 41 



32 27 



61 



30 108 38 22 



Average chiasma 18-17 19-24 19-04 20'06 19-90 17-65 17-56 17-83 17-20 16-41 
frequency per 0-20 0-37 0-61 iO'23 0-40 iO-16 ^0-39 0-20 0-34 0-26 
nucleus 



Per bivalent 



1-Sl 1'60 1-59 1-67 1-66 1-47 1-46 1-49 1'43 



1-32 



Diak = Diakinesis Meta-I = Metaphasc~I 
* Metap^a$e-I data not available. 



90 PS Ktrti and B G S Rao 

3-4* Species relationships 

Thus on the basis of the above cytogenetic observations on crossability, chromo- 
some pairing and meiotic events, S. integnfolium is closely related to 5. indicum 
and S. surattense (figures 4 and 5). The relationship with the former species 
seems to be much cloeer since they can be crossed in reciprocal directions and the 
recombination potential of the reciprocal hybrids is approximately the same as 
in the parents. The hybrid, S. integnfolium x S. surattense, could not be main- 
tained for long and extensive analyses on this hybrid could not be done. But 
to the extent possible, it was observed that univ^lents were more frequent and 
mean chiasma frequencies were comparatively lower. Also the cross was 
achieved with greater difficulty and only a solitary hybrid could be realised. The 
reciprocal cross was unsuccessful. But as far as possible, S. integrifoliurn and 
S. surattense are also closely related. While full bivalent pairing has been reported 
in hybrids of S. surattense and S. indicum, even though the hybrids are sterile 
(Bhadhuri 1951 ; Rajasekaran 1969), Raju etal (1981) observed higher chromo- 
some associations and a good amount of fertility in the hybrid, S. indicum x 
S. surattense. Thus it can be concluded that the three species S. integnfolium, 
S. indicum and S. surattense form a closely related group. With the reports of 
hybridization in spinous solanums of Bhadhuri (1951), Zutshi (1967), Rajasekaran 
(1969, 1970a,b, 1971), Rangaswamy and Kadambavanasundaram (1974), Veera- 
bhadra Rao and Rao (1977b), Khan et al (1978), Kirti and Rao (1982), 
S. integrifoliurn, S. rnelongena and its wild forms, S. surattense, S. indicum 
form a group of closely related taxa. 



Acknowledgements 

The first author is thankful to the CSIR and UGC, New Delhi, for the award of 
fellowships. 



References 

Baksh SJqbal M and Jamal A 197& Breeding systems of Solatium integrifoliurn with an emphasis 
on sex potential and inter-crassability ; Euphytica 27 SI 1-8 15 

Bhadhuri P N 1951 Interrelationships of non-tuberiferous species of solanum with some consi- 
deration on the origin of brinjal, S. melongena ; Indian J. Genet. 11 75-32 

Gamble G S 1957 Solaaaceae '. In Flora of the Presidency of Madras Bat. Survey of India, 
Calcutta, Vol. II, pp. 654-661 

Hagiwara T and lida H 1939 Interspecific crosses between Solanum integnfolium and the egg 
plant and the abnormal individuals which appeared in F 2 . Sot. Mag. (Tokyo} 50 (cited by 
Magoon et al 1962) 

Khan Reayat, Rao G R. and Baksh S 197S Cytogenetics of Solanum integrifoliurn and its 

possible use in egg-plant breeding; Indian J. Genet, 33 343-347 
Kirti P B 1978 Studies on the cytogenetic aspects of some spinous solanums. Unpublished Ph.D 

thesis submitted to. the Andhra University, Waltair. 
Kirti P B and Rao B G S 1978 Meiasis in the interspecific hybrid of two spinous 

and its bearing on their affinities ; Curr. $ci, 47 696-697 



Chromosome relationships of spirtous solanums 9\ 

Karti P B and Rao B G S 1981a Cytogenetic studies on fi hybrid Solatium indicum X S. torvum ; 

Theor. Appl Genet. 59 303-306 
KLirti P B and Rao B G S 1981b Chromosome pairing in reciprocal hybrids of Solatium 

integri 'folium and S. indicum var. multi flora ; Caryologia 32 289-296 
Kirti P B and Rao B G S 1982 Cytological studies on F x hybrids of Solanum integrifolium with 

S. melongena and S. melongena var. insanum ; Genetica (Accepted). 
ECrishnaiah K and Vijay O P 1975 Evaluation of brinjal varieties for resistance to brinjal shoot 

and fruit-borer, Leucinodes orbonalis ; Indian J. Hort. 32 84-86 
Magoon M L, Ramanujam S and Cooper D G 1962 Cytogenetical studies in relation to the 

origin and differentiation of species in the genus Solanum L.; Caryologia 15 151-252, 
lajasekaran S 1969 Cytogenetic studies on the interrelationships of some common Solanum 

species occurring in South India ; Annamalai Univ. Agric. Res. Annual 1 49-60 
lajasekaran S I970a Cytogenetic studies o.f the Fi hybrid, S. indicum x S. melong-ena and its 

arnphidiploid ; Euphytica 19 217-224 
fcajasekaran S 1970b Cytology of hybrid, S. indicum X S. melongena var. insanum ; Curr. Sci. 

3922 
lajasekaran S 1971 Cytological studies on the F x hybrid Solanum xanthocarjpum Schrad and 

Wendl x s. melongena L. and its amphidiploid ; Caryologia 24 261-267 
lajasekaran S and Sivasubramanian V 1971 Cytology of the FI hybrid of Solanum zuccagnfanum 

Dunn x S. melongena L. ; Theor. Appl. Genet. 41 S5-S6 
^aju D S S R, Moorty K V, Kirti P B and Rao B G S 1981 Studies on the origin of Solanum 

melongena L. (The egg plant) : I. Cytogenetics of interspecific hybrids between S. indicum 

L. and S. surattense Burm. F ; Indian J. Bot. 4 91-98 
langaswamy P and Kadambavanasundaram M 1974 A Cytogenetic analysis of sterility in 

interspecific hybrid, . indicum L. x S. melangena L. ; Cytologia 39 645-654 
lao B G S and Veerabhadra Rao S 1976 Some observations on the reproductive biology of 

a few spino.us solanums in relation to their crossability relationships; In Physiology of sexual 

reproduction in flowering plants (ed.) C P Malik (New Delhi : Kalyani Publishers) 
larvayya J 1936 The first generation of an interspecific hybrid cross in solanums, between 

Solanum melongena and S. xanthocarpum ; Madras Agric. J. 24 132-142 
Itebbins G L 1950 Variation and evolution in plants ; (New York : Columbia University Press) 
fatebe T 1939 Genetic and cytolagical studies OB the F x hybrid of scarlet or tomato egg plant 

(Solanum integrifolium Poir) X the egg plant (Solanum melongena L.) ; Bot. Mag. (Tokyo) 

50 457-462 
/eerabhadra Rao S 1977 Studies on the crossability relationships of some spinous solanums ; 

Unpublished Ph.D. thesis submitted to the Andhra University, Waltair. 
^eerabhadra Rao S and Rao B G S 1977a Screening of flowers in interspecific crosses of some 

spinous solatium species ; Curr. Sci. 46 123-124 
Veerabhadra Rao S and Rao B G S I977b Chromosome pairing in the Solanum surattense X 

S. melongena F heterozygote ; Curr. Sci. 46 124-125 
Veerabhadra Rao S and Rao B G S 1977c Chromosomal repatterning ia the differentiation of 

two spinous solanums ; Curr. Sci. 46 458-459 
ftitshi U 1967 Interspecific hybrids in solanum I. Solanum indicum L. and S. incanum 

L. Proc. Indian Acad. Sci. 65 111-113 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 2, April 1982, pp. 93-100. 
Printed in India. 



Groundnut rust its survival and carry-over in India 4 ' 

P SUBRAHMANYAM and D McDONALD 

Groundnut Pathology, International Crops Research Institute for the Semi-Arid 
Tropics, ICRISAT, Patancheru 502 324, India 

MS received 27 December 1980 

Abstract Groundnut rust has become an important disease in India, particularly 
in the South, probably because of extensive and continuous cultivation of the crop. 
Uredospores present on crop debris in the field, and on pods or seeds in storage 
at ambient temperatures, lost viability within 6 weeks. They retained viability for 
long periods when stored at 16 C. Neither teliospores nor any collateral or 
alternate hosts were found. Seeds heavily contaminated with viable uredospores 
and sown in sterile soil gave rise to disease-free seedlings. There should be no 
risk of spread of rust from properly treated seed samples. 

Keywords. Groundnut rust ; survival ; carry-over ; Puccinia arachidis Speg. ; 
Arachis hypogaea L. 

1. Introduction 

Rust of groundnut (Arachis hypogaea L.), caused by Puccinia arachidis Speg., was 
reported from Punjab, India, in 19619 (Chahal and Chohan 1971) and now occurs 
in most groundnut-growing Indian States (Snbrahmanyam et al 1979). The 
disease has become particularly important in South India, where groundnuts are 
grown for nrich of the year and where conditions favour development and spread 
of the pathogen. This paper deals with the survival of the rust fungus and presents 
results of investigations on possible carry-over of the disease in crop debris, on 
seeds, and on weeds. The biology of the fungus is discussed in relation to distri- 
bution of rust and groundnut cropping seasons. 

2. Materials and methods 

2.1. Survival of uredospores in crop debris 

Dried haulms of groundnut collected from rust-infected rain-fed and irrigated 
crops (cv. TMV-2) during 1976-78 were immediately exposed to weather by 
spreading them in shallow layers in the field at ICRISAT Centre farm. At 
intervals, uredospores were collected from the crop debris (dried haulms), suspended 
in sterile distilled water on glass slides, and incubated in the dark at 25 C. After 
6hr, 1,000 spores were checked for germination. 

* Submitted as Journal Article No. 125 by the International Crops Research Institute for the 
Semi-Arid Tropics (ICRISAT). 

93 



94 P Subrahmanyam and D Me Donald 

2-2. Effect of temperature on uredospore longevity 

Uredospores, freshly collectee from infected plants, were placed in glass vials and 
stored at temperatures of - 16, 6, 25 and 40 C. At intervals, they were sampled 
and tested for viability as described above. 

2-3. Presence of uredospores on pods and seeds 

Pods were collected from a crop with severe rust and separated into those with 
no shell damage and those with shells broken during threshing. Undamaged 
pods were shaken in distilled water to which Tween 80 (1 : 1000) had been 
added, and washings were centrifuged at 2,000 rev/min for 1 hr. The pellet 
obtained was examined microscopically for uredospores. t>amaged pods were 
carefully opened and seeds were removed with minimal contact with the outside 
of the shells. The seeds were washed and the washings examined as. described 
for undamaged pods. 

2-4. Longevity of uredospores on stored seed 

Seeds were dusted with freshly collected uredospores and stored in cloth bags in 
the laboratory at 25 to 30 C. Samples were removed at 5-day intervals and 
uredospores washed off the seeds and their viability tested as described above. 

2-5. Carry-over of rust on seed 

Seeds of rust-susceptible cultivar TMV-^2 were surface-sterilised by immersion for 
5 min in a 0-1% aqueous solution of mercuric chloride to which a small 
amount of Tween-80 had been added. They were then washed in repeated changes 
of sterile tap water. Isolation plant propagators (Burkard Manufacturing 
Company, England) were prepared containing steam-sterilised garden soil in 
pots which could be watered from below with sterile tap water. Into the pots 
in one unit, 200 seeds, were aseptically sown. In another unit, 200 seeds liberally 
coated with freshly collected uredospores were sown. A further 200 seeds were 
aseptically sown in a third unit, and after germination, the seedlings were dusted 
with freshly collected uredospores. Seedlings were checked for rust infection. 

2-6. Germination of uredospores on germinating seeds 

Two-day-old seedlings of the cultivar TMV-2 were carefully washed, testas 
removed, and 100 cotyledons and 50 radicles excised. These organs were surface* 
inoculated with a suspension of uredospores and placed in moist chambers for 
incubation in the dark at 25 C. Samples were removed after 24 hr, stained, 
and examined under the microscope. In another test, artificially-contaminated 
seeds were sown in sterile soil, and resulting seedlings were carefully removed 
and examined at intei-vals. 

2.7, Search for teliospores and collateral hosts 

A large number of specimens of rust-infected groundnut from different parts of 
the country were examined for the presence of teliospores. Some 2,000 entries 
from the ICRISAT groundnut germplasm collection were also examined at various 



Groundnut rust survival in India 95 

stages of development under severe rust infection. Attempts were also made to 
induce telial production by growing rust-infected plants of the susceptible TMV-2 
cultivar under the following combinations of temperature and day length in plant 
growth chambers. 

Treatment Day temperature Night temperature Day length 
(C) (C) (hr) 



1 


20 


10 


8 


2 


30 


10 


8 


3 


30 


20 


10 


4 


35 


25 


12 


5 


40 


30 


12 


6 


25 


25 


12 


7 


15 


15 


12 



Various common crop and weed plants growing in or near fields of rust-infected 
groundnuts on the ICRISAT farm and farmers* fields were examined for rust. 
Some were also subjected to inoculation with uredospores in greenhouse tests ; 
the groundnut cultivar TMV-2 was used as a susceptible check. 

3. Results and discussion 

3.1. Survival of uredospores in crop debris 

The high initial viability of uredospores decreased rapidly with exposure to weather 
(table 1). This was most marked in uredospores from irrigated crops, probably 
because of the higher temperatures experienced in May than in the November-to- 
January period following the rain -fed crop. Invariably, uredospores on exposed 
crop debris lost all viability within 30 days. Similar work in other parts of India 
also indicates that uredospores are short-lived in crop debris under field condi- 
tions (Lingaraju etal 1979 ; Mallaiah and Rao, personal communication). 

3-2. Effect of temperature on uredospore viability 

Spores remained viable for several months when stored at low temperature ( 16 C) 
while at 40 C they lost viability within 5 days (table 2). At the intermediate 
temperatures, viability decreased with time of storage and was completely lost 
within about 2 months. Mallaiah and Rao (personal communication) found that 
uredospores remained viable for up to 4 weeks when stored at temperatures 
below 30 C but lost viability within 2 weeks when stored at temperatures above 
35 C. It would thus appear that temperature is an important factor influencing 
viability and longevity of rust uredospores. 

3.3. Carryover and distribution on seed 

Garry-over and dissemination of uredospores on groundnut pods and seeds have 
been suggested. Peregrine (1971) indicated -that movement of contaminated 



96 P Subrahmanyam and D McDonald 

Table 1. Viability of uredospores after various periods of exposure to weather on 
infected crop debris. 



Percentage of uredospores viable* 


T> * J f 








jrenouL 01 exposure 








GO 


Rainy-season crops 


Post-rainy-season crops 




1976 1977 


1976-77 


1977-78 





65 90 


82 


89 


6 


36 74 


9 





14 


1 42 


1 


1 


20 


26 








22 


10 








26 











Period of test 


13-12-1976 7-11-1977 


4-5-1977 


2-5-1978 




to to 


to 


to 




7-1 -1977 2-12-1977 


30-5-1977 


28-5-1978 


RH% 0714 hr 


80-7 83-5 


60-7 


60-7 


1414 hr 


26-0 46-6 


26-9 


23-9 


Temp. C : Max. 


28-3 28-0 


37-6 


39-7 


Min. 


13-4 19-5 


24-9 


25-6 


* 1,000 spores per sample. 


Figures to nearest whole number 







Table 2. Effects 


of storage temperature on viability of uredospores. 


Storage 


Percentage* of uredospores viable after storage for 


















<C) 


Days 






5 


13 28 40 48 60 


70 78 99 


110 120 


16 88 


82 89 90 98 88 


92 93 92 


94 93 


6 84 


85 82 35 15 4 


00 


. 


25 81 


88 80 24 


000 


.* * 


40 


00000 


000 






* 1,000 spores per sample. Figures to nearest whole number. 



seed may have been involved in the spread of ru&t to Brunei . Pods from a rust- 
infected crop would be contaminated with spores during threshing and any 
damage to shells could well lead to contamination of seeds. Seeds could alsobe 
contaminated during shelling. Examination of pods from a severely rusted crop 
showed presence of uredospores on the shells. Where shells were broken, uredo* 
spores were found on the seed surfaces. 



Groundnut rust survival in India 97 

Table 3. Effects of storage at room temperature (25-30 C) on viability of uredosporcs. 



Percentage* of uredosporcs viable after storage for : 
Days 

5 10 15 20 25 30 35 40 45 50 55 
95 72 30 28 25 28 30 29 39 10 

*1,000 spores per sample. Figures to nearest whole number. 

The viability of uredospores on seed stored at room temperature for varying 
engths of time is shown in table 3. Viability decreased rapidly with storage 
:ime from an initial 95% to zero after 45 days. 

Surface-sterilised seeds of cultivar TMV-2 sown in sterile soil in isolation plant 
>ropagators gave rust-free seedlings. Seeds similarly treated, but coated with 
viable uredospores prior to sowing, also gave rise to rust-free seedlings. A ' check ' 
xeatment where the foliage of seedlings was dusted with uredospores resulted in 
severe rust disease within 25 days of sowing. This supports the argument that 
,urface contamination of seeds with uredospores is unlikely to result in rust 
nfection of seedlings. 

When excised cotyledons and radicles of germinating seedlings, were surface- 
noculated with uredospores and incubated in the dark, the spores germinated 
ind appressoria were produced, but there was no development of disease. Exami- 
lation of seedlings from seeds heavily contaminated with uredospores and sown 
n sterile soil again showed germinated uredospores with appressoria, but no rust 
leveloped. 

There would appear to be little danger of rust disease developing, from uredo- 
;pores carried on sown seed. Also, there is no authenticated report of the 
'ust fungus being internally seed-^borne. 

Although rust has spread rapidly to most parts of the world in recent years 
Hammons 1977 ; Subrahmanyam etal 1979), there are still some groundnut- 
growing areas where it is not present. Plant quarantine authorities and those 
Concerned with distribution of groundnut germplasmare understandably concerned 
vith the possible spread of the disease to these areas through contaminated seed 
amples. However, the practice of dressing seed with fungicides, the rapid loss 
>f viability of uredospores at ordinary temperatures and their inability to infect 
eeds or germinating seedlings below ground all indicate that disease spread 
hrough properly treated and handled seed samples is extremely unlikely. To 
>btain successful spread, viable uredospores would have to be carried to the 
urface of foliage of the susceptible plant under environmental conditions 
Conducive to infection. This is more likely to happen due to long-distance air 
lispersal or contamination on clothes and baggage of air travellers than on 
>roperly treated seed samples. 

J .4. Biology of the rust fungus 

Oie pathogen is known almost exclusively by its uredial stage. There are a few 
:ecords of the occurrence of the telial stage on cultivated Arachis hypogaea in 



P.(B)-2 



"38 P Subrahmanyam and D McDonald 

South- America (Spegazzini 1884 'J Hennen etal 1976) and on wild Arachis spp. 
(Guarch 1941 ; Bromfield 1971). In India, Chahal and Chohan (1971) recorded 
the occurrence of teliospores on groundnut leaves but gave no details of spore 
morphology and the disease has not recurred in Punjab. There has been no 
other authenticated report of the occurrence of teliospores of groundnut rust. 

We have examined many specimens of rust-infected groundnuts from different 
parts of India but have found only uredospores. Some 2,000 entries from the 
ICRISAT groundnut germplasm collection were examined at various stages of 
development under severe rust infection, but again only the uredial stage of the 
rust was found. 

Attempts were made to induce teliospore production by growing rust-infected 
plants under various combinations of temperature and day length but were 
unsuccessful It is not known if the fungus can produce pycnia and aecia or if 
any alternate host is involved in the life cycle, It would appear that uredospores 
are the main, if not the only, means of dissemination of the groundnut rust 
' fungus. 



Table 4. Plant species examined as possible collateral hosts of rust. 



"Leguminous crop plants 

Cajdnus cajan (L.)-Millsp. 

Canavalia gladiata DC. 

Cicer arietinum L. 

Crotalaria juncea L. 

Cyamopsis tetragonoloba (L.) Taub. 
' Gly cine max (L.) Men. 

Lablab purpureus (L.) Sweet 

Lens culinaris Medik. 

Phaseolus lunatus L. 

P. vulgaris L. 

Sesbania sp. 

Vidafaba L. 

Vigna mungp (L.) Hepper 
, y. radiata (L.) Wilcz. 

Leguminous weeds 

Aeschynomene aspera L. 

>A. Mica L. . 

Alysicarpus monilifer (L.) DC. 

.Cassia torn L. 

Ihdigofera hirsute L. 

Stylosanthes fruticosa (Retz.) Alston 

Tephrosia- hirta Ham. ... , . 

T. purpurea (L.) Pers. 

Zornia diphylla (L.) Pers. 



Non-legumes 

Acanthospermum hispidum DC. 

Achy rant lies asp era L. 

Aerva monsoniae (L.F.) Mart. 

Amaranthus viridis L. 

Anisomeles indica (L.) O. Ktze. 

Boerhaavia diffusa L. 

Cathamnthus pusillus (Murr.) G. Don 

Cor chorus aestuans L. 

Cypems compressus L. 

C. rotundus L. 

Dactyloctenium aegyptium (L.) Beauv. 

Digitaria ciliaris (Retz.) Koeler 

Eclipta alba (L.) Hassk. 

Euphorbia hirta L. 

Evolvulus alsinoides (L.) L. 

Ipomoea tridentata Roth 

Lactuca hastata DC. 

Lagascea mollis Cav. 

Leucas lavandulifolia Sm. 

Micrococca mercurialis Bth. 

Mollugo pentajphylla L. 

Ocimunt americanum L. 

Panicum sp. 

Phyllanthus niruri L. 

Portulcica oleracea L. ' 

P. quadrifida L. 

Sida sp. 

Trianthema portulacastrum L. 

Tridax procumbens L, 



Groundnut rust survival in India 



99 



There is no record of the occurrence of any collateral hosts of groundnut rust 
outside the genus Arachis, and in India wild Arachis spp. occur only in research 
centres and can hardly be involved in perpetuation of the disease. The possible 
occurrence of other hosts was considered, and various common crop and weed 
plants growing close to or within fields of rust-infected groundnuts (table 4) were 
regularly examined for the presence of rust, but no case of infection was found 
Some of these plants were also subjected to inoculation with rust urcdospores in 
greenhouse tests, but again no case of infection was recorded. 

3.5. Cropping seasons and rust survival and spread 

There is no uniform groundnut growing season in India. In some of the southern 
Sttates, particularly Andhra Pradesh, Tamil Nadu and Rarnataka, groundnuts 
are grown in some areas throughout the year (figure 1), presenting excellent 
opportunity for survival of rust. About 90% of the crop is grown in the rainy 
season, most of the rest is gjcown in the post-brainy dry season under irrigation. 
In some places a summer crop is grown. 

Rust attack is most severe on the rainy-season crops but can still be noticeable 
on dry^season crops. The disease has been seen on the summer crop in parts of 
Andhra Pradesh, but pustules developed very slowly and did not sporulate until 
the coming of the monsoon rains, when the disease developed rapidly on the 
maturing crop. 

On the rainy-season crop, the disease appears in July and August in South India, 
in September in Central India, and in October in North India (May^e et al 1977). 
In Central and North India normally only a rainy-season crop is grown, and it is 
thought that the groudnut crops in South India may act as a reservoir of rust 
disease from which spores are carried by the monsoon winds to infect the crops 
in the north. The present trend towards increased cultivation of groundnuts in 
southern India, particularly the irrigated dry-season crops, could result in more 
effective carry-over and spread of rust disease within the country. 




Figure 1. Groundnut cropping seasons in India. 



100 P Subrahmanyam and D McDonald 

References 

Bromfield K R 1971 Peanut rust A review of literature ; J. Am. Peanut Res. Educ. Assoc 

3 111-121 

Chahal D S and Chohan J S 1971 Puccinia rust on groundnut ; FAO Plant Prot. Bull. 19 90 
Guarch A M 1941 Communicaciones fitopatologicas ; Rev. Fac. Agron. Univ. Montevideo, 

23 14-16 (Abst. in Rev. Appl. Mycol. 1942 21 129-130) 

Mammons R O 1977 Groundnut rust in the United States and the Caribbean ; PANS 23 300-304 
Hennen J F, Figucredo M B, Riberio I J A and Soave J 1976 The occurrence of tcliospores 

of Puccinia arachidis (Uredinales) on Arachis hypogaea in Sao Paulo State, Brazil ; Sununa 

Phytopathol 2 44-46 
Lingaraju S, Siddaramaiah A L and Hegde R K 1979 Viability and survival of urcdosporcs 

of Puccinia arachidis Speg. in Dharwad ; Curr. Res. 8 68-69 
Mayee C D, Godbole G M and PatiJ F A 1977 Appraisal of groundnut rust in India : problems 

and approach ; PANS 23 162-165 

Peregrine W T J 1971 Groundnut rust (Puccinia arachidis) in Brunei ; PANS 19 318-319 
Spegazzini C L 1884 Fungi guaranitici I ; Anal Soc. Ci. Argentina 17 90 
Subrahmanyam P, Reddy D V R, Gibbons R W, Rao V R and Garrcn K H 1979 Current 

distribution of groundnut rust in India ; PANS 25 25-29 



Proc, Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 2, April 1982, pp. 101-106. 
Printed in Indif>. 



Correlated promotion of ray-floret growth in chrysanthemum 
by potassium chloride, gibberellic acid and sucrose 

P PARDHA SARADHI and H Y MOHAN RAM 

Department of Botany, University of Delhi, Dellii 110007, India 

MS received 7 October 1981 

Abstract. The role of 10~ 2 M, 2 X 10~ 2 M, 4 x 1Q- 2 M patassium chloride, gibberellic 
acid(10- G M; GA 3 ) and sucrose (5 x 10" 2 M) (used individually and in various combi- 
nations) in the elattgatio,n growth of excised ray-florets of Chrysanthemum mori- 
folium var. Jyothsna, was investigated. KC1 (10~ 2 M) caused 33-3% increase in 
elongation as compared to control (16-7%). With GA 3 and sucrose the percentage 
of elongation recorded was 39-8 and 28-9 respectively. Maximal growth response 
(2-8%) was recorded in KC1 (4 X 10~ 2 M) -f GA 3 (10- B M) -i-sucrose (5 X 10~ 2 M). 
When used in combination either with GA 3 or sucrose, KC1 showed an almost 
additive effect, whereas in the presence of bath it acted synergistically. It is 
inferred that the increased turgor resulting from sucrose-promoted potassium uptake 
along with GA 3 -caused tissue extensibility accounts for enhanced floret growth, 

Keywords. Chrysanthemum ; cell elongation ; flower growth ; gibberellic 

potassium chloride ; sucrose, 



1. Introduction 

Studies on the opening of flowers harvested at the immature stages of development 
(bud-cut flowers) have gained importance owing to several commercial advantages 
(Marousky 1971 ; Halevy and Mayak 1974). Sucrose and gibberellins have been 
used for promoting flower bud opening in chrysanthemums and gladioli (Marousky 
1971, 1972 ; Bravdo etal 1974 ; Rao and Mohan Ram 1979). In several bud-cut 
flowers, however, further opening is a serious problem. It is envisaged that the 
difficulties faced in causing the opening of bud-cut flowers can be overcome after 
the basic processes controlling petal growth have been understood. 

There has been a good amount of physiological work on flower initiation and 
senescence. In comparison, literature on flower growth is scanty. The investi- 
gations on cell and organ expansion have been largely confined to vegetative parts. 
These events are known to be under the control of turgor pressure, viscoelastic 
properties of the wall and cell wall synthesis. Ions such as K" 1 * are crucial in 
regulating osmotic potential of the cell sap (Haschke and Luttge 1975 ; Parrish 
and Davies 1977 ; Stuart and Jones 1977, 1978). Sugars have been shown to be 
involved in the synthesis of wall precursors, besides regulating osmotic potential 
and providing energy (Siegelman etal 1958). Gibberellins regulate the visco- 
elastic properties of cell wall (Kamisaka et al 1972 ; Adams etal 1975 ; Nakamura 

101 



102 P Pardha Saradhi and H Y Mohan Ram 

et al 1975 ; Kawamura et al 1976 ; Coartney arid Morre 1980) beside.s promoting 
the influx of solutes (Katsumi and Kazama 1978). With this information in the 
background, a study of the role of KC1, gibberellic acid and sucrose on ray-floret 
elongation in chrysanthemums was taken up. 



2. Material and methods 

Stocks of Chrysanthemum morifolium var. " Jyothsna " (Asteraceae) were procured 
from the National Botanical Research Institute, Lucknow, and were grown in the 
Botanical Garden of the Department. The plants flowered profusely in December. 
Capitula (measuring approximately 14 mm in diameter) in which the ray-floret 
would become visible the next day were used for experimentation. Ray-florets 
belonging to the outer two whorls and measuring 9 or 9-5 mm were excised from 
the capitula. For each treatment 20 ray-florets, in groups of five, were floated in 
petri plates containing 30 ml of the test solution and kept under continuous light 
(cool-white fluorescent tubes ; 1200 Lux) at 20 2 0. The test solutions con- 
sisted of distilled water (control) ; potassium chloride (KC1) at 10~ 2 M, 2 x 
10~ 2 M and 4 x 10~ 2 M ; gibberellic acid (GA 3 ) at 10~ 5 M ; sucrose (S) at 5 x 
10- 2 M ; KC1(10- 2 M) + S (5 x 10~ 2 M) ; KC1 (2 x 10~ 2 M) + S (5 x 10~ 2 M) - 
KC1 (4 x 10~ 2 M) + S (5 x 10~ 2 M) ; KC1 (10~ 2 M) + GA 3 (1Q- 5 M) ; KC1 
(2 x 10- 2 M) + GA 3 (lO^M) ; KCl (4 x 10~ 2 M) + GA 3 (10~ 5 M) ; KC1 
(10-2 M) + S(5 x 10~ 2 M) + GA 3 (10- 5 M) ; KC1 (2 x 10~ 2 M) -H S (5 x 10~ 2 M) 
+ GA 3 (10- 5 M) ; KC1 (4 x 10~ 2 M) + S (5 x 10~ 2 M) 4- GA 3 (10~ 5 M). Strepto. 
Aiycin (25 ppm) was added to all the solutions to prevent microbial infection. 
The length of the floret was measured every 24 hr to the nearest 0-1 mm. Each 
experiment lasted 10 days. 



3. Observations 

In all the treatments (including the control) the florets showed an increase in 
length with- time (table 1). Among the three concentrations of KCl used, the 
florets showed the maximum response with 10~ 2 M (33 3 %), followed by 2 x 10"" 2 M 
(22-2%) and 4 x 10" 2 M (20-0%) by day 5. 

The floret length increased by 28-9% with sucrose and by 49-8% with GA 8 , 
the -maximum length having been attained on day 6. When KCl was used in 
combination with GA 3 or sucrose, greater elongation of the florets than in the 
treatments with only KCl, sucrose or GA 3 was recorded. When KCl (all the 
three concentrations) and sucrose were present together, the elongation ranged 
from .46 -.2%. to 49-5%. In the presence of GA 3 , KCl (2 x 10~ 2 M) showed as 
high as 57 % increase in length. With KCl (1Q- 2 M) 4- GA 8 and KCl (4 x 1Q- 2 M) 
rf- GA S the corresponding values were 50-0 and 47-3% respectively. 

- The florets exhibited highest elongation when they were kept in a mixture 
containing -KC1, sucrose and GA 3 . The increase in length observed over the 
control was 82r8% when the concentration of KCl in the combination was 
4 x. iJOrfM. However, 10~ 2 M KCl and 2x 10- 2 M KCl also showed large 
increases up to 65 -6 and 72%, respectively. With the exception of KCl (10~ 2 M) 



Promotion of ray-floret growth in chrysanthemum 



I 



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104 P Pardha Saradhi and H Y Mohan Ram 

-t- GA 3 , in which the florets showed an increase in length till day 8, in the other 
combinations floret elongation continued until the termination of the experiment. 
It is interesting that the effect elicited by KC1 in combination with either sucrose 
or GA 3 was nearly additive and that in combination with both was synergistic. 



4. Discussion 

In the present work it was noted that KC1 caused greater elongation at low rather 
than high concentration. Potassium has been shown to play an important role 
in the elongation of vegetative tissues by altering the osmotic potential (Stuart 
and Jones 1977, 1978), by effecting wall loosening (Tagawa and Bonner 1957 ; 
Haschke and Luttge 1975), by acidification of the incubation medium (Thimann 
and Schneider 1938 ; Ordin etal 1956 ; Tagawa and Bonner 1957 ; Haschke and 
Luttge 1975) or by acting as a co-factor for stimulating certain enzymes (Mahler 
1961 ; Purves 1966). 

In the presence of sucrose the florets showed 12-2% higher increase in length 
than that observed in the control. Besides acting as a source of energy and in 
providing building blocks for cell wall synthesis, sucrose is probably involved in 
osmoregulation, providing the necessary force for the maintenance of turgidity 
in the elongating ray-florets of chrysanthemums (present work) as has been shown 
for other flowers (Winkenbach and Matile 1970 ; Dilley and Carpenter 1975). 
Additionally, in the present investigation, sucrose showed better response in combi- 
nation with KC1. There is evidence that the energy for K 4 ion uptake and for 
the probable production of carbon skeletons for organic anions that move with 
K+ ions is derived from sucrose (Satter et al 1976). Setter etal (1976) have 
suggested that the rhythmically controlled sucrose permeation in Samanea pulvini 
could regulate sucrose-H+ transport and thus, in order, membrane potential, salt 
flux and water flux resulting in increased turgor. 

The enhanced ray-floret elongation observed in the present study in the presence 
of GA 3 could emanate from its effect on the viscoelastic properties of the cell 
(i.e., cell wall extensibility) (Kamisaka etal 1972 ; Adams etal 1975 ; Coartney 
and Morre 1980), osmoregulation (Kazama and Katsumi 1973) or the synthesis 
of cell wall material (McComb 1966; Srivastava etal 1975) as demonstrated 
in several other systems. 

In the present work a greater elongation of the ray-florets was noted in response 
to KC1 + sucrose + GA 3 over that with KC1 + GA 3 or KC1 + sucrose. When 
used in combination either with GA 3 or sucrose, KC1 showed an almost additive 
effect, whereas in the presence of both it acted synergistically. A combined effect 
of sucrose and GA 3 has also been noted in the stimulation of elongation of 
bypocotyl segments (Purves and Hillman 1958 ; Kazama and Katsumi 1973), 
in the linear growth of staminal filaments (Murakami 1973) and in flower growth 
and opening (Rao and Mohan Ram 1979). 

GA 3 perhaps enhances ATPa.se activity. This could regulate K + ion and 
sucrose influx (Katsumi and Kazama 1978) thereby regulating osmotic potential 
and turgor pressure leading to ray-floret elongation in chrysanthemum, 



Promotion of ray-floret growth in chrysanthemum 105 

Acknowledgements 

The authors thank Dr I V Ramanuja Rao for his critical comments. Financial 
assistance provided by the Council of Scientific and Industrial Research, New 
Delhi, to one of the authors (PPS) is gratefully acknowledged. 

References 

Adams P A, Montague M J, Tepfer M, Raylc D C, Ikuma H and Kaufman P B 1975 

Effect of gibberellic acid on plasticity and elasticity of Avena stem segments ; PL PhysioL 

56 547-554 
Bravdo B, Mayak S and Gravrieli Y 1974 Sucrose and water uptake from concentrated sucrose 

solutions by gladiolus shoots and effect of these treatments on floret life ; Can. J. Sot. 52 

1271-1281 
Coartney J S and Morre D J 1980 Studies on the role af wall extensibility in the control of 

cell expansion ; Bat. Gaz. 141 56-62 
Dilley B R and Carpenter W J 1975 The role of chemical adjuvants and ethylcne synthesis on 

cut flower longevity; Acta Horticultiirae 47 117-132 
Halevy A H and Mayak S 1974 Improvement of cut flower quality, opening and longevity by 

pre-shipment treatments; Acta Horticultwae 43 335-347 

Haschke H R and Liittge U 1975 Stoichioraetric correlation of malate accumulation with auxin- 
dependent IC^-H* 1 exchange and growth in Avena coleoptile segments ; PL Physiol. 56 

696-698 
Kamisaka S, Sano H, Katsurni M and Masuda Y 1972 Effects of cyclic AMP and gibberellic 

acid on lettuce hypocotyl elongation and mechanical properties of its cell wall ; PL Cell 

Physiol. 13 167-173 
Katsumi M and Kazarna H 1978 Gibberellin control of cell elongation in cucumber hypoco-tyl 

sections : Bot. Mag. Tokyo Special Issue 1 141-158 
Kawanmra H, Kamisaka S and Masuda Y 1976 Regulation of lettuce hypocotyl elongation 

by gibberellic acid. Correlation between cell elongation, stress relaxation properties of the 

cell polysaccharide content ; PL Cell Physiol. 17 23-34 
Kazama H and Katsumi M 1973 Auxin-gibberellin relationship in their effects on hypocotyl 

elongation of light-grown cucumber seedlings : Responses of sections to auxin, gibberellin 

and sacro.se ; PL Cell Physiol. 14 449-458 
Mahler H R 1961 Interrelationships with enzymes ; in Mineral metabolism (eds.) C L Coxnar 

and F Bronner (New York : Academic Press) IB 743-879 
Marousky F J 1971 Handling and opening cut-chrysanthemum flowers from bud stage with 

8-hydroxyquinoline citrate and sucrose ; USDA Agr. Mktg. Res. Rept. p. 905 
Marousky F J 1972 Water relations, effects of floral preservatives on bud opening and keeping 

quality of cut flowers; ffortsci. 1 114-116 
McComb A J 1966 The stimulation by gibberellic acid of cell wall synthesis in the dwarf pea 

plant: Ann. Bot. 30 155-163 
Murakami Y 1973 The role of gibberellins in the growth of floral organs of Pharbitis nil; PI. 

Cell Physiol. U 91-102 
Nakamura T, Sekine S, Arai K and Takahas-hi N 1975 Effects of gibberellic acid and indole- 

3-acetic acid on stress-relaxation properties of pea hook cell wall ; PL Celt Physiol. 16 

127-138 
Ordin L, Applewhite T H and Bonner J 1956 Auxin induced water uptake by Avena coleoptile 

sections ; PL Physiol. 31 44-53 
Parrisli D J and Davies P J 1977 On the relationship between extracellular pH and the growth 

of excised pea stem segments ; PL Physiol. 59 574-573 



106 p Pardha Saradhi and H Y Mohan Ram 

Purves W K 1966 Monovalent cations and growth regulation. I. Growth responses in cucumber 

hypocotyl segments ; PL Physiol. 41 230-233 
Purves W K and Hillman W S 1958 Response of pea stem sections to indoleacetic acid, gibberellic 

acid and sucrose as affected by length and distance from apex ; Physiologia PL 11 29-35 
Rao. I V Ramanuja and Mohan Ram H Y 1979 Interaction of gibberellin and sucrose in flower 

bud opening in gladiolus ; Indian J. Exptl. BioL 17 447-448 
Satter R L, Applewhite P B and Galston A W 1976 Pfr phytochrome and sucrose requirement 

for rhythmic leaflet movement in Albizzia ; Photochem. Protobiol. 23 107-112 
Siegelman H W, Chow C T and Biale J B 1958 Respiration of developing rose petals ; PL 

Physiol. 33 403-409 
Srivastava L M, Sawhney V K and Taylor I E P 1975 Gibberellic acid induced cell elongation 

in lettuce hypocotyls ; Proc. NatL Acad. Set., USA 72 1107-1111 

Stuart D A and Janes R L 1977 The roles of extensibility and turgor in gibberellin- and dark- 
stimulated growth; PL Physiol, 59 61-68 
Stuart D A and Jones R L 197& The role of acidification in gibberellic acid and fusicoccin 

induced elongation growth of lettuce hypocotyl sections; Planta 142 135-145 
Tagawa T and Bonner J 1957 Mechanical properties of the Avena coleoptile as related to auxin 

and to ionic interactions ; PL Physiol. 32 207-212 
Thimann K V and Schneider C L 1938 Differential growth in plant tissues ; Am. J. Bot. 25 

627-641 
Winkenbach F and M.itile Pit 1970 Evidence for de novo synthesis of an invertase inhibitor 

in senescing petals of Ipomoea ; Z, Pfl. Physiol. 63 292-295 



Prac. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 2, April 1982, pp. 107-114 
Printed in India. 



Nuclear behaviour during heartwood formation in Acacia 
auriculiformis A. Cann. 



K V BHAT and J D PATEL 

Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 3S8 120, 
India 

MS received 15 September 1930 ; revised 23 March 1982 

Abstract. Nuclear behaviour is studied in Acacia auriculiformis A. Cann. with 
reference to aging in both axial and ray parenchyma cells (contiguous to vessels 
and away from the vessels). The size of the nucleus and nucleolus reduces gradually 
towards the inner sap wood and at last they disappear at the heartwood boundary. 
Nuclei show lobing, fissuring, fragmentation and contraction in this zone prior to 
their disintegration. Thus a gradual loss of vitality of parenchyma cells during 
aging is noted. The parenchyma cells contiguous to vessels seem to be more actively 
involved in formation of heartwood extractives. 

Keywords. Nuclear disintegration ; nucleolus ; nucleus ; slenderness ratio ; 
transition wood ; Acacia auriculiformis. 



I,'. Introduction 

Ths transformation of sapwood into heartwood during aging involves loss of proto- 
plasm of living cells and concomitant accumulation of the extractives. The 
parenchyma cells of the sapwood are known to play a major role in heartwood 
formation as in the majority of the species they are the only living cells in the 
sapwood (exceptionally living fibres occur in the sapwood). The physiological 
status of the living parenchyma cells during different stages of aging is still a matter 
of controversy. Evaluation of the vitality of the parenchyma cells and the rate 
of metabolic activities is very important and can be done only by adequate -methods. 
Bosshard (1966) considers that the form and dimensions of the nuclei are very 
valuable indications for tracing the activity of a cell. Alteration of nuclear con- 
figurations, in conifers, as a part of necrobiosis of storage tissue can be of great 
use in studying the heartwood formation. This can be studied in two ways : by 
determining the degree of slenderness ratio, and by calculating the nuclear surfaces 
(Bosshard 1965). Although nuclear behaviour in ray cells has been studied in 
detail, information on changes in shape and size of the nucleus in the parenchyma 
contiguous to the vessels is not available. As there is a remarkable difference in 
the nuclear, size and shape in, the cells contiguous to vessels and those away from 
the vessels, the nuclear behaviour is studied in relation to the* above situations in 
both axialand ray parenchyma cells of Acacia auriculiformis A/Cann. Thenuclear 
disintegration has also been studied. 

107 



108 K V Rhat and J D Patel 

2, Material and methods 

The wood material of A. auriculiformis A. Gann. was collected from the University 
botanical garden and was flxed in FAA (Sass 1958). The fixed material was 
divided into blocks of suitable sizs for microtomy. No intermediate block was 
discarded so that continuity was maintained from cambium to heartwood. The 
radial strips of wood were divided into outer sapwood, middle sapwood, inner sap- 
wood and sapwood-heartwood boundary or transition. Radial sections of 15 /mi 
thichness were cut from each of the blocte and stained with toluidine blue *O* 
(O'brien et al 1964), and pyron in -methyl green (Brachet 1953). Camera lucida 
drawings of 100 nuclei each from axial and ray parenchyma cells (contiguous as 
well as away from the vessels) from each zone of the wood were made on a piece 
of paper using an oil immersion object The length, width and sectional area of 
the nucleus were measured using area calculating device (ACD) (Chavan et al 
1979). The slenderness ratio of the nuclei was obtained by dividing the average 
length by average width of the nucleus. 



3. Observations 

The ray and axial parenchyma cells of sapwood contain nuclei but in fibres the 
nuclei disappear in the outermost sapwood. For brevity we avoid the terms axial 
and ray parenchyma cells in this paper, instead they are referred to as cells at many 
places except where the two types of cells are to be specifically mentioned. The 
chromaticity of the nucleus is comparatively higher in parenchyma cells contiguous 
to vessels (figures 1-3, 7-11) as compared to those away from the vessels (figures 
4-6, 12-14). However, the chromaticit} of the nuclei in general increases in the 
cells of the inner sapwood (figures 3, 6, 11, 14). At the heartwood boundary 
the nuclei show the highest chromaticity (figures 15-25). 

Usually the nuclei are uninucleolate, but occasionally bimicleolate nuclei are 
also observed (figure 1). The nucleolus is very prominent in parenchyma of the 
outer and middle sapwood, especiall} in the cells contiguous to vessels (figures 
1, 7- 10). The size and distinctness of the nucleolus are reduced towards the inner 
sapwood (figures 3, 6, 11, 14), and it becomes totally indistinct in the cells of 
heartwood boundary (figures i5-25). The nucleolus is larger in the nuclei of cells 
contiguous to vessels (figures I, 2, 7-10) than those of the cells which are not 
contiguous to vessels (figures 4, 5, 12, 13). 

The nuclei of the cells contiguous to vessels and those away from the vessels 
show a great difference in their cross-sectional area and slenderness ratio (SR) 
during various stages of transformation of sapwood into heartwood. 



3.1. Nuclear area 

The size of the nucleus as revealed by its sectional area differs from zone to 
zone. Cells contiguous to vessels possess larger ilplei (figures 1-3, 7-H) than 
the cells away from the vessel (figures 4-6, 12-14). 



Heartwood formation in A. auriculiformis 



.a. ............. a, : i|(jj|jiiBHilL 




Figures 1-14. 1-3. Nucleus in ray cells contiguous to vessels. 4-6. Nuclei in 
ray cells away from vessels. 7-11. Nuclei in axial parenchyma cells contiguous to 
vessels. 12-14. Nuclei in axial parenchyma cells away frora vessels. 1, 4, 7, 12. 
Outer sapwood. 2, 5, 8-10, 13. Middle sapwood, 3, 6, 11, 14. Inner sapwoo<t 
Inserted scale, 12 *5 microns, 



11.0 



K V Shat and J D Paid 




Figures 15-25. Nuclei at the heartwood boundary. 15-22. In ray cells. 23-25. 
In axial parenchyma cells. 15-18. Lobed nuclei. 19, 20. Fissuring of the nucleus. 
21. Condensed nuclei. 22. Nuclear fragments. 23. Lob ing of the nucleus. 24, Nuclear 
fragmentation. 25. Contracted nucleus. Inserted scale, 12 -5 microns. 



Heart-wood formation in A. auriculiformis 



111 



3. la. Nuclear area in parenchyma cells contiguous to vessels : Among the cells 
contiguous to vessels the nuclei are larger in axial parenchyma (figures 7-10) 
than those in the ray cells (figures 1, 2; table 1). There is a little and gradual 
reduction in the nuclear size of ray cells as traced from outer sapwood to the 
middle sapwood, but then the nuclei show sharp reduction in their size as traced 
towards the inner sapwood (table 1). Further reduction in nuclear size from inner 
sapwood to heartwood boundary is very little. On the other hand the nuclear 
size in axial parenchyma gets reduced slightly as traced towards the middle 
sapwood, and from middle sapwood to heartwood boundary through the inner 
sapwood the nuclear size diminishes rapidly and rather uniformly (table 1). 

3. Ib. Nuclear area in parenchyma away from vessels : The changes in the nuclear 
size are not prominent in these cells (table 1). However, as traced towards the 
heartwood boundary after a slight enlargement of the nucleus in ray cells from 
outer to middle sapwood a slight reduction in its size follows from middle to inner 
sapwood. Again at the heartwood boundary a slight increase in the nuclear size is 
observed (table 1). In the ray cells of the heartwood boundary region the nuclei 
are much lobed and slightly enlarged (figures 15-.18), On the other hand the size 
of the nucleus in axial parenchyma cells decreases from outer sapwood to middle 
sapwood. As traced from middle to inner sapwood the axial parenchyma cells 
do not show appreciable reduction in their nuclear size, but from inner sapwood 
to heartwood boundary there is appreciable reduction in nuclear size (table 1). 
Thus, reduction in the nuclear size is observed from outer sapwood to heartwood 
boundary except in ray cells away from the vessels. But, invariably there is a 
reduction in the size of the nuclei from middle to inner sapwood. 

3.2. The slenderness ratio (SR) 

The nuclear SR (length/width) differs with the increasing radial distance from the 
cambial zone. Slendcraess ratio serves as an index of the changes in nuclear shape 
occurring simultaneously with the changes in nuclear size. 



Table 1. Average sectional area (NA) in /nn 2 and slenderness ratio (SR) of 
nucleus in ray (RP) and axial parenchyma (AP) of various zones of wood. 



Zone Parenchyma contiguous to vessels 

nf 


Parenchyma not contiguous to. vessels 


wood Axial parenchyma Ray parenchyma 


Axial parenchyma Ray parenchyma 



NA 



SR 



NA 



SR 



NA 



SR 



NA 



SR 



OS 


78 


3-0 


70 


3-2 


23 


2-7 


16 


4-0 


MS 


75 


4-3 


58 


4-4 


19 


2-2 


20 


3-9 


IS 


51 


5-0 


40 


4-2 


17 


2-7 


15 


3-7 


HB 


32 


4-9 


37 


4-2 


13 


3-0 


20 


3-4 



HB, heartwood boundary ; IS, inner sapwood ; MS, middle sapwood ; OS, outer sapwood. 
Data based an measurements of 8,000 nuclei. 



112 K V Bhat and J D Fatel 

3-2a. SR of nutfei in cells contiguous to vessels : A gradual elongation accom- 
panied with a reduction in width is noted in the nuclei of ray (figures . 1,. 2) and 
axial parenchyma (figures 7-40) from outer to middle sapwood. Hence, the SR 
increases from outer to middle sapwood in ray and axial parenchyma cells (table 1). 
The SR in ray cells does not change appreciably thereafter, but in axial parenchyma 
it continues to increase up to the inner sapwood and then remains more or less 
constant (table 1). 

. 3.2b. SR in parenchyma celts away from the vessels : The SR of nuclei in ray 
parenchyma cells gets reduced gradually towards the heartwood boundary (table 1). 
Nonetheless, in the axial parenchyma the SR of the nucleus reduces abruptly as 
traced from outer to middle sapwood, and from middle sapwood. to heartwood 
boundary it increases in uniform and moderately steep manner (table 1). 

3.3. Nuclear disintegration 

In both ray and axial parenchyma cells the nuclei disintegrate at the heartwood 
boundary. The nuclei in axial parenchyma disappear slightly earlier than those 
of the ray cells. Prior to the disintegration the nuclei show some morphological 
variations. In some ray cells they appear variously lobed (figures 15-il8). Con- 
strictions appear along the length of the nucleus at one (figures 15, 16) or more 
loci (figures 17, 18). Thus, the nuclei appear enlarged and convoluted. Nuclei 
in some ray cells show longitudinal flssuring (figures 19, 20). whereas in others they 
appear dense, spherical and highly contracted (figure 21). A few cells atthe heart* 
wood boundary contain only fragments of nuclear material (figure 22). 

The nuclei in axial parenchyma rarely show lobing prior to their disintegration 
(figure 23), Fragmentation of the nucleus can be observed in some cells (figure 
24). In a few axial parenchyma cells the nuclei appear very small and highly 
wrinkled (figure 25). 



4. Discussion 

The cells with high activity and high vitality possess large nuclei and nucleoli 
(Bosshard 1966). The nuclei in sapwood near cambium have larger surface than 
in the sapwood adjacent to the heartwood (Bosshard 1965), Earlier studies on 
nuclei of storage tissue of wood have considered slenderness ratio of the nucleus 
and nuclear surfaces in cells in relation to their radial position from the cambium 
(Frey-Wyssling and Bosshard 1959; Bosshaid 1965, 1966). But, these are not 
studied in relation to their radial position from cambium, as well as in relation to 
their contiguity to the vessels. The present study clearly indicates that the nuclei 
of the cells contiguous to vessels are quite different in their size and morphology 
from those belonging to the cells away from the vessels. The axial and ray paren- 
chyma cells which are contiguous to vessels in the investigated species possess 
larger nuclei and nucleoli as compared to those which are ^way from the vessels* 
Further the chromaticity of these larger nuclei in cells contiguous to vessels is 
comparatively high. A similar situation was found in other angiosperm species 
investigate dearlier (Bhat and Patell980). It reveals that the cells in the contiguity 
of vessels are more active than other living cells of the wood. 



Heartwood formation in A. auriculifofmis -J-J3 

* From outer sapwood to the heartwood boundary through the middle and inner 
sapwood a gradual reduction in the size of the nuclei is observed, except in ray 
cells away from the vessels. The reduction in nuclear size is more prominent in the 
cells contiguous to vessels. The reduction in nuclear size is also accompanied by 
a gradual reduction in nucleolar size and loss of its distinctness. These facts 
suggest that the activity and vitality of the cells gradually dimmish during aging. 
In exceptional cases nuclear enlargement in ray cells away from the vessels at the 
heartwood boundary is not accompanied by a nucleolar enlargement. Bosshard 
(1966) considered the dimension ofnucleolusas one of the indications of cell vita- 
lity and reported increase in dimension of nucleoius in the transition zone between 
the sapwood and heartwood. Fufcazawa and Higuchi (1966) observed a gradual 
reduction in the RNA content from cambium to negligible amount at the inter- 
mediate wood. As we could not Snd either increase in nucleolar size or RNA 
content (as judged by staining reaction) at the heartwood boundary we consider 
the nucleolar enlargement at the heartwood boundary in ray cells away from the 
vessels to be merely a stage of its disorganisation and it is attributed to the intensive 
lobing of the nucleus. Enlargement of the nucleus at the heartwood boundary, 
prior to nuclear disorganization, was observed by us earlier in Ougeinia oojeinensis 
and Garuga pinnata (Bhat and Patel 1980). 

The data show that the degree of slenderness varies in different zones of the wood 
with the decreasing size of the nucleus. A more prominent increase in the slen- 
derness ratio is observed with the decreasing nuclear size in cells contiguous to 
vessels, while in cells away from the vessels the change in the SR was not very pro-* 
minent. In the ray cells away from the vessels the SR gradually decreases. In 
our earlier observations also we had noted that the SR either increases or decreases 
towards the heartwood boundary (Bhat and Patel 1980). The form of the nucleus 
does not appear to be significant for tracing the activity of the cell. It should be 
regarded together with the size (Bosshard 1966). Higuchi et al (1967) found a 
gradual elongation of the nucleus in conifers with the increasing distance from the 
cambial zone, and in angiosperms the variation in the shape of the nucleus was 
found to be more or less dependent on the species. In the present species varia- 
tion in nuclear shape appears to depend on the type of the cell and/or contiguity 
of the cell to the vessels. 

Histochemical observations in other angiosperm species have revealed that the 
extractives are formed earlier in the cells contiguous to vessels than the others 
(Bhat 1981). Thus, it appears probable from the data that the cells in contiguity 
with the vessels are more actively involved in the metabolic activities involved in 
the formation of heartwood extractives . 

Acknowledgements 

This work is supported by the University Grants Commission by a departmental 
research project. KVB acknowledges a fellowship. 

References 

Bhat K V 19S1 Studies on heartwood formation in some Angiosperm trees ; Ph.D. thesis, Sardar 
Patel University, Gujarat, India 



114 K V Mat and J D Patel 

Bhat K V and Patel J D 1980 Nuclear studies in relation to heartwood formation in Ougeinia 

oojeinensis and Garuga pinnata ; Caryologia 33 519-526 
Bosshard H H 1965 Aspects of the aging process in cambium and xylem ; Holzforschmg 19 

65-69 
Bosshard H H 1966 Notes on the biology of heartwood formation ; Int. Assn. Wood Anatomists, 

News Bull. 1 11-14 
Brachet J 1953 The use of basic dyes and ribonuclease for the cytochemical detection of ribo- 

nucleic acid ; Q. J. Micros c. Sci. 94 1-10 
Chavan R R, Kothari I L and Patel J D 1979 A simple area calculating device (ACD) for 

biological systems ; Curr. Sci. 48 792-793 

Frey-Wyssling A and Bosshard H H 1959 Cytology of the ray cells in sapwood and heart- 
wood ; Holzforschung 13 129-137 
Fukazawa K and Higuchi T 1966 Studies on the mechanism of heartwood formation IV. RNA 

content in the ray parenchyma cell ; /. Jpn. Wood Res. Soc. 12 221-226 
HigucaiT, Fukazawa Kand Shimada M 1967 Biochemical studies on the heartwood formation ; 

Res. Bull. Coll. Expt. Forests 25 167-192 
O'brien T P, Fedder N and McCully M E 1964 Polychromatic staining of plant cell wall by 

"toluidine blue 4 O' ; Protoplasma 59 367-373 
Sass J E 1958 Botanical microtechnique ; Iowa State Univ. Press, USA 



>roc. Indian Acad. Sci. (Plant 3ci.), Vol. 01, lumber 2, April 1932, pp. il5-12$. 
0) Printed in India. 



[dentity of Ficus macrocarpa Wt. ex King (== F. amplocarpa nom. nov.) 
md JF. guttata (Wt.) King A reinvestigation with anatomical 



evidence 



E GOViNDARAJALU and P MASILAMONEY 

Department of Botany, Presidency College, Madras 600005, India 

MS received 4 April 1981 

Abstract. The two interesting and endemic but hitherto taxonomically indistin- 
guishable and confused south Indian species of Ficus (F. guttata and F. macrocarpa) 
were reinvestigated both exomorphologically and anatomically. With the help of 
data thus obtained their original specific status instead of recently reduced ranks 
has been restored, better and dependable distinguishing characters have been blocked 
out and the existing confusions, inaccuracies and inconsistencies in literature have 
all been rectified. Revised descriptions, illustrations and a workable key are 
presented. Vegetative anatomy and the descriptions and illustrations of male 
flowers are given for the first time. F. amplocarpa is proposed as a new name 
for F. macrocarpa in view of its being a later homonym. It is established that 
these two taxa belong to section Neomorphe King and not to Rhizocladus EndL 
Anatomically F. amplocarpa is considered to be less specialized than F. guttata. 

Keywords. Ficus guttata \ Ficus amplocarpa; morphology; anatomy ; revised 
descriptions ; taxonomy. 



L. Introduction 



the south Indian species of Ficus, F. guttata and F. macrocarpa are two 
iteresting taxa which are rather uncommon and restricted in distribution even 
a the localities of their occurrence. They resemble each other so closely that 
heir respective identity and taxonomic status have hitherto been confused and 
aisunderstood. This unfortunate situation in the first place seems to have 
temmed from the circumstances that the establishment of these two taxa was 
originally based on just one or two complete, incomplete or badly preserved 
pecimsns eventually resulting in recording wrong observations and conclusions 
y earlier authors. Secondly the subsequent treatments of these taxa in all 
ndian Floras [King, Ann. R. Hot. Qard. Calc. 1 (1888) 166, 167 pi. 208, 209 ; 
t in Hoot/. Fl. Br, Ind. 5 (1888) 534 ; Brandis, Ind. Tr. (1921) 621 ; Fischer in 
Jamble'sFl. Pres. Madras 3 (1928)951 ; Fyson, Fl. s. Ind. Hill Stn. 1 (1932) 
41 et 2 (1932) /. 473] perpetuate what is contained in the protologue without 
dding any new points for further improvement Thirdly the illustrations as 
provided by Wight [Ic. 6 (1853) t. 1965, 1966] and King (I.e.) reflect not only few 
aaccuracies in respect of the delineation of certain characters but both their illus* 

US 
P.(B)-3 



116 E Govindarajalu and P Masilamone'y 

trations are not comparable. Lastly the differentiating key characters between 
these two taxa have not been sufficiently emphasized but instead more number 
of overlapping and common characters camouflaging their respective identity is 
repeatedly given. 

King (/.c.) has placed these two taxa under his section Neomorphe. Corner 
[Gard. Bull Sing. 18 (I960) 7, 32] on the other hand has transferred them to 
altogether a different section Rhizocladus Endl. to which F. laevis belongs as they 
happen to be root climbers and possessing leaves similar to those of F. laevis 
but differing from the latter by the cauliflorous condition only. Furthermore 
he has reduced F. macrocarpa as a variety of F. laevis [F. laevis var. macrocarpa 
(Miq.) Corner] and F. guttata as synonym of the variety. 

The aim of the present investigation in the first place is to wipe out all the existing 
wrong and inconsistent details of these two species and to restore their original 
specific status and section utilizing new and overlooked exomorphic characters 
combined with anatomical data hitherto unavailable, and secondly to provide 
revised descriptions and illustrations based on more and better samples and liquid 
preserved materials. 

The information on general and wood anatomy is meagre in general for the 
family as a whole and still less in particular for this large tropical genus Ficus 
(Solereder 18.98 ; Metcalfe and Chalfc 19-50). Furthermore no anatomical infor* 
mation is available on F. amplocarpa and F. guttata. In the present work the 
vegetative anatomy of both these species is undertaken from this standpoint and 
also to apply the anatomical data thus obtained as supplementary evidence towards 
the elucidation of the taxonomtcal problems mentioned above. 

2. Materials and methods 

For exomorphological studies both liquid preserved and herbarium materials 
cited under each species were used. Likewise both dried and materials pickled 
in FAA were used for the anatomical investigations. The dried materials were 
revived by boiling in 2 : 1 mixture of glycerol and ' Det ' which is a commercial 
reagent (Govindarajalu 1966). After washing in water the revived materials were 
stored in FAA. Epidermal peelings were prepared by treating the laminal bits 
taken from the midregions of the lamina with 5% Jeffrey's maceration reagent 
for about 6 hr at room temperature. When the appropriate stage is reached 
the abaxial and adaxial epidermal layers were separated carefully and the unwanted 
mesophyll cells gently removed with the help of a fine brush. Finally they were 
stained with safranin and mounted in glycerine. Serial microtome sections were 
prepared following the conventional methods of dehydration, clearing and 
embedding (Johanson 1940). The old in tern odes were repeatedly boiled in water 
and later softened with hydrofluoric acid of commercial strength for 4 hrs. After 
thorough washing in running water the sections at a thickness of 14 /m were 
taken with the sledge microtome and they were stained with safranin and permanent 
slides prepared following the customary methods (Johanson 1940). The early 
secondary xylem was macerated using Jeffrey's maceration reagent (10%) for 
about 3 hrs at 60 C for talcing the measurements of vessels, librifonn and septate 
fibres. 



identity of Ficus macrbc'ar/a 117 

& Observations 
3.1. Descriptions 
Ficus amplocarpa, Horn. nov. 

F. macrocarpa Wt. ex King, Aan. R. Rot. Qard. Calc. 1 (1888) 166, pi. 208 et in 
Hook. /. Fl. Br. IndL 5 (1888) 534 Brandis, Ind. Tr. (1921) 610 ; Fischer in Gamble's 
Fl. Madras 3 (1928) 951,955 ; Fyson, Fl. s. Ind. HillStn. 1 (1932) 541 et 2 (1932) 
t. 473 ; nan Bl. Cat. Gewass Buitenz. 36 (1823) et Beijdr. (1825) 459, nee L6vill< 
and Vaniot in Mem. Acad. Barcelona 6 (1907) 152 Pogonotrophe macrocarpa 
Miq. in Book Lond. J. Bot. 7 (1848) 74 ; Wight, Ic. 6 (1853) t. 1965 ; King 
in Hook /. Fl. Br. Ind. 5 (1888) 534 ; Brandis, Ind. Tr. (1921) 610 ; Fischer in 
Gamble's Fl. Pres. Madras 3 (1928) 955. Ficus vagan$v&x. macrocarpa Miq. Ann. 
Mus. Bot. Lugd. Bat. 3 (1867) 293. F. laevis var. macrocarpa (Miq.) Corner, 
Gard. Bull. Sing. 18 (1960) 7 [excl. Coveltia guttata Wt. and F. guttata (Wt.) 
King] figure 1 N, 2 A-G. 

Tall scandent tree. Twigs of first few internodes densely hairy, ftstular, rooting 
at nodes. Leaves broadly elliptic ovate, rounded at base with entire margin^ 
membranous (subcoriaceous), usually trinerved (quintinerved), densely or sparsely 
tomentose beneath, glabrous above, abruptly acuminate, 10-45 x (5-) 7-9 cm ; 
tomentum over the veins brown or rusty brown and the remainder colourless ; 
lateral veins 3 (-4) pairs, slightly raised beneath, curvipinnate, arcuate; petiole 
subterete, tomentose, ultimately becoming glabrous, 3 -0-4 -5 cm long ; stipules 
ovate lanceolate, acute (glabrous) 1 5-2 cm long. Receptacle ramiflorous, in fas- 
cicles on naked pendent and/or horizontal cable like branches, globose, purp- 
lish red with white streaks and patches, densely pubescent, later becoming glabrous 
containing all 3 kinds of flowers without hispid hairs in the interior, 3-0-<6-5cm 
across ; basal bracts absent ; peduncles up to I cm long with few small bracts at 
base. Male flowers dark brown, orange yellow (when fresh), 4-gonous, pedicellate, 
closely arranged behind ostiolar scales in 4 rows, 1-8-2 -Omm long ; pedicels 
flattened, membraneously winged, conspicuously 1 nerved, widening towards and 
hairy at base, up to 2 mm long; tepals 4-5, tannin punctate, dissimilar in size 
and shape, concave ; outer pair narrowly elliptic oblong, 2-4-2-5 x 1-4 mm ; 
inner pair elliptic ovate, 2-0-2-4 x 1-0-1-4 mm ; stamens 2, free, almost sessile, 
surrounded by a dense tuft of stiff erect, brownish hairs, up to 1-5 mm long ; 
anthers fleshy, more or less falcate or erect, mucronate, distinctly 4 lobed, 
trigonous, more or less basifixed or basally adnate, up to 1-3 mm long ; filament 
0-2 mm long. Female flowers few, intermingled with male, 2-2 mm long (excl. 
pedicel, incl. style), pedicellate, rare ; pedicel nearly 1-5 mm long, hairy at base 
and top, membraneously winged, flattened, strongly 1 nerved ; tepals 4, elliptic 
ovate, inflated, purplish red, obtuse, tannin punctate, equalling or shorter than 
ovary, concave, 1-5-1-6 x 1 mm ; ovary subglobose, up to 1-5 mm long; style 
excentric, exserted, glabrous, slightly widening towards top, 0-5- 0-6 mm long; 
stigma slightly dilated. Gall flowers abundant, 2- 5 mm long (excl. pedicel, incl. 
style), pedicellate; pedicel hairy at base, 3-0-6-5 mm long; tepals 6 as long as or 
longer than ovary, black, narrowly spathulate, attenuating towards base, acute- 
sutjacute, 2-8r-3-0 x 05 mm J ovary subglobose containing pupa, 2 mm long f 



118 E Govindarajalu and P Masitamoney 

style short, lateral, included, erect, adherent with ovary wall, black, 0-5 mm 
long, widening into discoid stigma. Acherte sub spherical. 

Specimens examined : Govindarajalu 8161, on the way to Thandikudi, Kodai- 
fcanal, Madurai Dt. ; Govindarajalu 15894, on the river banks* of Amngallar, 
Pannaikadu, Kodaikanal, Madurai Dt., alt. 1300-1400 m (PCM) ; Joseph 12312, 
Waverly estate, Anamalai, Coimbatore l>t. ; Vajravelu 37026, Kanakarai R.F., 
Nilgiris Dt. (MH). 

Distribution : Coonoor ; Lamb's Rock Road, Pulneys ; Sholas on Church 
Cliff, Kodaikanal. Seems to be an endemic to south Indian Hill stations. 
F. guttata (Wt.) King 

F. guttata (Wt.) King, Ann. R. Bot. Card, Calc. 1 (1888) pi. 209 ; King in Hook. 
/. Fl. Br. Ind. 5 (1888) 534 (sub F. guttata Kurz, sphalm.) ; Fischer in Gamble's 
Fl. Pres. Madras 3 (1928) 951 and 955 ; Brandis, Ind. Tr. (1921) 610 ; Fyson, 
Fl. s. Ind. Hill Stn. 1 (1932) 542; Covellia guttata Wt, Ic. 6 (1853) 8, /. 1966, 
figures 1 A-M. 

Tall scandent tree. Twigs densely hairy, sometimes glabrous, solid, rooting at nodes. 
Leaves coriaceous, (subcoriaceous), petiolate, broadly cordiformis, cordate or 
subcordate at base with entire margin, usually trinerved (quintinerved), densely 
tomentose throughout beneath and glabrous above, abruptly acuminate, (8-1) 
10-15 x 6-tl2cxn; lateral veins 3 (-4) pairs, raised beneath, curvipinnate, arcuate ; 
petiole subterete, tomentose ultimately becoming more or less glabrous, 2-4 (-^8) 
cm long; stipule ovate-lanceolate, acute, rusty brown tomentose, 1 -0-1 -5cm long. 
Receptacle short peduncled occurring in fascicles from the tubercles on the bran- 
ches of main stem, globose-sub globose, densely rusty tomentose later becoming 
glabrous, 2-5-3*5cm across ; interior of receptacles with hispid hairs ; basal 
bracts 3, broadly ovate. Male flowers dark brown, trigonous, tannin punctate, 
sessile, closely arranged behind ostiolar scales more or less in 2 rows, 2- x 1 5~ 1 6 
inm ; tepals brownish yellow, 2+2, similar, elliptic ovate, 2 -Ox 1-8-2 -Omni. 
Stamen 2, basally united; up to 2 mm long; anthers fleshy, obcuneate, trigonous, 
erect, mucronate, basally surrounded by a dense tuft of dark reddish brown stiff 
erect hairs, 1-5 x 0-8^0-9 mm ; filament 0-5 mm long. Female flowers few, inter- 
mixed with male, 2 7->3 - (incl. pedicel) x I 5 mm; pedicellate ; pedicel 8-1 -0 
inm long, hairy at base, flattened ; tepals 5-6 (-7), narrowly elliptic ovate or oblong, 
purplish red, subequal, obtuse, shining, longer than ovary, concave, tannin punc- 
tate, 1-5-1-6 x 0-6-0 -7 mm ; ovary obovoid,up to 1 mm long ; style excentric, 
erect, distantly hairy behind stigma, exserted, l'0->l-2 mm long ; stigma peltate, 
or discoid with crenulate margta (sometimes oblique). Gall flowers abundant, pedi, 
ceilate, 2*5mmlong(excl. pedicel, incl. style) ; pedicel hairy at base, <S*5~i7-Omm 
long, rest as in female flowers ; tepals 6, dissimilar, narrowly obovate or spathu- 
late attenuating towards base, acute-subacute , 2 5^3 -0x0- 6-0 8 mm ; ovary sessile 
globose-subglobose containing pupa, 2' 0-^2-2 mmlong; style short, glabrous, in- 
cluded, lateral, curved, adherent with ovary ending in dilated stigma, 0- 5~0 6 mm 
long. 

Specimens examined : Fyson 6448, sine loco ; Fyson, $.n., Shembaganur, Kodai- 
kanal, Madurai Dt. ; Fischer, s.n. 9 Coonoor, Nilgiris ; Govindarajalu 15720, 
Tiger shola, Kodaikanal, Madurai Dt. et 15930, Thandikudi, Kodaikanal, Madurai 
Dt. (all PCM) J Joseph 12312, Waverly estate, Anamalai, Coimbatore Dt 



Identity of Fiats macrocarpa 119 

(1333m) ; Sebastine 18343, Lockhart gap, Kottayam Dt. (1700m) et 24988, 
Perumalmalai, Kodaikanal, Madurai Dt. (1700m) ;Vajravelu 26131, Silent Valley 
R.F., Palghat Dt (950 m) et 35010, Curzon estate, Nilgiris (1925 m) et 37026, 
KonakaraiR.K, Nilgiris ; Shetty 37613, Avalanche, Nilgiris (2000m) ; Vajravelu 
39645, Kodanad, Nilgiris (1850m) all MR. 

Distribution : Districts of Coimbatore, Madurai, Nilgiris and Palghat seems 
to be restricted to south Indian Hill stations, 

3.2. Anatomy 

Ficus amplocarpa 

Lamina Abaxial surface : Cells hexagonal, variable in size and shape ;. cell walls 
thin, straight (undulate). Stomata (L. 18-22- 5 /im ; W. 9-13 -5 /mi), thin-walled, 
anomocytic, elliptic (figure 2M). Trichomes (L. 348-512 /on), unicellular (2-3 
celled), erect or reclinate, acute (figure 21), the basal cells of which containing 
colourless nodular deposits abundant throughout (figures 3 B i 4 G) ; unicel- 
lular microhairs (figure 4 B)and bicellular microhairs (L. 18-0-22-5 jton) with basal 
rosette of cells common in the interveinal areas as in F. guttata (figure 3 E) ; shortly 
stalked multicellular capitate or peltate glandular trichomes common over the 
veins (figure 2 J); erect, unbranched, pointed, multicellular hairs (L. 232-406 //m) 
abundant (Qgure 3 C). Idioblasts containing deposits of calcium carbonate of 
variable shapes and sizes usually occurring in groups of few excessively thick- 
walled cells commonly present as in F. guttata (figure 2 K, L). 

Adaxial surface : Cell walls excessively thick-walled, straight. Stomata, cal- 
cium carbonate containing idioblasts absent. Other details, see abaxial surface. 
T.S. Lamina : Width of lamina examined 0*4-0-45 mm. Cuticle thin over 
adaxial and thick with undulations over abaxial surface. Keel "U'-shapcd, adaxi- 
ally grooved in the midrib ; margin subacute sloping, down wards containing 3-4 
layers of collenchyma and one small submarginal vb. Adaxial epidermal cells 
Isodiametric with a tendency to become subdivided in certain places. Abaxial 
epidermal cells tangentially elongated, dissimilar in size, some of them containing 
granular materials in the form of nodules. Palisade 2-3 layered ; spongy meso- 
phyll conspicuously lacunose with large intercellular spaces and abundant tannin ; 
rnesophyll cells tangentially elongated, lobed, reticulately arranged. Cyatoliths 
common beneath abaxial epidermis (figure 4 B). Abaxial hypodermis 4-5 
layered, collenchyma tous. Midrib : vascular strands deeply crescentiform adaxially 
closed by strap-shaped single strand (figure 3 G) ; central ground tissue paren- 
chymatous containing few phloem bundles ; vascular strands subtended by 4-5 
discontinuous layers of gelatinous fibres (figures 2H ; 3 G) j nests of sclereids 
with rounded outline and gelatinous wall layers belonging to convolute type 
(Type 'A', Hoster and Liese, 1966) less common. Girders adaxia], incomplete, 
collenchymatous, 8-10 celled in height and 4-5 celled in width present for all 
vb's. Laminal vb's somewhat circular in outline surrounded by a single layer of 
parenchymatous bundle sheath. Tannin abundant throughout. 

T.S. Petiole (Distal) ; Outline circular with adaxial median groove (figure 
4 A). Diameter of petiole examined 2-8-3-0 mm. Epidermal cells isodiametric 
containing tannin. Hypodermis single layer of isodiametric tannin free cells. 
Qijter cortex consisting of 10-12 layers of lamellar collenchyma ; inner cortex 



120 E Govindarajalu and P Masitamoney 




Figure 1. 809 for caption page 138. 



Identity of Ficus macracarpa 



121 




Figure 2. See for caption page 128 f 



122 



Govindarajalu and P Masilamoney 




Figure 3. See for caption page 128, 



Identity of Ficus macrocarpa 

CQ 



123 




H. 



PH. 





Figure 4, See for caption page 129, 



124 E Govtndarajalu and P Masilamoney 

broad consisting of thin-^walled polygonal parenchyma cells arranged with inter* 
cellular spaces. Vb's 16-18 in number of different sizes, discrete arranged in a 
ring with a gap confronting adaxial groove (figure 4 A). Central ground tissue 
parenchymatous containing 7-8 phloem bundles in abaxial half (figure 4 A). Tri- 
chomes (L. 348-928 /no), erect, multicellular, brown (due to tannin), unbranched, 
pointed, thick-walled present ;; club shaped unicellular trichomes (L. 58-11 6 /on), 
excessively thick-walled containing tannin also present ; peltate or spherical 
multicellular, shortly stalked glandular trichomes present and all of them inter- 
mixed. Cubical crystals and irregularly shaped crystalline bodies less common 
in ground and cortical parenchyma cells. Basal and mid-regions of petiole 
differing by containing 25 vb's subtended by discrete units of gelatinous fibres 
(Type A). Basal and midregtons of petiole, see distal region of petiole. 

T.S. Node : Pentalacunar, each one of three adaxial gaps confronted by a 
single trace except two abaxial gaps containing split traces in each (Howard 1974). 

TJS. Internode (early secondary xylem) : Diameter of internode examined 
7-8 mm. Phellem superficial. Phelloderm broad. Sclereids tangentially elon- 
gated or elliptic with lumen and ramiform pits (figure 4 H), 2-3 layered forming 
a continuous ring present beneath phellem ; brachysclereids with reduced lumina 
and gelatinous wall layers occurring in several tangential units through out secon- 
dary cortex and secondary phloem (figure 4 E). Growth rings present. Librifora 
fibres thin-walled (L. 1160-1508 //m) J septate fibres not common (L. 580-812 
/on). Parenchyma paratracheal banded, 2-3 layered (figure 4 E). Pores solitary, 
circular-oval or in short radial multiples of 2-3. Vessels (L. 348- 580 /an), oblique 
porous (transverse) with alternate intervascular pittings, non-storied. Tannin 
rare in secondary cortex. Starch grains abundant in cortex, xylem parenchyma 
and rays. 

F. guttata 

Lamina -Abaxial surface ; Cells polygonal, thin^walled J cell walls straight. 
Stomata (L. 21-2-26- 5 /on ; W. 15 -9-21- 2 /*m), thick-walled, anomocytic,broadly 
elliptic (figure 3 F). Trichomes (L. 337- 5-540 0m), unicellular, erect or reclinate, 
thick-walled, pointed, mounted on dilated calcified base abundant over veins as 
in F. amplocarpa (figures 3 B, 4 C) ; trichomes surrounded by 1 concentric row of 
cells, the latter usually containing smooth (figure 4 F) or unevenly striated calca- 
reous, deposits and groups of such cells variable in number and arrangement abun- 
dantly present independent of trichomes also (figures 2 K, L). Calcareous de- 
posits usually of elongated form (smaller rounded ones) occurring in discontinuous 
rows over larger veins also present (figure 3 A) j shortly stalked multicellular 
capitate or peltate glandular trichomes common as in F. amplocarpa (figure 2 J) ; 
multicellular trichomes (L. 348-928 /^m), see F. amplocarpa j prickles (L. 22-5- 
27-0 /an), 2 celled, excessively thick-walled, pointed, present usually in crypts 
opposite to cystoliths (figure 3E), 

Adaxial surface : Cells variable in size with moderately thick walls. Trichomes 
less common. Other details, see abaxial surface. 

T.S. Lamina ; Width of lamina examined 0-4-^0 -47 mm. Adaxial epidermis 
usually tending to become 2-3 layered due to subdivisions. Midrib vasculature 
as in F. amplocarpa but adaxially confronted by (2-) 3 (-4) discrete strands and 
enclosing within variously oriented 3-5 additional strands (figure 3 H), Sclereids 



Identity of Ficus rnacrocarpa 125 

with gelatinous wall layers as in F. amplocarpa (figure 2 H) characteristically and 
abundantly present in regular radial rows subtending the strands (figure 3 H). 
Central ground tissue heterogeneous containing parenchyma, sclerenchyma and 
few groups of gelatinous fibres. Phloem bundles absent. Cubical crystals pre- 
sent in phloem parenchyma and in central ground tissue. Other details as in 
F. amplocarpa. 

T.S. Petiole (distal) : Diameter of petiole examined c. 4mm. Vb's 18-20 
in number of different sizes, discrete, arranged in a ring without adaxial gap, en* 
closing within phloem bundles (figure 4C). Trichomes (L. 290-464 ^m), 1-2 
celled, erect, thicJowalled, uribranched, pointed, containing tannin abundant J 
trichomes of other types found in F. amplocarpa absent. Basal and mid regions 
differing by way of reduction in the number of phloem bundles and for other 
details see distal. Other details as in F. amplocarpa. 

T.S. Node : See F. amplocarpa. 

T.S. Internode (early secondary xylem) : Pores usually in radial multiples of 
(2-) 3-4. Vessels (L. 174-324/zm), storied. Tyloses common. Secondary 
phloem rather broad and radially traversed by dilated rays (figure 4 D). Other 
details, see F. amplocarpa. 



4. Discussion 

The present study clearly indicates that Ficus amplocarpa and F. gut tat a are two 
distinct species and in this respect the former is characterized by usually membra* 
nous broadly elliptic ovate leaves with rounded base, fistular internodes, rami- 
florous larger receptacles on naked pendent and/or horizontal cable like branches, 
ebracteate receptacles tetragonous pedicellate male flowers, flattened membra^ 
nously winged 1 nerved pedicel widening and hairy towards base, unequal tepals 
in male flowers, almost sessile free stamens with somewhat falcate anthers, smaller 
female flowers with longer pedicels and 4 tepalled perianth, sub globose ovary, 
glabrous style slightly widening towards top with slightly dilated stigma, gall 
flowers with elliptic linear tepals and discoid stigma. With reference to the 
above mentioned characters JP. guttata differs from F. amplocarpa by coriaceous 
(subcoriaceous) leaves, broadly cordiform solid internodes, usually smaller 
receptacles developing from tubercles of stem and branches, bracteate receptacles, 
sessile trigonous male flowers with similar tepals, stamens more or less united at 
base with longer filaments., obcuneate anthers, larger female flowers with shorter 
pedicels and perianth of 5-6 (-7) tepals, obovoid ovary, style distinctly hairy 
behind stigma, peltate or infundibuliform stigma with crenulate margin, gall 
flowers with narrowly ob ovate or spathulate tepals and dilated stigma. 
Wight (1853) has given the illustrations of both F. amplocarpa ( = F. macro* 
carpd) and F. guttata under Pogonotrophe rnacrocarpa Miq. (Ic. 1. 1965) and Covellia 
gutiataVJt.Qc. t. 1966) respectively, the former without the analysis of floral 
parts and the latter accompanied by them. Furthermore the green colour of the 
receptacles, glabrous condition of young twigs, petioles and stipules, emarginate, 
subacute or attenuating base mentioned by Wight (1853) for F. macrocarpa&tQ 
all quite contrary to the present observations * 



126 E Govindarajalu and P Masilamoney 

King (Ann. Roy. Bot. Gard. Calc. 2 (1888) pi. 209) has adopted the figures of 
female flowers of F.guttata as given by Wight (I.e.) with some modifications. But 
the figures of vegetative organs and the receptacles as given by Wight on the one 
hand and those of King on the other show no agreement at all. Wight has shown 
the infundibuliform stigma to be hairy at the margin and also said that the 
receptacles do not contain any male flowers which are not correct 

The present observations which are based on more number of herbarium speci- 
mens and liquid preserved materials differ from those of King (1888) in respect of 
F. amplocarpct ( = F. macrocarpd). King reports the absence of male and gall 
flowers in the receptacles but in fact the former are present in fewer numbers just 
behind the ostiolar scales and the latter are abundant in the rest of the receptacles. 
The perianth of female flowers are said to be made up of 6 tepals instead of only 
4 tannin punctate tepals. He says fertile female flowers are sessile or pedicellate 
and the style hairy. On the contrary they are always pedicellate and style glabrous. 
The stigma is distinctly entire and dilated and not bilobed as mentioned by King 
(1888). The ovary is not stipitate as shown in pi. 208, f. 5, 7 and 9. Ficus macro- 
carpa as illustrated by King shows more number of primary lateral nerves, glabrous 
twigs, leaves and stipules, receptacles without white patches, hairy style, bilobed 
stigma and elliptic ovate tepals which prove to be otherwise according to our obser- 
vations. In the legend for figure 1 it is stated that it is part of fascicle of recep- 
tacle from the stem below the leaves but actually they are arranged in an elongated 
cable like pendent and/or horizontal branches. It is shown that fertile female 
flowers are pedicellate (figures 5 and 7) and sessile (figure 6) but the present 
observation reveals only pedicellate condition. 

King (Ann. Roy. Bot. Gard. Calc. 2 (1888) 167) has said that in F. guttatahz 
could not observe the male flowers in the only receptacle that was available to 
him and also as in the case of F. macrocarpa he could not conie across any gall 
flowers since all the flowers in the single receptacle appeared to be fertile female 
flowers. In fact, the receptacles are found to contain all the three kinds of 
flowers. According to King (I.e.) the fertile female flowers are said to be sessile 
which is contrary not only with reference to his own figure (pi. 209, f.6) but our 
observations. Eachtepalis shown to be strongly medianly 1 nerved instead of the 
nerveless condition. The ovary is obovate and the style is distinctly hairy behind 
stigma unlike those of the figures in which the former is shown as subglobose and 
the latter perfectly glabrous. Another interesting situation in this respect is that 
Wight's figure 4 (Ic. t 196161) representing the female flowers of F.guttata 
( = Covellia guttatd) is given as such with slight enlargement by King (i.e. pi. 
209, figure 6) but the stigmatic margin shown to be hairy (Wight's figure 4) has 
been shown as perfectly glabrous (King's figure 6). Furthermore King's figures 
6 and 7 (pi. 209) are labelled as female flowers under different stages of develop- 
ment but they all look so different that one has got nothing to do with the other. 

Corner (Gard. Bull. Sing. 18 (I960) 7) has reduced F. macrocarpa as a variety of 
F. laevis Blunder sect Rhizocladus Endl. and F. guttata as a synonym of this 
variety. The reasons given by him for this procedure are the existence of root 
climbing habit and the shape of the leaves which are similar to those of F. laevis. 
Nevertheless F. laevis differs from F. amplocarpa (= F. macrocarpd) by its climbing 
habit ? dentate margin and deeply cordate base of the teaf ? axillary solitary 



Identity of Ficus macro carp a 127 

greenish yellow receptacles on long peduncles subtended by 3 basal bracts with 
densely hispid hairy interior, 5 linear lanceolate tepals in male and female flowers, 
glabrous stamtnal filament, dorsifihced n on fleshy anthers with sub sagittate base and 
acute apex, terminal style equalling the length of achene, bifid stigma and elongated 
ovoid achene. Furthermore when the characters of both F. amplocarpa and 
F. guttata agree well with those of the section Neomorphe King in that the flowers 
are unisexual, male and gall flowers in one set of receptacle, fertile female flowers 
in another set of receptacle, male flowers with 2 stamens, inflated perianth with 

3 or 4 membranous tepals, fertile female flowers smaller than male or gall flowers* 
receptacles often very large in fascicles from tubercles on the stem and longer 
branches, trees rarely scandent shrubs, never epiphytal, they are allowed to retain 
their berth in the sect. Neomorphe itself. Furthermore as the specific differences 
between K amplocarpa and F. guttata are many and clear cut (see above) and each 
one of them in turn widely differs from F. laevisin respect of several characters 
the original specific status of the former two taxa is restored. 

As mentioned by King (Ann. Roy. Bot. Gard. Calc. 2 (1888) 129), Miquel 
(Ann. Mus. Lugd. Bat. 3 (1867) 278) has considered Pogonotraphe macrocarpa 
(Wt. Ic. t. 1965) as referable to F. vagans Roxb. but Roxburgh's manuscript 
drawing of the latter in Hb. CaL shows that it is clearly identical with authentic 
specimens of F. laevis BL 

Fyson (PL s. Ind. Hill. stn. i. (19-32) 541) distinguishes F. amplocarpa from 
F. guttata only by the glabrous young parts of the former and the hairy young 
parts of the latter and not on the basis of any other characters. Although the 
peduncle bearing the receptacle and leafy branch as illustrated by Fyson I.e. 2 
(1932) appears to be satisfactory the shape of the leaves has greater resemblance 
to F. guttata than to F. amplocarpa. Likewise the female flowers illustrated by 
him differ from the present observations. 

The anatomical differences between F. amplocarpa and F. guttata are so signifi- 
cant and convincing that these two taxa can be maintained undoubtedly as two 
distinct species on this ground also. F. amplocarpa differs from K guttata by the 
absence of 2-celled thick-walled prickles in the leaf, midrib with crescentiform 
vascular strand adaxially closed by single strap-shaped strand, occurrence of gela- 
tin/oils fibres in 4-5 discontinuous layers subtending the midrib vascular strands, 
central ground tissue of midrib containing phloem bundles, less common occur- 
rence of sclereids with gelatinous wall layers subtending the midrib strands, 
presence of club shaped and peltate or spherical glandular trichomes in the petioles, 
non-storied vessels, longer vessels with oblique porous perforations, absence of 
tyloses, nondevelopment of radially traversing dilated rays in the secondary 
phloem, longer libriform and septate fibres. Incidentally it is also interesting to 
observe that F. amplocarpa is found to be less specialized than F. guttata since the 
vessels of the former are longer with oblique porous perforations and non-storied, 
and septate and libriform fibres are longer than those of the latter. 

Leaves broadly elliptic, membranous, rounded at base; receptacle large 3-0- 
6' 5 cm across with white blotches, ebracteate and without hispid hairs in the 
interior ; stamens free with somewhat falcate anthers ; tepals of female flowers 

4 ; equalling or shorter than ovary ; ovary subglobose ; style glabrous, stigma 
dilated F. amplocarpa (=F. macrocarpa) 



128 E Govindarajalu and P Masilamoney 

Leaves broadly cordiform, coriaceous, cordate or subcordate at base ; 
receptacles small, 2 -5-3 -5 cm across without white blotches, bracteate with hispid 
hairs in the interior ; stamens basally united with obcuneate anthers ; tepals of 
female flowers 5-6-(-7) ; longer than ovary ; ovary obovoid ; style hairy behind 
stigma ; stigma usually peltate or discoid F. guttata. 



Acknowledgement 

We are highly thankful to Dr N C Nair, Botanical Survey of India, Southern 
Circle, Coimbatore, for loaning the specimens cited here as M H. 



References 

Goviadarajalu E 1966 The systematic anatomy of South Indian Cyperaceae : BulbostylisKunth; 

jr. Linn. Soc. (Bat.) 59 289-304 
Howard R A 1974 The stem node-leaf continuum of the Dicotyledoneae ; /. Arnold. Arb. 55 

125-173 
Hoster H R and Liese W 1966 Ober das Vorkommen von Reaktionsgewebc in Wurzeln und 

Asten der Dikotyledonen ; Holforsch. 20 $0-90 
Johanson D A 1940 Plant microtechnique (New York : McGraw-Hill) 
Metcalfe C R and Chalk L 1950 Anatomy of the Dicotyledons 2 vols. (Oxford) 
Solereder H 1908 Systematic anatomy of the dicotyledons 2 vols. (Oxford) 



Figures 1 A-N. Ficus guttata. A. abaxial surface of leaf x 1/5 ; B. male flower 
bud x 8 ; C. tepal of male flower x 14 ; D. stamen x 16; E. and F. different forms 
of stigma (diagrammatic) ; G. female flower with tepals removed showing one form 
of stigma x 12 ;H. gall flower x 7 ; L ovary of gall flower x 8 ; J, tepal of female 
flower x 16 ; K. female flower (entire) with another form of stigma x 11; L. and M, 
tepals of gall flower (two types) x 13 ; N. F. amplocarpa abaxial surface of 
leaf x i. 

Figures 2 A~M. A-J. F. amplocarpa. A. male flower bud X $ J B. outer tepal 
of male flower x 11 ; C. inner tepal of male flower x 13 ; D. staminal pair of a 
single male flower x 16 ; E. gall flower x 5 ; F. female flower X 10 ; G. another 
type of stamen X 14 ; H. cortical gelatinous fibres (type A) x 170 ; I. unicellular 
hair with a basal rosette of cells x 107 ; J. peltate glandular hair X 170 ; K. and L. 
F. guttata leaf epidermal cells containing different forms of calcareous deposits 
X 170 ; M. F. amplocarpa surface view of stoma x 450. 

Figures 3 A~H. A. Ficus guttata portion of lateral vein showing different forms of 
calcareous deposits x 170 ; B. and C. F. amplocarpa : B. part of leaf epidermal hairs 
showing calcareous deposit containing basal cell x 170 ; C. rnulticellular hair aver 
veins x 100 ; D-F. F. guttata : D.cystolith containing idioblast x 170 ; E. 2-celled 
microhair with cystolith containing basal idioblast x450; F. surface view of stoma 
x 450 ; G. F. amplocarpa transection of midrib x 22 ; H. F. guttata transectioa of 
midrib X 22. 



Identity of Ficus macro carpa 129 

Figures 4 A-H. A. and B. Ficus amplocarpa : A. trans ectioni of petiole at distal end 
(semi diagrammatic) ; B. cystolith opposed by 1 -celled microhair x 170 ; C. and D. 
jp. guttata: C. transection of petiole at distal end (semidiagrammatic); D. transection 
of oldinternode (semi diagrammatic) ; E. F. amplocarpa transection of old internode 
(semiciiagrammatic) ; F. F. guttata leaf epidermal hair base encircled by calcareous 
deposit containing cells x 170; G. and H. F. amplocarpa : G. surface view of cystolith 
containing hair base x 170 ; H. brachysclereid from cortex x 65. 

Abbreviations:. C. cystolith CO. cortex; D. calcareous deposit; E. epidermis ; 
G. gelatinous fibres G.C guard cell ; H. hair; L. lenticel; P. xylem parenchyma; 
PD. phelloderrn; PH. phloem ; PM. phellem ; R. rays ; S. sclereids ; T- tannin 
idioblast ; VB. vascular bundle ; XY. xyiem ; c. circa ; D. diameter ; Ht. height ; 
L. length ; vb. vascular bundle (pi. vb's) ; W. width. 



Proc. Indian Acad. Slci. (Plant Sci.), Vol. 91, Number 2, April 1982, pp. 131-137. 
Printed in India. 



The genus Jackiella in South India 

RAM UDAR and ADARSH KUMAR 

Department of Botany, University of Lucknow, Lucknow 226007, India 

MS reoeived 8 April 1981 

Abstract. Jackiella ceylanica Schiffn. ex St. and /. javanica var, cordifolia SchifFn. 
are being reported for the first time from India. The taxonornic details of the 
two taxa together with a discussion of the tenabiiity of two varieties of J. javanica 
SchifFn. namely var. cordifolia and var. cavifolia have been given. 

Keywords. Bryophyta; hepaticae; Genus Jackiella; South India; taxonomic details* 



1. Introduction 

In a recent contribution (Udar and Kumar 1981) the range of distribution and 
diagnostic characteristics of the marsupial genus Jackiella together with the taxo-* 
nomic details of J. javanica Schiffn. from eastern India had been given. However, 
during a survey of south Indian liverworts, which have not yet received proper 
attention, plants resembling J. javanica in all characters, except in habit of plant, 
siz3 and shape of leaf, degree of development of trigones in cells of leaf and stem 
and in structure of capsule wall, were discovered. Among all these characters, 
the development of trigones seems to be ecologically influenced but the habit of 
the plant, the shape of the leaves and the structure of capsule wall show more or 
less stabilised features which merit recognition of varietal ranks for the east Indian 
and south Indian plants. Schiffner (1900) had already recognised two varieties of 
Jackiella javanica Schiffn, primarily based on vegetative characters viz., J. javanica 
var. cavifolia Schiffn. from Java, Sumatra and /. javanica var. cordifolia Schiffn. 
from Java (see also Bonnet 1966). The former variety is stated to be characterised 
by small and creeping plants with leaves broader than long and the latter by more 
elongated, uprising to almost erect plants with leaves longer than broad. The 
plants of east Indian territory could be referred to as J". javanica var. cavifolia and 
those from south Indian territory as J. javanica vat. cordifolia. Our study based 
on east Indian (from Sikfcim and Shillong) and south Indian (from Wellington 
and Dodabetta) plants reveals that sporophytic differences are also present 
between these two varieties hitherto not recognised. 

A survey of plants from Kodaikanal (Palni Hills) iu the Indian peninsular region, 
nearer to Ceylon, revealed plants which resemble 3. ceylanica Schiffn. ex St. a 
taxon which has so far been considered endemic to Ceylon. The discovery of this 
species from India constitutes a transoceanic disjunct distribution for this taxon 

131 



132 Ram Udar and Adarsh Kumar 

In the present state of our knowledge the genus Jackiellais represented in India 
by /. ceylanica, J. javanica var. cavifolia and /. javanica var. cordifolia. The 
taxonomic details of /, ceylanica and /. javanica var. cordifolia, occurring in south 
India, are being described in the present paper. The remainin g taxon from eastern 
India has already been reported elsewhere (Udar and Kumar 1981). 



2. Materials and methods 

The plants of J. javanica V&T. cordifolia were collected from Wellington and Doda- 
betta and /. ceylanica from Kodaikanal. The taxonomic details of the former 
taxon are based on plants collected from Wellington except those relating to the 
capsule wall which are drawn from the plants of Dodabetta. The plants were 
stretched in water and placed in incubator for 24 hr at 40 C. The slides were 
prepared in 70% glycerine. 



3. Key to the Indian species of Jackie lla 

1. Plants erect, deep brown. Leaves with curved margin. Male inflorescence 
with 7-15 (or more) pairs of male bracts ; male bracts oblong with flat dorsal 
lobe /. ceylanica 

1. Plants prostrate^suberect, light green. Margin of leaves not curved. Male 
inflorescence with 3-10 pairs of male bracts; male bracts globose- subglobose 
with saccate dorsal lobe. . /< javanica 2 

2. Plants prostrate. Leaves broader than long, concave, with decurrent antical 
margin. Inner layer of capsule wall in surface view with incomplete 
thickening ban da /. javanica var. cavifolia 

2. Plants suberect. Leaves longer than broad, without decurrent an tical margin. 
Inner layer of capsule wall usually with complete thickening bands 
/. javanica var. cordifolia 



4. Observation 

4.1. Jackiella ceylanica Schiffn. ex St. Species Hepaticarum 3 :272, (1908). 
Figures 1-48. 

Plants dioecious, dark brown, up to 15 mm long, rigid, erect, in dense mat. 
Branches ventraHntercalary, large, ascending upward, brownish green to deep 
brown. Stem up to 8(-10) cells across, 140-210 fan in diam., cortical cells isodia- 
metric or variable deep brown, thick-walled, 18-33 x 18-25 jam ; medullary 
cells usually isodiametric, yellowish brown, thin to thick-walled, 12-03 x 18-25 
//m. Leaves succubous* entire, ovate to oblong, usually broader than long, or 
occasionally longer than broad, 0-65-0*97 (-1*2) x 0-8-1 -Omm, deep brown, 
sub-opposite, insertion oblique, apex obtuse, base broad J antical margin slightly 
decurrent, curved ; postical margin arched, sometimes curved, marginal cells 
longer than broad, broader than long or as long as broad, 15-30 x 15-30 //m J 
middle and basal cells longer than broad, occasionally broader than long, (18- 



The genus Jackiella in South India 



133 




Figures 1-18. Jackiella ceylanlca 1. Female plant with marsupium. 2-4. T. S. 
of axes (2, 3 axes of vegetative plant ; 4. axis of male inflorescence). 5-7. Leaves: 
8. Marginal cells of leaf. 9. Middle cells of leaf. 10. Underleaf . 11. Portion 
of male plant with male inflorescence. 12. Male inflorescence. 13-16. Male 
bracts. 17. Young marsupium. 18. Female bract. 



21-44 (-51) x 18-44 /mi ; cell walls thick, sometimes thin in younger leaves, tri* 
gones prominent and bulging. Gemmae not seen. Underleaves highly reduced, 
only near apex of stem, (1-) 2-3 cells long, 2-3 cells broad, connate at base with the 
leaf of one side, with 1-celled marginal teeth ; cells thin-walled, trigones feebly 
developed. Rhizoids scarcely present on axis, usually restricted at base of under- 
leaves, with swollen tips harbouring mycorrhiza. Male plant with short, spicate, 
ventral branches, androecia terminal or intercalary consisting of 7-J5 or more pairs 
of male bracts ; male inflorescence usually with 5-celled thick axis having indis- 
tinct cortical and medullary regions ; male bracts bilobed, oblong, 180-260 x 
105-155 im : ventral lobe oblong, comparatively larger, saccate : dorsal lobe 
oblong, smaller, flat, enclosing single anthericjium ; male bractegles yestrjjctecl 



134 Ram Udar and Adarsh Kumar 

only at base of male inflorescence. Female plant with 2 or more short, ventral, 
female inflorescence having 1-2 pairs of female bracts at mouth of marsupium ; 
female bracts saccate, apex obtuse, sometimes subacute to acute j perianth absent ; 
marsupium deep brown, cylindrical, 0' 5-1 -Omm long, with numerous rhizoids 
on its surface ; archegonia up to 3->5 at mouth of the marsupium with short neck. 

4- la. Specimens examined : LWU 79/66, 95/66 ; Coll. R. Udar and S. C. Sri-, 
vastava ; Loc. Kodaikanal (Palni Hills, South India), alt ca 1200m ; t>ats 
January 6, 1966 ; Det. R, Udar and A. Kumar. 

4-lb, Ecology : The plants grow on rocky soil in association with Pogortatum 
sp. on moist and exposed surface. 

4. Ic . Discussion : J. ceylanica Schiffn . ex St. was originally described by Stephani 
(1906- 1909) from Ceylon, who provided a short Latin diagnosis only along with 
some illustrations in his unpublished Icones No. 2024. Later Abeywickrama 
(1959) reported it from the same island with a meagre description and wrong 
illustration (see fig, I6c, p. 48). The adequate taxonomic details of this plant do 
not seem to have been published so far. The characters of this species from Kodai- 
fcanal reveal a great deal of diversity and plasticity in vegetative and reproductive 
characters which tend to approach J.javanica. However, /. ceylanica differs from 
the latter in larger, ascending to erect and deep brown plants, and with curved 
margins in leaves. The androecia are very characteristic in being terminal or 
intercalary and consist of 7-il,5 or more pairs of oblong male bracts with flat 
dorsal lobe. In /. javanica, however, the plants are smaller, prostrate and light 
green with more or less flat leaves. The androecia are terminal with 3~ 10 pairs 
of male bracts which are globose to subglobose with saccate dorsal lobe. 

Considerable variation occurs in the leaves in /. ceylanica which may be ovate 
to oblong, broader than long (figures 6, 7) or longer than broad (figure 5) but 
characteristically with curved margin. The uaderleaves are highly reduced 
(figure 10) and apparently confined near the apex of the young shoot. The rhizoidal 
tipsi are swollen and harbour mycorrhiza. They may perform similar function of 
nitrogen fixation as reported for the east Indian plants by Udar and Kumar (1981). 

4.2. Jackiella javanica var. cordifolia Schiffn. 

Denkscher. Mat. Nat. Cl. Kais. Acad. Wiss. Wien 70 :217 ( : 115), (1900). 
Figures 19-38, Plants dioecious, light green, occasionally yellowish brown, up to 
10mm long, delicate, prostrate to suberect, in dense mat. Branches ventral inter- 
calary, small, ascending, light green. Stem prostrate or with ascending apex, 
up to 6-7 cells across, 100-160 /*m in diam., cortical cells isodiametric, light to deep 
brown, thick-walled, 7-28 x 7-28 //m ; medullary cells more or less isodiametric, 
light brown to yellowish brown, thick to thin-walled, trigonous, 10-25 x 10-23 /*m, 
Leaves succubous, entire, ovate or cordate or subquadrate to slightly oblong, usually 
longer than broad or as long as broad or occasionally broader than long, light 
.green to light brown, 0-45-0 -62 x 0' 44-0 -54 mm at middle; at plant apex 
usually broader than long, light green, (0-5-) 0-64-0-75 x (0-6-) 0-75-0 -9 
(-1-0) mm, sub-opposite, insertion oblique, apex obtuse, occasionally slightly retuse, 
entire (to wavy), not ctecurrent ; marginal cells usually as long as broad, 



The genus Jackiella in South India 



135 




Figures 19-38. Jackiella Javanica var. cordifolia. 19. Female plant with marsupium. 
20, 21. Vegetative plants. 22. T.S. of axis. 23-25. Leaves. 26. Marginal 
cells of leaf showing attached gemmae. 27. Gemmae. 28. Marginal cells of leaf. 
29. Middle cells of leaf. 30, 31. Underleaves. 32. Marsupium with young 
sporophyte. 33, 34. Female bracts. 35. Outer layer of capsule wall. 36. Inner 
layer of capsule wall. 37. Spores. 38. Elater. 



longer than broad or broader than long, 18-28 (-38) x 18-28 (-33) //m ; middle 
and basal cells isodiametric, more or less longer than broad, 20-44 x 15-30 /on ; 
cell walls thin, trigones prominent, bulging ; base broader in younger leaves. 
Gemmae occasional, at apical margin of young leaf, hyaline to yellowish brown, 
1 -celled, spherical, 10-15 jum in diam., 2-celled, 18-35 x 12-15 //m. Underleaves 
reduced, up to 4-5 cells long, 4-5 cells broad, bifid, lobes uniseriate, margin 
dentate, connate at base with leaves of one side, cell walls thin, trigones feebly 
developed. Rhizoids numerous on prostrate axis, particularly near the base of 
Underleaves, with swollen tips harbouring mycorrlvza. Male plants absent, 



136 Ram Udar and Adarsh Kumar 

Female plant with 1-2 short, ventral, female inflorescence having 1-2 pairs of 
female bracts at mouth of marsupium ; female bracts saccate, apex obtuse to 
subacute with entire to wavy margin ; perianth absent ; marsupium light to deep 
brown, cylindrical to sickel-shaped, narrowed at base, 1-2 mm long, 0-4-0 -55 mm 
wide, with some rhizoids at surface, archegonia up to 6 at mouth of marsupium 
with short neck. Sporophyte young, enclosed within marsupium. Seta small. 
Capsule cylindrical, deep reddish brown, 540-760 /mi long, 2 15-325 /on wide, 
with obtuse apex on 1-celled thick capsular disc, dehiscence in two valves each 
with a small cleft at apex; wall bis-tratose ; cells of outer layer quadrate to 
subquadrate to elongated, 23-44 x 12-24 /mi with nodular to confluent thickenings 
on radial walls, occasionally also on transverse walls ; cells of inner layer sub- 
quadrate to elongated, 25-50 (-60) x 20-25 jum with complete, sometimes in- 
complete thickening bands on inner tangential wall, sometimes semiannular bands 
bifurcate on inner tangential wall. Spores spherical, light yellowish-brown, 
6-9 /mi in diam., exine with small papillae. Elaters 75-210 jum long, 11-1 6 /on 
broad at middle, reddish brown, bispiral. 

4-2a. Specimens examined: LWU 375/71 ; Coll. R. Udar and party ; Loc. 
Wellington (Nilgiri Hills, South India), alt. ca 1350m ; Date December 31, 1971. 
LWU 184/72 ; Coll. R. Udar and party ; Loc. Dodabetta (Nilgiri Hills, South 
India), alt. ca 2670m ; Date January 5, 1972 ; Det. R. Udar and A. Kumar. 

4-2b. Ecology : The plants grow on laterite soil on rock surface in dense mats 
at moist places in association with Jungermannia sp. and Pogonatum sp. 

42c. Discussion : J. javanica var. cordifolia Schiffn. was instituted by SchifFner 
(1900) from Java and is being described from the bryoflora of Wellington and 
Dodabetta (South India) for the first time from India. The plants differ from 
/. javanica var. cavifolia in habit and in shape of leaves (see Schiffner 1900). How- 
ever, the capsule wall structrae a sporophytic character, earlier not known, also 
provides additional differentiating features for the two varieties. The former 
variety has prostrate plants with ascending apex (figure 21) or ascending branches 
(figure 20) which are prostrate in the latter. The leaves in var. cordifolia, similar 
to J. ceylanica, show plasticity in size and shape as they are longer than broad, 
flat, hardly decurrent and comparatively smaller at middle part of the axis whereas 
in var. cavifolia these are broader than long, concave and antically decurrent. In 
addition to these vegetative features, the development of thickening bands on inner 
tangential wall of the inner layer of capsule wall is usually complete in var. 
cordifolia but normally incomplete in var. cavifolia. 

The leaves show a great deal of variation in their size and shape. These are 
longer than broad and cordate to ovate or sometimes subquadrate to slightly oblong 
at middle and basal part of the axis but broader than long towards apex. The 
margin may be entire or wavy with obtuse to retuse apex (figure 24) which is due to 
formation of gemmae. The gemmae are 1-2 celled (figure 27) and formed exo- 
genously from the marginal cells of the leaves (figure 26). The rhizoids are mostly 
restricted on prostrate axis and also on the surface of marsupium (figure 19) with 
swollen tips harbouring mycorrhiza. The marsupium is either cylindrical or sickel- 



The. genus Jackiella in South India 137 

shaped and the female bracts are with truncate to obtuse apex and entire 
margin (figure 32) or with acute to subacute apex and wavy to dentate margin 
(figures 33, 34). 



Acknowledgements 

The authors wish to acknowledge their gratitude to the late Dr Herman Persson 
for Stephani's unpublished Icones and to the Department of Science and Techno- 
logy (SERC), Government of India, for financial support. 



References 

Abeywickrama B A 1959 The genera of the liverworts of Ceylon ; Ceylon /. Sci. {Bio. Sc.) 

2 33-81 

Banner C E B 1966 Index Hepatlcarum VI (Germany : Verlag Von. J. Cramer) 4S1-739 
Schiffner V 1900 Die Hepaticae der flora von Buitenzorg (Leiden : E J Brill) 1-220 
Stephana F 1906-1909 Species Hepaticarum III (Geneve : Georg and C* Libraires-Editeurs) 

1-693 
Udar R and Kumar A 1981 Jackiella javanica SchifTn. a rare and interesting taxon from India ; 

/. Indian Bot. Soc. 60 105-111 



Proc. Indian Acad. Scj. (PJant Sc/.), Vol. 91, Number 2, April 19&2, pp. 139-143 
Printed in India. 



Geocalyx Nees-a rare marsupial genus from India 



RAM UDAR, S C SRIVASTAVA and BHlRENt)RA KUMAR 
Department of Botany, University of Lucknow, Lucknow 226 007, India 

VCS received 11 Miy 1981; revised 23 April 1982 

Abstract Taxonamic details of Geocalyx graveolens Nees recently discovered from 
tlvj Valley of Flty.vers in western Himalayas (altitude ca 4670 meters) have been 
provided. Tiie discovery of this genus in the above area not oaly extends its range of 
distribution in the Himalayas but also constitutes a new record of this taxon In Indian 
bryoflora. The plants are monoecious and characterized "by undulating stem, bifid 
leaves which are flat or ascending, and bifid underleaves. The androecial and 
g/aoccial branches are ventral and axillary. The marsupium arises initially as tuber- 
like structure (often 2-S in number on the ventral surface of a plant) and, at matu- 
rity becomes cylindrical with numerous rhizoids studded on its surface. 

Key wards. Bryophyta; Hepaticae ; Jungermannialcs ; Geocalycaceae ; Geocdyx\ 
now record for India, 



1. Introduction 

Geocalyx Nees is a small genus represented by only seven species, viz., (?. cakdo* 
nicus St. from New Caledonia, G. cantor tuplicatus Mont, et Nees from San 
Domingo, G. graveolens (Schrad.) Nees from Europe, Siberia, Japan and North 
America, G. orientals Besch.ef Spr., G. borbonicus St. (c.f. orientals) from Re- 
union, G. novazelandiae Efcrz. from New Zealand and G. yakusimensis Hatt. from 
Japan (Stephaui 1906-1909 ; Macvicar 1926 ; Herzog 1935 ; Hattori 1948 ; 
Hodgson 1958;Bonner 1965). 

This genus is remarkable in having the young sporophyte deeply sunk in a fleshy, 
cylindrical sac-like, pendulous, marsupium arising from the postical surface of 
the stem. Although several marsupial taxa are known from Indian sub-cor.ti- 
nentbutthe details of the marsupium are available only in Jackiella javanica 
Schiffn. and /. ceylanica Schiffn.. luxuriantly distributed in eastern Himalayas and 
south India (Udarand Kumar 1981, 1982). The present paper provides details of 
Geocalyx graveolens, another marsupial taxon , recently collected from the Valley 
of Flowers. The discovery of this genus in the above area not only extends its 
rangfc of distribution in the Himalayas but also constitutes a new record of this 
taxon in Indian flora. Evans (1939) treated this genus under the family Harpan* 
thaceae (see also Mtiller 1951-1958 ; Hodgson 1958). Schuster (1972), on the 
other hand, erected a new suborder Geocalycinae to accommodate Geocalycaceae 
to which this plant belongs, along with other families. 

139 

P.(B)-5 



140 Ram Udar, $ C Srivastava and Dhirendra Kumar 

2. Taxonomic description 

Geocalyx graveolens (Schrad.) Nees, Eur. Lab. II. p. 397 1836 (figures 1-25) 

Plants yellowish -green to green, sparingly branched, creeping with distinct undula-* 
tions. Stem l-l-5(-i2)cm long, 9-11 cells across, cortical and medullary cells 
alike. Leaves succubous, sub-opposite or alternate, obliquely inserted, flat or 
ascending, lamina up to 24 cells broad, bifid, occasionally tri-tetrafid, sinus broad, 
extending 1/4 to 1/3 of leaf length, lobes ovate, ICM4 cells long, 9-14 cells broad* 
682^770 x 561-605 jum, apex acute to subacute (1-celled or 2-celled long), antical 
margin decurrent, postical margin straight, leaf cells polygonal with inconspicuous 
trigones, 15-23 x 10-18 /on at the margins, 19-29 (39) x 21-33 (45)^m towards 
the middle and the basal region. Underleaves bifid, with narrow sinus extending 
to 1/2 or more of length, lobes 5- 10 cells long and 5-7 cells broad, 275-407 
(605) x 198-275 /mi, with smooth margin, apex acuminate, usually uniseriate, 
cells polygonal, without conspicuous trigones. Rhizoids numerous, usually in 
fascicles at the bases of the underleaves and also scattered on the stem ventrally. 
Monoecious. Antheridial shoot ventral, in the axil of underleaf, 495-715/un long, 
spiiate occasionally showing proliferation; antheridial bracts in 4-61 pairs, imbri- 
cate, shortly bilobed, saccate, ventral lobe withlaciniate margin, incurved ; brae- 
teoles bifid, one per pair of brae ts, usually 1/2 bifid with 3-celled uniseriate acuminate 
apices ; antheridium single in the axil of each bract, body sub-globose, 56-67 
(141) x 64-67 (l47)/mi with irregularly arranged jacket cells, stalk biseriate, 
21-33 /jm long. Archegonial branches ventral, in the axil of underleaves, arche- 
gonia 3-4 at the apex surrounded by small perichaetial leaves. Marsupium ini- 
tially small, tuber lifce, later becoming fleshy and cylindrical, 2-2- 5 mm long, with 
narrow base, studded with numerous rhizoids. Sporophyte with an anchor-shaped 
multicellular foot buried at the base of the marsupium ; seta cylindrical, 7-8 cells 
across, up to 2cni long, hyaline ; capsule cylindrical, deep brown, dehiscing in 
four equal valves extending up to the base of the capsule, capsule wall bistratose, 
outer layer of cells quadrate to elongated, with 2-phase development, thickenings 
nodulose on the radial walls, extending slightly on the tangential walls, marginal 
cells of each valve lacking any such thickenings ; inner layer of quadrate to elon- 
gated cells with complete-incomplete bands on the inner tangential walls usually 
connecting the radial bands at both ends. Spores yellowish to reddish-brown, 
globose to sub-globose 8-12 #m in diameter, exine minutely papillose. Elaters 
reddish-brown, 9 6- 192 /on long, 7-8 ^m broad, bispiral to occasionally trispiral 
with blunt to tapering ends. 

2.1. Distribution 

Europe, America, Russia, Japan and India. 

2 2. Specimens examined : 

LWU 4279, 4390 Coll. S. C. Srivsstava, Dinesh Kumar and D. K. Singh : Loc : 
Ou way to Hemkund from Ghangaria (Valley of Flowers), alt. ca 4670 meters 
Date 22 May 1980. E>et. R. Udar, S. C. Srivastava and D. Kumar. 



Geocalyx. a rare marsupial genus 



141 




Figures 1-25. Geocalyx, graveolens. 1. Plant showing a male branch, a mature 
marsupimn (with exserted sporophyte) and several tuberous young marsupia. 
2. A male branch showing proliferation. 3. T.S. stem. 4, 5. Bifid leaves. 
,6, 7. Apices of the le?f lobes. 8. Leaf cells from middle and base. 9-12. 
Underleaves. 13. Male bract. 14. Antheridium. 15. Male bracteole. 16. L.S. 
marsupium with sporophyte. 17. T.S. seta. 18. Base of capsule. 19. T.S. 
capsule wall. 20. Cells of the outer layer of capsule wall. 21, 22. Cells of the 
inner layer of capsule wall. 23, Spore. 24, 25. Elaters, 



142 Ram Udar, S C Srivastava and Dhinndra Kumar 

3. Remarks 

This taxon grows in the alpine or subalpine zone of the western Himalayas (ca 
46?a m) with low temperature and long seasonal periods of snow cover. It exhi" 
bits a variety of habitat preferences as well as liverwort associates because of consi" 
' derable variation in the microclimate where these plants grow. Growth of the 
plants is favourable under diffused sunlight on soil covered rocks, on decaying 
forest litter or at the base of small bushy trees at different locations along with 
Lophocolea minor Nees, Lopkozia incisa (Schrad.) Dam., Haplomitrium hookeri 
(Sm.) Nees, Blepharostoma trichophyllum (L.) l>um., Jungermannia (Plectocolea) 
limbatifolia Amak. and species of CalypogeiaR&ddi, Plagiochila Dum. and Jame* 
sonlella (Spruce) Schiffn. A more or less similar habitat preference as well as the 
liverwort associates at the generic level have been reported by Schuster (1953) in 
American population. Haplomitrium hookeri has. been found for the first time 
growing in association with Geocalyx graveolens. 

The stem, bearing a number of marsupia on the ventral surface, is highly 
undulated and internally un differentiated (figures 1,2). Succubously arranged 
leaves are obliquely inserted and slightly raised above the substratum forming a 
continuous, gutter (channel) over the dorsal surface rather characteristic of this 
tax: on. Both leaves and underleaves are typically bifid and the margin is usually 
entire except in few underleaves where a tendency of developing additional lobe 
on one or both sides is present. 

The sexual branches are usually short and arise from the postical surface In the 
axil of underleaves (figure 1) as also in Jackiella javanica. The antheridial branches 
som'tiims proliferate (figure 2). The bracteoles are similar to amphigastria and 
are relatively smaller, sometimes bearing rhizoidal outgrowth (figure 15). Owing 
to the absence of perianth the archegonia having short necks are surrounded by 
small pcrichaetial leaves which persist till the capsule remains embedded within 
the marsupium. In the early stage of development there is a rapid elongation of 
the lower side of the stem of the archegonial branch and eventually the apex bends 
upwards. After fertilization the tissue beneath the archegonial group undergoes 
rapid msristematic activity causing the formation of initially a sm? 11 ventral tubei> 
like marsupium (figure 1). According to Schuster (1966) the development of the 

marsupium takes place " due to an auxin secreted by the embryo or at least 

by something derived from the embryo the embryo factor/' However, there is 
no experimental proof regarding this contention. Simultaneously with the forma- 
tion of marsupium the development of sporophyte also takes place. As the growth 
continues further the marsupium becomes pendulous and cylindrical, enclosing the 
developing sporophyte. The mar&upium grows downwards into the substratum 
and remains studded with dense rhizoids of the simple type. The sporophyte even 
up to a late stage of development remains embedded within the marsupium. When 
the sporophyte is mature the seta elongates considerably and the capsule is pro- 
truded out of the calyptra as well as the marsupium. The foot is niulticellular, 
anchor-shaped and remains embedded in the basal tissue of the marsupium (figure 
16) unlike that of Jackiella which has a large haustorial collar consisting of nume* 
rous uniseriate septate filaments (coiinate at base) arising from the junction of 
foot and seta. The outer layer of capsule wall shows biphasic development with 
nodulose thickenings on radial walls slightly extending over the tangential walls 



Geo calyx a rare marsupial genus 143 

(figure 20). The cells of the inner layer have incomplete or semiannular bands 
formed by the fusion of thickenings on the radial walls and the tangential walls 
(figure 21). Such thickenings appear T-shaped or very rarely c L'-shaped in the 
outer layer and c U'-shaped in the inner layer of cells in a transverse section (figure 
19). 

The plant has been discovered from high altitude zone of the western Himalayas 
the liverwort flora of which still remains unexplored. A cursory survey of the 
entire collect! on of liverworts revealed that the area is extremely rich in rare 
taxa which are either poorly known or still not described in Indian bryology. 

Acknowledgements 

The authors are thankful to the University Grants Commission, New l>ethi, for 
financial assistance. Contribution, from the Department of Botany, University 
of Lucknow, Luc know, New Series (Bryophyta) Ho. 140. 



References 

Banner C E B 1965 Index Hepaticanim VI Germany 

Evans A W 1939 Classification; of the Hepaticae ; JBot. Rev. 5 45-96 

Hattori S 1948 Contributio. ad floram hepaticarum yakusimcnsem III ; J. Hattori JBot. Lab. 3 

1-35 

Hferzog Th. 1935 Description of new species of N,Z. Hepaticae; Trans. R. Soc. N.Z. 65 365 
Hodgson E A 1958 New Zealaad Hepaticae (Liverworts) X-Marsupial Genera of N.Z. ; Trans. 

R. Soc. N.Z. 85 656-6S4 

Macvicar S M 1926 The students handbook of British hepatics (London: Eastbourne) 
Miiller K 1951-58 Die Lebermoose Europas-An Rabenhorst's Kryptagamen-Flora, ed. 3 

(Leipzig) 
Schuster R M 1953 Boreal Hepaticae ; A manual of the liverworts of Minnesota and adjacent 

regions ; The Am. Middle Nat. 49 257-684 
Schuster R M 1966 The Hzpaticae and Anthocerotae of North America Vol. I (New York and 

London : Columbia University Press) 
Schuster R M 1972 Phylogenetic and taxonomic studies on Jungermanniidae ; J. Hattori Bot. 

Lab. 36 321-405 

Stephani F 1906-1909 Species Hepaticarum III (Geneve) 
Udar R and Kumar A 1981 Jackiella javanica SchifTn. A rare and interesting taxon ; J. Indian 

Bot. Soc. 60 105-111 
Udar R and Kuimr A 19S2 The Genus Jackiella in. south India; Proc. Indian Acad. Sci. 91 131-137 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 2, April 1982, pp. 145-152. 
Printed in India. 



Ontogeny of the paracytic stoma: Variations and modifications 

PARVEEN FAROOQUI (nee KIDWAI) 

Department of Botany, The University, Allahabad, India 

Present address : Regional Forest Research Centre, Jabalpur 482 020, India 

MS received 6 March 19S1 ; revised 2 April 1952 

Abstract. It was generally believed that the topography of the cells surrounding 
the guard cells in the mature condition indicate their mode of development. How- 
ever, it has now been established that more than one ontogenetic type may corres- 
pond to a single mature type, or it may lead to the development of varied stoma tal 
types. The paracytic stoma was studied from this viewpoint. It was found that 
it may be formed through one of at least eight different modes. These are classified 
and reviewed. The need to undertake studies on. the ontogeny of this type of stoma 
in various groups of plants has been emphasized. 

Keywords. Paracytic ; stoma ; ontogenetic types ; variations. 



1. Introduction 

Metcalfe and Chalk (1950) defined the paracytic stoma as a stoma "accompanied 
on either side by one or more subsidiary cells parallel to the long axis of the pore 
and guard cells ". Earlier, Vesque (1881, 1889) named such stomata as " Rubia- 
ceous " and cited the family Rubiaceae as a typical example. Florin (1931, 1933, 
1934), based on his studies of gymnosperms alone, proposed the term " syndeto- 
cheilic " for such stomata, but the name also implied a mesogenous development 
of the subsidiary cells. Developmental studies on stomata of the Rubiaceae by 
Tognini (1897) and Pant and Mehra (1965) confirmed that the parallel subsidiaries 
in these plants were formed from the same mother cell as the guard cells. 

It was, therefore, generally believed that paracytic stomata always developed in 
a mesogenous or syndetocheilic manner. However, it has been well established 
that the developmental type should not be inferred from a study of the mature 
stomatal complex alone but actual developmental studies should be carried out. 

Recent work (Stebbins and Jain 1960 ; Stebbins and Khu&h 1961 ; Pant and 
Kidwai 1966 ; Tomlinson 1974) on monocotyledons has brought to light the fact 
that here the paracytic stomata develop perigenously and the subsidiary cells are 
formed from lateral cells adjacent to the guard cells. 

Indeed, the paracytic stomata, formed by these two entirely different modes, viz, 
mesogenous and perigenous, look so similar that it has aroused a great deal of 

145 



146 Parveen Farooqui (nee Kidwai) 

controversy and various authors have described their formation by different onto- 
genetic pathways in one and the same plant. In Equisetum, Duval-Jouve (1864) 
and Chatterjee (1964) mentioned that the stomatal meristemoid first divides by a 
periclinai wall, each of these segments then divides by an anticlinal wall to form 
two overlapping subsidiaries from the outer cell and two sunken guard cells from 
the inner cell. However, other authors (Strasburger 1866-1867 ; Johnson 1933 ; 
Hauke 1957 ; Pant and Mehra 1964a ; Pant and Kidwai 1968) found no periclinal 
division and the stomata according to them are of the ordinary paramesogenous 
type. 

In Gnetum, tie development of stomata was described as haplocheilic or peri- 
genous by some authors (Maheshwari and Vasil 1961a, b ; Inamdar and Bhatt 
1972) whereas, others on the basis of division figures have concluded that the two 
parallel subsidiary cells are formed inesogenously (Takeda 1913b : Kaushik 1974 ; 
Nautiyal et al 1976 and others), but as they fall short of the poles, the stomata may 
even be termed mesoperigenous. 

The paracytic stoma is perhaps the most common among plants, occurring in 
pteridophytes, gymnospenns, monocotyledons and dicotyledons. Dilcher (1974) 
has pointed out that " more than one ontogenetic type may correspond to a single 
mature stomatal type ". This is perhaps nowhere better illustrated than in the 
paracytic stoma and a detailed study of its development based on published litera- 
ture and illustrations has brought to light a number of different ontogenetic path- 
ways which ultimately result in the formation of a paracytic stoma in the adult 
condition, the three main types being the perigenous, the mesoperigenous and the 
mesogenous types. Some of these types have not actually been reported but as 
the possibility of their discovery at a later date remains, they have been included 
here. Some types have been reported only as variations of other predominant 
types and not as characteristic of any particular taxon. The terminology according 
to any particular author is indicated by appropriate reference. A few new terms 
are also being introduced for the first time here. The following main categories 
have been found (figure 1). 



2. Ontogenctic pathways leading to the formation of pamcytic 

2-1. Perigenous type (Pant and Mehra 1964b) 

Here a protoderrn cell becomes directly converted into the guard cell mother cell 
and divides once to form the two guard cells. The parallel subsidiary cells are 
formed from cells on the side of the guard cells (figure 1-1). 

(a) Hemipam-perigenous type : Only one lateral subsidiary cell is formed from a 
perigene cell on the side of a guard cell and the stoma is of the hemiparacytic 
type in the mature condition (figure Ma). Fryns Classens and Van Cotthem 
(1973) named it the mono-perigenous type but here it has been termed the hemipara- 
perigenous type in keeping with their hemipara mesoperigenous type. Although 
this type has not been described so far, Fryns Classens and Van Cotthem (1973) 
suggested that some stomata on Polygonum lanigerum (Inamdar 1969b) may follow 
this developmental pathway. 



Ontogeny of the paracytic stoma 



147 



ONTOGENETIC MODES OF PARACYTIC STOMA FORMATION 



FIRST 
DIVISION 



SECOND 

DJVIfilOk 



THIRD 
DIVJSIOJM 



FOURTH flFTH MATURE 
DIVISION 



D 



c 



(GO 



D 



1-2 



D- 



(D 



ccn 






a 



CD 



CDj 



(IDS 



SO 



D 



(D 



o 



am 



1-3 



a 



OB 



a 



CD 



GB 



a 



D 



00 



Figure i. Diagrammatic representation of the different ontogenetic modes of 
paracytic stoma formation. The perigene cells and their divisions are represented 
by dotted lines. 1-1. Perigenous. a. Hemi-para perigenous. b. Paraperigenous. 
1-2. Meso perigenous. a. Herni-para-mesapcrigenotis. b. Para-meso perigenous 
(Type I), c. Para-mesoperigenous (Type II). 1-3. Mesogenous. a. Para-msso- 
genous. b. Hemi-parallelo-mesogenous. c. Parallelo-mesogenous. 



(b) Para-perigenous type : The stoma is formed in the perigenous manner and the 
two parallel subsidiaries are formed by asymmetric divisions in the cells lying on 
the two lateral sides of the guard cells (figure 1 . Ib). This has been termed the 
biperigenous (Paliwai 1969) or the diperigenous (Fiyns Classens and Van Cotthem 
1973) type but for the sake of uniformity the term paraperigenous is preferred here- 
This type is common in the monocotyledons and has been described in the Musa- 
ceae, Gramineae, Juncaceae, Cyperaceae, Alismataceae, Marantaceae (Strasburger 
1866-1867 ; Porterfield 1937 ; Flint and Moreiand 1946; Stebbins and Jain 1960 ; 
Metcalfe 1960), twenty-four families of monocotyledons (Stebbins and Khush 1961), 
Araceae (Pant and Kidwai 1966), Commelinaceae, Zingiberaceae (Tomlinson 
1969), Centrolepidaceae and Phydraceae (Paliwai 1969) and others. 

2.2. Mesoperigenous type (Pant and Mehra 19646) 

The cells surrounding the guard cells in the adult condition have a dual origin, 
some being formed from the same meristemoid as the guard cells while the others 
are perigenous in origin (figure 1.2). 



P.(B)-6 



148 Parveen Farooqui (nee Kidwai) 

(a) Hemipara-mesoperigenous type (Fryns Classens and Van Cotthem 1973) : The 
single lateral subsidiary cell is formed from the stomatal meristemoid (figure 1 -2a) 
before the formation of the two guard cells, e.g., Lophosoria (Kondo and Toda 
1956, 1959), some Rubiaceae (Pant and Mehra 1965), Nyctaginaceae (Inamdar 
1968), Bigonia (Inamdar 1969a), Crotalaria (Shah and Gopal 1969), Kalanchoe 
(Inamdar and Patel 1970), Polemoniaceae, Boraginaceae and Solanaceae (Patel and 
Inamdar 1971) and some stomata of Rauwolfia and Tabernae montana (Trivedi and 
Upadhyay 1976) and Annona (unpublished observations). 

(b) Para-mesoperigenous type I or Para-eumesoperigenous type : The adult para- 
cytic stonia is surrounded by two parallel subsidiaries, one of which is formed from 
the stomatal meristemoid and the other by a division in the perigene cell on the 
other side of the guard cells (figure l-2b). This type is the true mesoperigenous 
paracytic type (s/type l-2c) and although it has not been described so far, it has 
been included here as the possibility of its occurrence exists. According to Payne 
(1970), some stomata in Liriodendron tulipidera may be formed in this manner. 

(c) Para-mesoperigenous type II or Para-pseudomesoperigenous type : The adult 
stoma is paracytic and its two lateral subsidiary cells are formed from the same 
initial as the guard cells (mesogenous). However, they do not meet at one or 
both poles of the guard cells and the neighbouring perigene cells abut on the polar 
ends of the guard cells (figure l-2c). Due to this falling short of the mesogene 
lateral subsidiaries towards the poles, the stoma is considered as mesoperigenous. 
This view has been expressed by Pant and Mehra (1964), Pant (1965), Fryns Clas- 
sens and Van Cotthem (1973) and Nautiyal et al (1976), e.g., Wdwitschia (Takeda 
1913a), Gnetum (Takeda 1913b; Nautiyal et al 1976), Drimys (Bondeson 1952), 
Linum (Paliwal 1961), Borreria and Oldenlandia (Pant and Mehra 1965), Bignonia 
(Inamdar 1969a), Polygonaceae (Inamdar 1969b), Jasminwn (Inamdar et al 1970), 
Claytonia (Payne 1970), Casuarina (Pant et al 1975), Xylonymus and Brassianthus 
(Den Hartog and Baas 1978), and some stomata in Zornia (Kannabiran 1975a), 
Abrus precatorius (Kannabiran 1975b), Dipteria (Khare 1978) and Annona (un- 
published observations). 

If the mesogene subsidiaries meet at only one of the poles, the stoma is frequently 
surrounded by three cells and appears aniso- or tricytic, eg. some stomata in Rumex 
(Verma 1975), Zornia (Kannabiran 1975a), Brassiantha (Den Hartog and Baas 
1978) and Annona (unpublished observations). 

2-3. Mesogenous type (Pant and Mehra 1964ft) 

The lateral subsidiary cells are mesogenous in origin and they completely flank 
the guard cells so that no other cells immediately surround the guard cells (figure 
1.3). 

(a) Para-mesogenous type (Fryns Classens and Van Cotthem 1973) : There are 
only two lateral subsidiary cells, parallel to the guard cells and these are meso- 
genous (figure l-3a). Recorded from Cheiropleuriaceae, Dipteridaceae and 
Dicksoniaceae, Phorbitis, Basella and Opuntia (Strasburger 1866-1867), Convol- 
vulus, Euphorbiaceae, Impatiens and C<^a(Tognini 1897). Magnoliaceae (Paliwal 



Ontogeny of the paracytic stoma 149 

and Bhandari 1962 ; Pant and Gupta 1966), Cheiroplueria and Cibotium (Kondo 
and Toda 1959), Convolvulaceae (Pant and Banerjee 1965), Rubiaceae (Pant and 
Mehra 1965), Strapleonema (Stace 1965), Portulaceceae, Trianthema (Ramayya and 
Rajagopal 1968), Dicksoniaceae, Dipteridaceae and Lopho&oriaceae (Van Cotthem 
1970), Nyctaginaceae, Polygonales, Centrospermae, Zygophyllaceae, Simerubaceae, 
Salvadoraceae, Bigonia and Ipomea (Inarndar 1968, 1969a, b, c, d, e), Leguminosae 
(Shah and Gopal 1970; Bora and Baruah 1979; Farooqui 1979), Zornia (Kannabiran 
1975a), Abrus precatorius (Kannabiran 1975b), Rauwolfia and Chatharanthus 
(Trivedi and Upadhyay 1973, 1976, 1977), Bhesa sp. and Hedraianthera (Den 
Hartog and Baas 1978), Dipteris (Khare 1978) and many others. 

(b) Hemiparallelo-mesogenous type : Three cells are cut off from the stomatal 
meristemoid in an alternate fashion so that the stoma is surrounded by 2 inner 
subsidiary cells and an outer encircling cell (figure l*3b). This, was first described 
by Payne (1970) under the parallelocytic type. However, as only one additional 
cell is formed, it has been separated here, e.g., some stomata of Cinchona 
succimbra (Pant and Mehra 1965), Magnoliaceae (Pant and Gupta 1966) and 
Rauwolfia serpent ina (Trivedi and Upadhyay 1976). 

(c) Parallelo-mesogenous type (Fryns Classens and Van Cotthem 1973): The meriste- 
moid, instead of forming only two parallel subsidiary cells divides further in the 
same alternate manner and forms two or more additional cells (figure 1 . 3c) which 
surround the subsidiaries and axe parallel to them (encircling cells), e.g., Cactaceae, 
Convolvulaceae, Euphorbiaceae, Leguminosae, Portulacaceae, Rubiaceae and 
Umbelliferae (Payne 1970), Euonymus globularia (Den Hartog and Baas 1978). 

The paracytic stoma may also show variations by further divisions in its sub- 
sidiary cells. If one of the subsidiary cells divides the stoma may appear tricytic 
or anisocytic in the adult condition. If both the subsidiaries divide, the stoma may 
appear tetracytic or anomocytic. All these variations have been noticed in 
Strychnos (unpublished observations). Therefore, although their adult form may 
differ, they begin their development in essentially the same manner. 



3. Discussion 

The paracytic stoma may be formed by many different ontogenetic pathways. It 
is, therefore, essential that actual developmental studies should be carried out 
before their ontogeny can be determined. 

Of the eight categories described, the most common are the para-perigenous, 
para-pseudomesoperigenous and the para-mesogenous types. The others repre- 
sent variations of these and their occurrence is limited or not reported so far. 
A study of development in varied groups of plants may lead to their discovery at a 
later date. 

Metcalfeand Chalk (1950) have listed 105 dicotyledonous families in which the 
paracytic type of stoma is predominant. In addition a number of monocotyledons' 
pteridophytes and gymnosperms also have the paracytic stoma. There is an 
urgent need to work out the exact mode of stomatal development in all these taxa. 

P.(B)-7 



150 Parveen Farooqui (nee Kidwai) 

There are other paracytic stomata, like those of the Cycadeodales, whose develop- 
ment will probably never be determined because they occur only as fossils. 

According to Takhtajan. (1969) the paracytic type of stoma is the most likely 
basic type of stomatal apparatus in the evolution of flowering plants. Its occur- 
rence in such diverse groups as the pteridophytes, gymnosperms, monocotyledons 
and dicotyledons may support such an assumption. 

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and Spathiflorae ; Senck. biol. 47 309-333 

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Takeda H 1913a Some points in the anatomy of the leaf of Welwitschia mirabilis : Ann Bot 
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152 Parveen Farooqui (rtee Kidwf) 

Takeda H 1913b Development of stomata in Gnetum gnemon\ Ann. Bot. 27 365-366 
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Prac. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 2, April 1982, pp. 153-158. 
Printed in India. 



Growth response of some thermophilous fungi at different 
incubation temperatures 



S SINGH and D K SANDHU 

Department of Biology, Guru Nanak Dev University, Amritsar 143005, India 

M3 received 4 September 19S1 ; revised 17 March. 19S2 

Abstract. Growth response of 21 thermaphilous fungi at 10 different tempe- 
ratures from 15-62 C has been studied. These fungi could be categorized into 
three groups, i.e., microtkermophiles, thermotolerant and true thermophiles. The 
temperature relations of 6 thermophilous fungi namely Asjpergiltiis tamarii, A. terreus 
v&T.aureus (microtliennophiles), A. nidulansv&x.echinulatus, A. viridi-nutcMS, A.fumi- 
gatus var. ellipticus and A. caespitosus (thermotolerant) are being reported for the 
first time. The growth rates of different fungi varied from 0-19-1 -25 mm/hr at 
their optimum temperatures. Penicittium sp. the slowest and Thermoascus sp. were 
the fast growing fungi. 

Keywords. Mtcrotherfnophiles ; thermotolerant ; thermophiles ; temperature ; 
growth rates. 



I. Introduction 

Thermophilous fungi are those which show temperature optima in the range of 
25 to 55 C (Apinis 1963), These fungi play a significant role in degradation of 
plant material, composting and humification (Fergus 1971 ; Cailleux 1973 ; Tansey 
and Brock 1978 ; Jain etal 1979). The occurrence of thermophilous fungi is 
now known to be ubiquitous (Tansey and Brock 1978). In India these fungi 
have also been reported from various substrates like compost (Maheshwari 1968 ; 
Qureshi and Johri 1972), stored grains (Mehrotra and Basu 1975), coal mine 
soils (Thakre and Johri 1976), nesting materials of birds (Satyanarayana etal 1977) 
and from air (Thakur 1977). Recently, Sandhu et al (1980) and Sandhu and Singh 
(1981) studied the occurrence and some ecological features of thermophilous fungi 
associated with decomposing sugarcane bagasse, and forest soils. The present 
study describes the effect of different incubation temperatures on the growth rates 
and development of these fungi. 



2. Materials and methods 

Twenty-one fungi isolated from bagasse and forest soils were studied for their 
response to different temperatures, The culture media used for the identification 

153 



154 S Singh and D K Saridku 

of ^spergilU and Penicillium sp. were czapek's agar, malt extract agar and yeast 
glucose agar respectively (Raper and Fennell 1973). Yeast starch agar (Cooney 
and Emerson 1964) was used for the remaining fungi belonging to mucorales, 
ascomycetes and hyphomycetes. Mycelial discs of 3 mm diameter from the peri- 
phery of growing colonies were used as the inoculum and the plates were incu- 
bated at 15 C, 20 C, 27 C and 32-62 C with 5 C interval. The petri plates 
kept at 42-62 C were placed in polythene bags and a beaker of water was also 
placed in each of these incubators to avoid desiccation. The bags were opened 
twice daily to aerate the cultures. Growth response was recorded in terms of 
colony diameter daily. Sporulation and cleistothecia production were noted 
visually and confirmed by observing under the binocular and by slide preparations. 
The growth rate of each fungus was calculated on yeast starch agar as follows : 

Average diameter in mm of four petri plates 
rate "" Total time period in h 

3. Results and discussion 

Based on colony diameter the growth response of 21 fungi to 10 different incuba- 
tion temperatures yielded 2 microthermophiles, 15 thermotolerant and 4 true 
thermophiles (table 1). A comparison of our results with the literature revealed 
that the temperature relations of 6 thermophilous fungi namely Aspergillus tamarii, 
A. terreus var. aureus (micro thermophile), A. rtidularts var. echirtulatus, A. viridi- 
nutans, A. fumigatus var. ellipticus and A. caespitosus are being reported for the 
first time. The temperature relations of the other fungi studied, in general, fall 
within the range described by Tansey and Brock (1978). Temperature responses 
of Thermomyces lanuginosus, Humlcala grisea var. thermoidea and Mucor pusillus 
are more close to those reported by Fergus (1964) and Evans (1971) respectively 
than Apinis (1963) and Qureshi and Johri (1972). However, Humicola grisea var. 
thermoidea differed from Evans (1971) since our isolates could not grow at the 
minimum ^temperature of 20 C. The cardinal temperatures of Aspergillus fumi- 
gatus and Sporotrichum thermophile varied widely from Qureshi and Johri (1972) 
and almost fell within the range given by Evans (1971) and Tansey and Brock 
(1978). In the case of Aspergillus nidulans and A. terreus the cardinal temperatures 
were similar to those reported by Evans (1971) but had more variation of minimum 
temperature than maximum. This may be due to the fact that in our study 
minimum temperature was recorded up to 15 C only. The temperature variations 
reported above may be due to strain variations, nutritional requirements and 
temperature study at wider temperature intervals. 

There was also variation in temperature regarding the optimum and the range 
among the species and their varieties, e.g., Aspergillus fumigatus and A. fumigatus 
var. ellipticus had their optimum temperature at 37 C and 42 C respectively 
(figure 1). In the case of A. terreus and A. terreus var. aureus both had their 
optimum temperature at 37 C. The former could grow up to 47 C while the 
limit for the latter was at 42 C (figure 1 and table 1). There was a variation, in 
optimum temperature for the development of the perfect and imperfect stage as 
in the case of A. nidulans and A. nidulans var. echinulatus, maximum cleistothecia 
formation was observed at 42 C while the sporulation was maximum at 37 Q, 



Growth response of terhmophilous fungi 155 

Aspergillus fumigatus r A. fumigatus var. ellipticus 



A. mdulans var. echinuLatus 
32 C 




24 48 72 95 120 U4 160 24 48 72 96 120 1U 168 

HOURS OF INCUBATION 

Figure 1. Growth response o.f ane microth.ermoplule (Aspergillus terreus var. 
aweus) and 5 thermatolcramt fungi at different temperatures. 



Similarly, abundant superficial cleistotkecia like bodies of A. caespitosus were 
seen at 42 C while the optimum temperature for sporulation was .37 G The 
temperature range for speculation and reproductive structures in all the groups 
was narrower tha,n that for mycelial growth. 

The growth rates of optimum temperature of 21 fungi varied from 0- 19- 
l-25inm/hr (figure 2). Penicillium sp. was the slowest and Thermoapcus sp. the 
fastest growing species. The fungi having growth rates of 0-19-0-39, 0-40-0 -75 



156 



Singh and D K Sctndhu 



Table 1. Optimum temperature and raage for growth and sporulation of 21 
thermopliilous fungi, 



Group 



Temperature Temperature Optimum 

rarige (C) for range (C) temperature 

mycelial for ( C) 

growth sporulation 



Fungi 



Microthennophile 15-42 

TJierrnotolerant I 15-47 
(psyclirotoleraitt) 



11 15-52 



True thermophiles I 20-52 

II 27-52 

III 32-52 

IV 32-57 



20-37 32 Aspergillus taniarii(S*) 

37 A. terreus var. aureus (B) 

27-42 32 Paecilomyces varioti (B) 

37 Aspergillus nidulans (S), A. nidulans 

var. echinulatus (S), A. teneus (S), 

A. caespitosus (B), Acrophialo- 

phora fusispora (B), Penicilliurn 

$P. (B) 
42 Thielavia seped&nium (S), Absidia 

carymbifera (B) 
27-47 37 Aspergillus fumigatus (B), A. viridi- 

nutans (S), Sporatrichum thermo~ 

phile (B) 
42 Aspergillus fumigates var. ellipticus 

(S), Absidia spinosa (B), Rhizopus 

microsparus (S) 

27-47 42 Mucor pusillus (B, S) 

32-47 47 Humicola grisca var. thermoidea (B) 

37-47 42-52 Thevmoascus sp. (S) 

37-52 47 Thermornyces lanuginosus (B r S) 



Source of fungi. B, bagasse and S, forest soil. 



and 0-86-1-25 mm/hr were categorized as slow, moderate and fast growing 
respectively. Comparison of growth rates at the optimum temperatures of these 
fungi show that 8 are slow growing and out of these 2 are microthermophiles and 
6 thermotolerant. Nine species belong to moderately growing group, 7 of which 
are thermotolerant and 2 true thennophiles. In the fast growing group 2 are true 
thermophiles and 2 thermotolerant. 

The growth response of these fungi to diiferent temperatures was determined 
on the basis of colony diameter. According to Hawker (1950) and Cochrane 
(1958) the colony diameter alone does not account for density height and depth 
of the colony. However, it has been justified for studies in which only one 
environmental variable is studied for example temperature (Branc^to and Golding 
1953 ; Cochrane 1958 ; Evans 1971 ; Trinci 1971 ; Tansey 1972). 



(tnwth respvnxf <*f thrmvphihus fungi 




I* growth rites at optimum temperature of 2! 

fiiiiil fell, moderate and slow growing groups. Figures 

llti bar* af inoubttion In hours. 



Apiitti A I! t%l cif iliritiplilliiiti micrafui^gi In certain alluvial soils near Notting- 

ham ; /tow S 57-7* 



I 1 I 1 und (folding H S i**ll Th diametor of the mold colony as a reliable measure of 
growth ; 4S 

Caiileux R tf II Mtyaitlur^ du compo&i dnUin6 t It culture du champignon dc Couche ; jRev. 

A(f>f*/, 17 14-35 
C\lifiiw V W t^H PhwMogv ff**l (Hew York : John Wiley) 

l> Cl mid i'.morvm H IW4 fhtrmopkUtefitiig: An account of their biology, activities and 
el*i*l/tr*tkm {San Fratdco and i*mdi : W H Frdwnan) 
II C W7t Thermnphitoii fungi of coal %poll tips* IL Occurrence, distribution and tempe- 

relationship : Tram* Mr, Myp#L $c> 57 255-266 
C L 1971 Ttitrmtiphific tad thermotolerant molds and actinomycetes of mushioom 

ttring ; W 267-284 

Hawker L I! iWQ //mrf : Univ. of London Press) 

Jain M K, K 1C md M M 1979 C^llul^e activity, degradation of cellulose and 

httmu formitl0n by ilttffii^tiilie funfi; Thvtf. Br. M^c<?/. *Soc. 72 85-89 






S Singh and D K Sandhu 

Maheshwari R 1968 Occurrence and isolation of thermophilic fungi ; Curr. Sci. 37 277-279 
MTehrotra B S and Basu M 1975 Survey of the microorganisms associated with cereal grains 

and their milling fractions in India. Part I. Imported wheat ; Int. JBiodeterior. Bull. 11 56-63 
Qureshi A R and Johri B R 1972 Temperature relationship of some thermophilic fungi ; Bull. 

Bat. Soc. Univ. Sagar 19 28-33 

Raper K B and Fennell D I 1973 The genus Asjpergillus (New York : Robert E Krieger) 
Sandha D K, Singh S and Waraich M K 1980 Thermophilous fungi of decomposing sugar-cane 

bagasse; Can. J. Bot. 58 2015-2016 
Sandhu D K and Singh S 19S1 Distribution of thermophilous microfungi in forest soils of 

Darjeeling (Eastern Himalayas) ; Mycopathologia 74 79-86 
Satyanarayana T, Johri B N and Sakessena S B 1977 Seasonal variation in mycoflora of nesting 

materials of birds with special reference to thermophilic f ungi ; Trans. Br. Mycol. Soc. 68 

307-309 

Tansey M R 1972 Effect of temperature on growth rates and development of the thermophilic 

fungus Cheetomium thermophile ; Mycologia 64 1290-1299 
Tansey M R and Brock T D 1978 Microbial life at high temperatures : ecological aspects ; in 

Microbial life in extreme environments (ed.) D J Kashner (London : Academic Press) 
ThakreRP and Johri B N 1976 Occurrence of thermophilic fungi in coal mines soils of Madhya 

Pradesh; Curr. ScL 45 271-273 

Thakur S B 1977 Occurrence of spores of thermophilic fungi in the air at Bombay ; Mycologia 
69 197-199 

Trinci A P J 1971 Influence of the width of the peripheral growth zone on the radial growth 
rate of fungal colonies an solid media ; /. Gen. MicrobioL 67 325-344 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 2, April 1932, pp. 159-174. 
Printed in. India. 



Studies on Beggiatoa : Distribution and growth in aquatic 
habitats of Visakhapatnam 

M R R MOHAN and A NARAYANA RAO 
Department of Botany, Andhra University, Waltair 530 003, India 

MS received 19 June 1981 

Abstract. Bzggiatoa is distributed in 11 of the 12 polluted aquatic habitats occurring 
on the sediment and decaying leaves as a thick white scurn at 2, in detectable popu- 
lation at 6, and in very less population at 3 habitats where H 2 S necessary for its 
growth is produced. The Beggiatoa spp. present in these habitats are considered 
as belonging to the 6 species described in Sergey's manual. J9. minima with less 
than 1 -0, B. leptomitiformis with 1 - 66, B. alba with 3-32 and 4-9S ju wide trichomes 
are distributed in 2, 6, 11 and 2 habitats respectively, which are fresh water, brackish 
and marine habitats. B. arachn&idea with 9-96, B. mirabilis with 17-0 and 
B. gigantea with 28- 22 & wide trichomes are present in only one brackish habitat. 
B. alba (3-32 fji) occurs alone at 2 habitats, and in association with either one or 
more of the other Beggiatoa species at the remaining 9 habitats. Except B. mirabilis 
and B. gigantea, the others distributed in the 11 habitats could multiply in enrich- 
ment culture media that contained extracted rice straw pieces and waters from 
their respective habitats, and aggregate into visual white colonies, or loose clumps 
in a thick film on the surface of medium, rice straw pieces and glass. B, mirabilis 
and B. gigantea seem to be halophilic, truly autotrophic and more exacting than 
the other species that exhibited differences in salt tolerance when grown in crude 
enrichment culture media of differing salt content. It appears that physiological 
strains or groups differing in salt tolerance may be existing in these species of 
Beggiatoa. 

Keywords. Beggiatoa ; distribution ; growth ; aquatic habitats ; Visakhapatnam * 9 
enrichment culture medium. 



1. Introduction 

Beggiatoa is a large, colourless, filamentous, gliding bacterium, and easily recogni- 
zable. It is usually considered as carrying on chemolithotrophic nutrition, oxidizing 
H 2 S, and depositing sulphur intracellularly as droplets. It is widespread in 
distribution, occurring in lake bottoms, stream beds, ponds, sulphur springs, 
brackish and marine habitats; even in the rice rhizosph ere (Pitts etal 1972) and 
water-saturated soils of rice fields (Joshi and Hollis 1977), playing an important 
role, the detoxification of H 2 S formed due to putrefaction, and activities of 
anaerobic organisms such as Desulfovibrio spp. It is associated with, and also 

159 



160 M R R Mohan and A Narayana Rao 

often described as indicative of strongly polluted polytrophic habitats. Ever 
since the investigations, and the concept of chemolithotrophy by Winogradsky 
(1887, 1888, 1889) attempts have been made (Keil 1912 ; Cataldi 1940 ; Faust 
and Wolfe 1961 ; Lackey 1961 ; Scotten and Stokes 1962 ; Pringsheim 1964 ; 
Kowallik and Pringsheim 1966 ; Jorgensen 1977) to increase the small number of 
Beggiatoa trichomes present in samples from natural habitats into a rich popu- 
lation by reliable enrichment culture techniques for isolation and cultivation in 
pure culture media ; to study its distribution, morphology, growth and nutrition. 
Pringsheim (1964) studied in detail the species differentiation of Beggiatoa, and 
pointed out among other problems that the number and types of forms found 
under a variety of ecological conditions are obscure. 

The review of the literature showed the need for detecting and isolating as 
many forms of Beggiatoa as possible for taxonomic differentiation, and studying 
its nutrition ; and more so because there are no reports from India. Since 
Visakhapatnam abounds with a variety of aquatic habitats that are continuously 
polluted with urban refuse, sewage and industrial effluents, detailed investigations 
are undertaken to study the distribution, growth, nutrition and species differen- 
tiation of Beggiatoa. This paper presents 4 the observations and experimental 
results on distribution and growth of Beggiatoa isolated from natural " polluted " 
habitats, for the first time from India. 



2* Materials and methods 

Twelve sites of different aquatic habitats in Visakhapatnam were surveyed for 
Beggiatoa. Their location and some relevant features are as follows : 

Site 1 : The edge of a shallow one feet wide canal of a septic tank near the 
Botany department garden, Andhra University campus. The entire edge is covered 
with a thick white scum. The decaying leaves present in the canal and at the edge 
are also covered with a white scum. The sediment is black silt. pH of water : 7, 
salinity : 58 ppt. 

Site 2 : The edge of a shallow sewage canal near Rama Krishna Mission, 
Mahaianipet. The sediment is fine sand. pH of water : 7, salinity : 0-72 ppt. 

Site 3 : The edge of a shallow sewage canal near Visakha women's college, 
Maharanipet. The sediment is black silt. pH of water : 7, salinity : 1 07 ppt. 

Site 4 : The edge of a stagnant pond receiving sewage water near " convent 
junction". The sediment is black silt. pH of water: 7, salinity: 1-07 ppt. 

Site 5 : The edge of a sewage stream near " naval coast battery ", Maharanipet. 
The sediment is light black fine sand. pH of water : 7, salinity : 1-08 ppt. 

Site 6 : The edge of a sewage stream near the wooden bridge, ferry road-old 
post-office junction. The stream joins the harbour channel, and the sample site 
is at about 200 metres from the channel. During high tide there will be a back 
flow of sea water. The sediment is black silt. pH of water : 7, salinity : 1 4 ppt. 

Site 1 : The edge of a stream adjacent to oil-loading tanks near naval head- 
quarters. The bank is muddy, and covered with a thick asphalt-like layer mixed 



Distribution and growth of Beggiatoa 161 

with cakes and clumps of resin-like material, a conversion product of oil. pH 
of water : 7-5, salinity : 4- 5 ppt. 

Site 8 : The edge of the sea water canal carrying effluents from the Coromandel 
fertilizers factory. The site is located near the Hindustan Petroleum Oil Refineries 
entrance gate. The sediment is light red silt. pH of water : 5 and 2, salinity 
19-0 ppt. 

Site 9 : The edge of a stagnant brackish water pond near ' Chavulamadam '. 
The sediment is black silt. pH of water : 8-5, salinity : 20 ppt. 

Site 10 : The edge of one of the Harbour channel terminals near sulphur- 
unloading berth of the harbour. The bank is fine sandy covered with a thick 
asphalt-like layer mixed with cakes and clumps of a resin-like material, a conversion 
product of oil. pH of water : 7-5, salinity : 28-0 ppt. 

Site 11 : The edge of the sea water canal carrying effluents from Hindustan 
Petroleum Oil Refineries. The bank is muddy and covered with a thick asphalt- 
like layer mixed with cakes and clumps of a resin-like material like above, but 
covered with a thick white scum. pH of water : 7, salinity : 31-25 ppt. 

Site 12 : The edge of the sea coast near the fishing outer harbour. The sedi- 
ment is light black fine sand. pH of water 7, salinity : 34-0 ppt. 

At these sites the sediment samples along with some water were collected with a 
thoroughly washed stainless steel spoon into 250 ml Erlenmayer flasks. Decaying 
leaves, or decaying leaves covered with white scum were also collected into separate 
flasks. The surface water samples were collected into 500 ml flasks. The flasks 
were plugged with cotton. 

The pH of surface water samples was noted using pH indicator papers (BDH) 
supplied by the Chemicals Division, Glaxo Laboratories (India) Ltd., Bombay. 

The salintiy of surface water samples was estimated after centrifuging at 
4000 r.p.m. for 15 min by conductivity method using direct reading conductivity 
meter-303 (Systronics, Ahrnedabad), and conductivity of different concentrations 
of common salt solution as standard. 

To detect the occurrence of Beggiatoa, the sediment, decaying leaf and water 
samples on arrival at the laboratory, immediately and also after 24 hr incubation 
in darkness at room temperature, ca 28 C, were repeatedly examined micro- 
scopically. 

To ascertain the distribution of Beggiatoa the samples were subjected to crude 
enrichment culture technique described by Faust and Wolfe (1961) with some 
modifications. Instead of seasoned roadside winter grass, seasoned rice straw 
was used. The procedure to extract and dry rice straw pieces of 2 cm in size 
was the same as that of Faust and Wolfe. The enrichment medium for sample 
of each site consisted simply of 0-6 g of extracted rice straw bits and 70 ml of 
surface water collected at the site. The surface water used is referred to as site 
water. The media were taken into 150 ml flasks, and plugged with cotton to 
prevent evaporation. The media were not sterilized, because sterilization in the 
initial studies proved to be less effective in increasing the population of Beggiatoa 
trichomes. The unsterilized media were inoculated with sediment of about 1 ml 
in volume, or 2 decaying leaf bits of about 1 cm 2 in size, or sediment plus decaying 
Jeaf bits. O$e set was left without adding either seciimei^t or decaying leaf bits, 



162 M R R Mohan and A Narayana Rao 

They were incubated at room temperature, ca 28 C, for 30 days in darkness. 

To study their growth, and salt tolerance the same crude enrichment culture 
technique was followed with some alterations. The enrichment medium for 
Beggiatoa of each site consisted of the same quantity of rice straw bits, and 70 ml 
of either respective unfiltered site water or filtered site water, or filtered and stored 
sea water (pH 7-0 ; salinity 35-0 ppt), or tap water (pH 6-8 ; salinity 0-31 ppt), 
or metal distilled water (pH 6-5 ; salinity 0-016 ppt). The media were taken in 
150 ml flasks. These were also not sterilized. They were inoculated with sedi- 
ment only, and incubated as above. The site and sea waters were filtered 
through Whatman No. 1 filter paper. 

The crude enrichment cultures were observed directly, and also examined micro- 
scopically. The amount of growth was estimated visually because of the tendency 
of the organisms to form aggregations of trichomes, fragmentation of trichomes, 
and due to certain practical difficulties in estimating the exact amount of growth 
by other methods in vogue 

To test sulphur, the trichomes were transferred on to microscope slides, and 
treated with aceto-carmine (Ellis 1932). 

To identify the species of Beggiatoa, the width of the trichomes, which was 
uniform, was considered and measured at x 970 magnification. 

To observe the gliding movement on agar surface, the method of Faust and 
Wolfe (1961) was followed using their modified Cataldi's agar medium dried at 
60 C for about 10 min. 



3. Results and discussion 

The species differentiation in Beggiatoa is mainly based on the width of trichomes. 
According to the usual nomenclature of Beggiatoa forms (Buchanan and Gibbons 
1974), the forms with 1# or less wide are called B. minima Winogradsky ; those 
between 1 and 2 /j, wide trichomes are called B. leptomitiformis Trevisan ; those 
between 2-5 and 5 ju wide as B. alba (Va.uch.er) Trevisan ; those between 5 and 
14 fi wide as B. arachnoidea (Agardh) Rabenhorst ; those between 15 and 21 p 
wide as B. mirabilis Cohn ; and those between 26 and 55ju wide as B. gigantea 
Klas. However, Pringsheim (1964) while discussing the species concept in 
Beggiatoa concluded that after dropping for taxonomic purposes the use of the 
arrangement of the sulphur droplets etc., one has to di!Op that of the width also, 
and there is no feature to replace it. In the present study as there is no other 
feature except width for species identification, width alone considered for identi- 
fying the species of Beggiatoa, and the usual nomenclature is followed in mentioning 
them. 

In the present study, all the above mentioned 6 species of Beggiatoa are identi- 
fied in the aquatic habitats of Visakhapatnam (figure 1). The trichomes of 
B. minima and B. leptomitiformis are less than 1-0 and 1-66/f in width respec- 
tively. Of B. alba, two distinct kinds or strains of consistent and uniform width 
were observed. The trichomes of one are 3 32 jx, and those of the other are 4 98 // 
wide. The trichomes of B. arachnoidea, & mirabilis and B. gigantea are 9 "96, 
17 -Q and 28-22/J wide respectively, 



Distribution and growth of Beggiatoa 



163 






e.arachnolka 



Figure 1. The Beggiatoa spp. occurring in the aquatic habitats of Visakhapatnam. 



Table 1. Distribution of Beggiatoa spp. in the sites. 



Site No. B. minima B.lepta- B.alba B.arach-B.mirabilisB.gigantca 

mitiformis noidea 

(1-0^) (I -66ft) (3 -3 2/*) (4-98/1) (9*9^) (IV-O^) (28-22**) 



1. 


4- 4: 


4* 


_ 


2. 


_ 


4- 





3. 


- 4- 


4- ' 


_ . _ . _ 


4." 





4- 


_L 


5. 


+ 


H- 





6. 


_ 


4- 





7. 





+ 


4- _ 


8. 


_ 





_ _ 


9. 


4- 


4- 


. . _. 


10. 


- 4- 


4- 


-4-4-4- 


11. 


- 4- 


4- 


_-. 


12. 


4. 


4- 


M 



- indicates presence and indicates absence of Beggiatoa group. 



The distribution of these 6 species in the 12 sites given in table 1 shows that 
teggiatoa is distributed in all the sites, except site 8 of the stream carrying 
[fluents from Coromandel Fertilizers Factory. In the sediment, the. decaying 
iaf and the water samples *6f- this site, which is at a distance of about 2km 
'om the factory, except a few minute unicellular bacteria, neither Beggiatoa nor 
ay other associated organisms were found even when the samples were subjected 
> enrichment culture technique (tables 1, 2, 3). Further, when the sediment was 
isturbed for collection there was no odour of H 2 S, indicating the absence of 
ay kind of organisms responsible for H 2 S production. It appears that the high 
cidity of water (pH 5 to 2) <h*e to the efflueitfs released is the main cause for tfye 



164 



M R R Mohan and A Narayana Rao 



Table 2. Growth of Beggiatoa spp. in, crude enrichment culture media containing 
Site waters. 



Site Beggiatoa Spp. 
No. 



Culture medium and inoculum 



Site Site water Site water Site water 
water + + + 

Scdiment decaying sediment and 

leaf decaying leaf 



1. B. minima 

B. leptomitiformis 
B. alba (1- 12 p) 

2. B. alba (!' 12 n) 

3. B. leptomitiformis 
B. alba (3'32ju.) 

4. B. alba (1- 12 p) 
B. alba (4- 98,*) 

5. B. leptomitiformis 
B. alba (1'12^) 

6. B. alba (3 -32 ^) 

7. B. alba (!' 12 p) 



+ + + 

+ + + 
+ + + + 



+ -H- + 
4 + 



4 + + 



+ 4- 



8. 

9. B. leptomitiformis 
B. alba (1- 12 & 

10. B. leptomitiformis 
B. alba (1'12 n) 
B. arachnoidea 
B. mirabilis 

B. gigantea 

11. B. leptomitiformis 
B. alba (I* 12 n) 

12. B. minima 

B. alba (3 -32^) 



+ + + 

+ + + + 
+ + + + 



4- + 



- =Nil, + = Very poor, ++ =Poor, +++ ==Fair, ++++ = Good. 

* No growth, but survived for 7 days after irtcubatior,. 



Distribution and growth of Beggiatoa 



165 



Table 3. Growth of Beggiatoa spp. in enrichment culture media containing 
different waters. 



Site 

Site water 
No. Salinity Beggiatoa spp. 



Culture media with 



Unfiltered Filtered Tap water Distilled Filtered 
sits site (salinity water sea water 

water water 0-31 ppt (salinity (salinity 
pH 6-8) 0-016 ppt 35-0 ppt 
pH 6-5) pH 7-0) 



1. 0*58 B. minima 

B. leptomitiformis 
B. 



2. 0-72 B. 



3. 0-72 B. leptomitiformis 
B. 



4. 1-07 B. 

B.alba (4 -98^) 

5. 1 *08 B. leptomitiformis 

B. alba (3-32fi) 



6. 1-40 B. 

7. 4-50 B. 

B. 

8. 19-0 



9. 20-0 B. leptomitiformis 
B. 



10. 28-0 B. leptomitiformis 

B. arachnoidea 
B. rnirabilis 
B. gigantea 

11. 31-25 B. leptomitiformis 

B. 



12. 34 '0 B. minima 
B. 



+4-4- 



+ 4+4 
4-44 



444 + 
44++ 



+4++ _ 



4-4+ 



++++ 
++ + 

++++ 
4-4-4- 



+4++ _ 

++ + + 
+++ + 



+4-4-4- 



444 
44 



+-++ - 



4-4-4-4- 



+++f- 
++++ 



++ 

+++ 
++ 



4-4-4- 



= Nil, + = Very poor, 4+ = Poor, 4 + + = Fair, 4444 
* No growth, but survived for 7 days after incubation. 
@ Not survived for even 1 day after incubation. 



Good. 



166 M R R Mohan and A Narayana Rao 

absence of not only Beggiatoa but also other associated organisms. On the 
contrary, the samples of the site 11 of the nearby stream carrying effluents from 
Hindustan Petroleum Oil Refineries, when examined immediately or after 24 hr 
incubation on arrival at the laboratory, were teeming with trichomes of B. lepto- 
mitiformis followed, in number, by those of B. alba (3-32 //), in association with 
some protozoans, Oscillatoria filaments and even nematodes. Further, the entire 
bank of the stream is covered with a thick asphalt-like layer mixed with cakes 
or clumps of resin-like material, a conversion product of oil, which in turn are 
covered with a white scum in which the two Beggiatoa spp. are predominant. In 
addition, when the scum was disturbed for collection there was a strong odour 
of H 2 S also. The same is almost the case with site 1 of the narrow and shallow 
drain of a septic tank, where B. leptomitiformis trichomes followed, in population, 
by those of B. alba (3-32 #) and B. minima are present in association with Thiospira 
and some other minute bacteria, protozoans, Oscillatoria filaments, nematodes, 
and even insect worms.' At the remaining sites, no such white scum was observed. 
However, in the samples of the sitps 4, 5,, 6, 7,, 9 and 10 were found the Beggiatoa 
spp. listed against each site in table 1. These are also associated with some 
minute bacteria, protozoans, Oscillatoria filaments, diatoms, nematodes and 
insect worms. The samples of site 10 contained, in addition, aggregations of 
many actively gliding spiral trichomes of Spirulina sp. At these sites also, when 
the sediment was disturbed for collection, there was a feeble odour of H a S. On 
the other hand, in the samples of the sites 2, 3 and 12, Beggiatoa spp. alone were 
not detected in the initial microscopic examination. However, there was a feeble 
odour of H 2 S at the sites, when the sediment was disturbed for collection. 

The trichomes of these Baggiatoa spp. present in the samples collected were 
full of intracellularly deposited sulphur globules, and in active gliding condition. 
In samples incubated for 24 hr in darkness, the trichomes present in the sediment, 
or on the decaying leaves, or in the white scum aggregated into loose white clumps 
on the surface of the glass, and as a white film at the surface of water. It appears 
that the trichomes had moved out of the sediment, or decaying leaves, or white 
scum, and aggregated on the surface of the glass, and water by gliding over the 
surface of glass. However, in samples from sites 2, 3, 8 and 12 no such aggre- 
gations were found. But, when the samples of these sites were subjected to 
enrichment culture technique, B. minima, B. leptomitiformis and B. alba (3-32/0 
trichomes had cropped up in considerable population in the case of samples from 
sites 2, 3 and 12 only. This reveals that* even a thorough direct microscopic 
examination of samples immediately and after 24 hr incubation on arrival at the 
laboratory will not suffice to detect Beggiatoa, if they occur in very small numbers 
between the detritus particles. However, ithis is not the case if they are subjected 
to a reliable enrichment culture technique, or inoculated into a suitable enrichment 
culture medium that is favourable for the small numbers of Beggiatoa trichomes 
to multiply into a rich population^ ' 

The availability of a reliable enrichment culture technique is an important step 
in dealing with any problem concerning Beggiatoa, either its natural distribution, 
or isolation and cultivation in pure culture media, or taxonomic differentiation, or 
nutrition, or tolerance to salinity, etc. Some of the techniques available are the 
Butomus-ihizome technique, the technique, with decaying bay and sulphate, and 
the sulphur spring technique of Winogradsky (1887) ; the macerated hay ami 



Distribution and growth of Beggiatca 167 

sulphate technique of Cataldi (1940) ; for isolation of marine strains of Beggiatoa 
the hay medium with inorganic salts and artificial sea water technique of Pring- 
sheim (1946) ; and the extracted roadside winter grass technique, a modification 
of the method of Cataldi, and the weathered Corncob technique of Faust and 
Wolfe (1961). Of these, as the extracted roadside winter grass technique of Faust 
and Wolfe is reported as a reliable and the most satisfactory method by Pringsheim 
(1964), and very simple, it was used to ascertain the occurrence of Beggiatoa spp. 
in the sites. However, as the dried roadside winter grass is not available in our 
place, dried roadside seasoned grass was used initially with either tap water or 
site waters as the enrichment culture medium, and inoculated with sediment, or 
decaying leaf bits. The media did not increase the population of Beggiatoa 
trichomes. And even when green or dried Ulva bits, or dried dicot leaf bits were 
used the result was same. As Beggiatoa occurs in rice rhizosphere (Pitts etal 
1972) and water-saturated soils of rice fields (Joshi and Hollis 1977) seasoned rice 
straw was next used with site waters as the enrichment culture medium. It has 
promoted the growth of Beggiatoa fairly well (table 2). 

Clear visual white colonies of the type described by Faust and Wolfe (1961) 
appeared on 6, 8, 10 and 15 days after incubation in the media with site waters, 
and inoculated with sediment or decaying leaf bits from the sites 1, 4, 6 and 7 
respectively (figure 2). These colonies were of various sizes ; the average was 
about 1-0 mm in diameter. They appeared both on rice straw bits and on the 
surface of the flasks. Further, bundles of trichomes radiated from the centre of 
each colony producing a starlike appearance. On the other hand, in the remaining 
cultures of the samples from the other sites, instead of such colonies, a thick white 
film was formed at the water surface, and on the rice pieces in all the cultures, 
except in those of site of the stream carrying effluents from Coromandel Fertilizers 
Factory. The microscopic examination of the colonies or the white film revealed 
the Beggiatoa spp. listed in table 1. The amount of growth of species of each 
site, estimated visually, was also recognizable (table 2). In general, the trichomes 
of B. leptomitiformis were more in number than those of others present among 
them. Of the remaining, the trend in density of population was first those of 
B. alba (3-32/j), followed sequentially by those of B. alba (4-49//), B. minima, 
B. arachnoidea, B. gigantea and B. mirabilis, when present in association with each 
other. 

In cultures of samples from sites 1, 7, 9 and 11, recognizable amount of growth 
occurred within 5 to 8 days ; increased fairly well till 20 to 25 days after incu- 
bation, and declined thereafter. However, in cultures of site 7, there was total 
lysis of trichomes from the 24th day after incubation. On the contrary, in those 
of sites 2 to 6 and 12, recognizable amount of growth was evident from 10 to 15 
days ; increased fairly well till 21 to 23 days after incubation, and declined there- 
after. Total lysis of trichomes, however, occurred after 28 and 25 days after 
incubation in the case of cultures of sites 6 and 12 respectively. Whereas, in 
cultures of site 10, in which B. alba (3 32 /*), B. leptomitiformis, B. arachnoidea, 
B. mirabilis and B. gigantea were present the growth of B. alba and B. leptomitiformis 
was evident from 14 days, and of B. arachnoidea from 7 days, and continued 
to increase till the 30th day after incubation. On the other hand, in B. mirabilis 
and B. gigantea even one day after incubation aggregations of trichomes as loose 
appeared QIJ the serfage of glass, and in the film forraed at titte surface of 



168 M R R Mohan and A Narayana Rao 

water. It appears that the trichomes present in the inocula had crept on to glass 
surfa.ce and into the film. Further, the trichomes started lysing from the 4th day, 
and completely dissolved by the 7th day after incubation revealing that they 
failed to multiply in the enrichment cultures. 

The microscopic examination of colonies, or loose clumps, or white film placed 
on microscope slides, clearly showed the gliding movement of the trichomes of 
all the species from the centre of the aggregations or colonies ; and folding of the 
trichomes back into the aggregate (figure 3). The dispersed trichomes of all the 
species also exhibited active gliding movement. The speed of movement, however, 
seems to be different in different species. The trichomes of B. alba, B. leptomitiformis, 
ajad B. minima seem to be fast moving, whereas those of B. arachnoidea, B. mirabilis 
and B. gigantea slow moving. The repeatedly washed colonies of site 9 cultures 
containing a mixed population of B. alba and B. leptomitiformis trichomes on 
Cataldi's agar medium solid surface exhibited not only the characteristic circular 
flow pattern (figure 4) but also other patterns that resembled rivers. Such patterns 
were also reported earlier by Faust and Wolfe (1961) and Pringsheim (1964). 

The intracellular deposition of sulphur as droplets was also observed in the 
trichomes of all the species of Beggiatoa multiplied in the crude enrichment culture 
media. The treatment to trichomes with aceto-carmine resulted in dissolution of 
sulphur droplets, and crystallisation of sulphur into flat octohedra outside the 
trichomes (figure 5). The arrangement of sulphur droplets in the trichomes of 
the different species of Beggiatoa was, however, different. It appears that the 
arrangement of sulphur droplets is a characteristic feature of each species irrespective 
of its distribution. But in enrichment cultures, sulphur-free trichomes were 
also observed. These were in small numbers, and present in declining cultures 
only. 

It is evident from the above observations made on the samples collected, and 
the growth of Beggiatoa spp. in enrichment culture media, that Beggiatoa is 
distributed in 11 sites out of the 12 investigated, and favourable conditions for 
its existence prevail ^t these sites, except at site 8. The pH of waters of these sites 
containing Beggiatoa is not much varied, about 7-0 or above, the optimum for 
most aquatic bacteria. The same wa,s also observed earlier by Lackey (1961) in 
his studies on occurrence of Beggiatoa relative to pollution. However, with 
regard to salt content there are considerable differences between the waters of 
different sites, ranging between very, low (0-58 ppt) and very high (34ppt) salt 
content. Sites 1 to 5 with waters having 0-58, 0-72, 1-07, 1-07 and 1-08 ppt 
salt content respectively are fresh-water habitats. Sites 6 and 7 with waters having 
1-4 and 4- 5 ppt salt content respectively are soft brackish water habitats. Sites 
9 and 10 with waters having 19 and 20 ppt salt content respectively are hard 
brackish water habitats, whereas sites 11 and 12 with waters having 31-25 and 
34 ppt salt content respectively are truly marine habitats. As shown in tables 
1 and 2, Beggiatoa of equal diameter exist in all these habitats. Further, there 
is no difference between them in appearance. If any difference existed, it would be 
physiological only, either in nutrition, or growth rate, or indifference to variation 
in salt content etc. To study the growth and tolerance to low or high salt content 
the sediment samples of all the 12 sites were, therefore, subjected to enrichment 
culture technique using the rice straw medium with respective site waters, or tap 
water of pH 6-8 ^d 0*31 ppt salinity, or met^l distilled. w^ter of pH 6-5 



Distribution and growth of 



169 




and 



3 2 Visual colony growth of Begglatoa spp. of site 7, in erwrichment 



the aggregate (x 700), b Edge af the aggregate showing trichomes filled wUh 
sulphur droplets also (x 860). 



170 M R R Mohan and A Narayana Rao 




Figures 4-6. 4. Circular gliding pattern of a mixed population of B. alba and B. 
leptomltiformls trichomes on modified Cataldi's agar medium surface (x 860). 
5. Octahedral crystals of sulphur outside the trichomes treated with aceto-caramine 
(x 1940). 6. Visual colony growth &i Beggiatoa spp. of site 7 in enrichment culture 
media containing a Unaltered site water (good growth), b Filtered site water 
(good growth), c Tap water (poor growth), d Distilled water (poor growth), 
e Sea water (nil growth) ; the white sctrm contained no Beggiatoa. 



Distribution and growth of Beggiatoa 171 

0-016ppt salinity, or sea water of pH 7 and 35 ppt salinity. The results are 
shown in table 3 and figure 6. 

In these cultures of samples from site 8 Beggiatoa was not detected, confirming 
the above results that it is not occurring at this site. In the remaining cultures 
of samples from 1 1 sites, the Beggiatoa spp. listed in tables 1 and 2 were again 
observed. In general, by 6, 8 10 and 15 days after incubation clear visual 
white colonies described above also appeared in the culture media favourable for 
growth of Beggiatoa from the sites 1, 4, 6 and 7 respectively (figure 6). On the 
other hand, in the remaining cultures, whichever was favourable for growth, 
Beggiatoa trichomes except those of B. mirabilis and B. gigantea increased in 
population between 8 and 15 days after incubation and aggregated into loose 
clumps in the white film that was formed at the surface of water and glass. The 
microscopic examination of colonies or loose clumps showed actively gliding 
trichomes filled with sulphur droplets. It appears that enough H 2 S is produced 
in the media due to putrefaction of sulphur containing proteins that were left in 
the extracted rice straw pieces, and supported the growth of Beggiatoa. Further, 
oxidation of H 2 S must have taken place, and probably organic nutrients were 
also utilized for growth. 

Regarding B. mirabilis and B. gigantea, the trichomes as in the previous enrich- 
ment cultures aggregated by one day after incubation on rice bits, surface of glass, 
and at the surface of medium with site waters or sea water only. They remained 
for 3 days in active gliding state ; thereafter started declining, and completely 
lysed and dissolved by 7th day after incubation revealing that they failed to grow 
in the media. Whereas in tap water and distilled water media the trichomes did 
not even appear either on rice bits, or on the surface of glass, or at the surface of 
water ; indicating that they lysed within one day after incubation. Further, 
when the trichomes of these two species were suspended IB tap water, or distilled 
water, or sea water, or their site water for microscopic examination, those suspended 
in tap water, or distilled water have bulged and burst releasing colloidal mass of 
cytoplasm and sulphur droplets. This confirms the earlier observations by 
Lackey (1961), Pringsheim (1964) and others that they are restricted to brackish 
or marine habitats and appear to be strictly autotrophic and rather delicate and 
exacting. These two largest species, hence, have not yet been grown successfully 
either in crude enrichment cultures or in pure cultures by anybody. 

The remaining Beggiatoa spp. of each site, as shown in table 3, could grow in 
enrichment culture media, but their amount of growth was different in diiferent 
media, ranging between good to nil growth. Further, the lag period was also 
different. Where the amount of growth was good and fair, recognizable growth 
occurred by 5 to 8 days ; increased steadily till 20 to 25 days after incubation 
and thereafter declined. On the contrary, where it was poor and very poor recog- 
nizable growth appeared by 10 to 15 days ; increased very slowly till 20 to 25 
days after incubation ; and afterwards declined. Thus, these differences in the 
growth of Beggiatoa spp. in different enrichment culture media suggest that 
strains or groups differing in salt tolerance may exist within a species. B. minima 
occurs in 2 sites, a. freshwater site 1 and a marine site 12. The nil growth in sea 
water medium of that occurring in the fresh water habitat indicates that it has a 
low salt tolerance. Whereas the growth, though very poor, in filtered site water, 
sea water and tap water media of that occurring in the marine habitat shows that 



1.72 M R R Mohan and A Narayana Rao 

it is indifferent to variation in salt content, and has a wide salt tolerance. Further, 
the nil growth of this marine form in distilled water medium may be due to lack of 
enough mineral salts for its growth in the medium. 

B. leptomitiformis is distributed in 6 sites ; 3 freshwater sites (1, 3 and 5), 
2 brackish water sites (9 and 10), and 1 marine water site (11). The forms occurring 
in freshwater habitats failed to grow in sea water medium, indicating that they 
have a low salt tolerance ; and seem to be restricted to freshwater habitats only. 
On the other hand, of the two occurring in brackish water habitats, the one from 
site 9 of a stagnant brackish water pond failed to grow in sea water medium, 
whereas the other from site 10 of one of the harbour channel terminals near 
sulphur-unloading berth could grow in all the media indicating that the former 
has a low salt tolerance and the latter has a wide salt tolerance. On the contrary, 
that occurring at site 11 of the sea water canal carrying effluents from the Hindustan 
Petroleum Oil Refineries by its good, fair, poor and nil growth in site waters, tap 
water, sea water and distilled water media respectively, shows that it is indifferent 
to variation in salt content, and can thrive well if enough mineral salts were 
available for its growth. Thus, these B. leptomitiformis forms, though similar 
IB appearance and morphology, may be different physiologically ; the 3 occurring 
in sewage containing freshwater habitats formed one group ; the two occurring 
in sewage containing brackish water pond, and oil containing and probably sulphur- 
rich harbour channel terminal formed two distinct groups ; and the one occurring 
in sea water canal carrying effluents from the oil refineries formed another group. 

B. alba of 3 32 // wide is distributed in 1 1 sites, suggesting that it is ubiquitous 
in distribution. The forms occurring in freshwater habitats, sites 1 1o 5, by their 
nil growth in sea water seem to be restricted to fresh water habitat, and have a 
low salt tolerance. It appears that physiologically these may belong to one group. 
The one from site 6 of a sewage stream that joins the harbour channel receiving 
sea water during high tides, by its nil growth in tap water or distilled water, and 
poor growth in sea water media seems to be possessing high salt tolerance. Further, 
it appears to belong to a separate group. On the other hand, those occurring in 
sites 7 and 9 exhibited nil growth in sea water medium only. It appears that 
these two could not tolerance high salt content and prefer lower levels, and may 
belong to one group physiologically. Whereas that occurring in site 10, a hard 
brackish water habitat, by its growth in all the media indicates that it is indifferent 
to variation in salt content, and has a wide salt tolerance. Most likely it may 
belong to another group. And those occurring in sites 11 and 12 truly of marine 
habitats, showed growth in all the media, except in distilled water medium. It 
shows that these two are also indifferent to variation in salt content with, however, 
a preference for at least some amount of mineral salts for growth, and may belong 
to one group. Thus, B. alba forms distributed in the 11 sites, though similar 
in appearance and morphology are different in salt tolerance, and may belong 
physiologically to 5 different groups. 

The B. alba strain with 4-98 p wide trichomes iis distributed in two sites, 4 and 7* 
Their nil growth in sea water medium indicates that they have a low salt tolerance. 
Further, the poor and nil growth in tap water and distilled water respectively by 
the one from site 4, and very poor growth in tap water and distilled water by the 
other from site 7 indicate that the two may be different physiologically. 



Distribution and growth of Beggiatod 173 

' The remaining widest forms of Beggiatoa, viz., B. arachnoidea, B. mirabilis and 
B. gigantea are detected in only one site (10). Of these, B. arachnoidea &lo&e could 
grow both in tap water and sea water indicating that it is indifferent to variation 
in salt content. However, its nil growth in distilled water may be due to lack 
of enough mineral salts in the medium. On the contrary, B. mirabilis and 
B. gigantea by their nil growth in any of the media seem to require more exacting 
conditions for growth in culture ; halophilic, and strictly autotrophic. 



4. Conclusion 

The present study reveals that Beggiatoa is widespread in distribution ; B. minima, 
B. leptomitiformis and B. alba are ubiquitous occurring in both freshwater and 
marine habitats ; B. arachnoidea, B. mirabilis and B. gigantea are restricted to 
brackish and marine habitats ; and conditions supporting the growth of these are 
prevailing in 11 sites, out of the 12 investigated. Further, the enrichment culture 
medium containing extracted rice straw bits and the respective site waters proves 
to be a reliable and satisfactory medium for increasing the small number of Beggiatoa 
trichomes into a rich population, which is a prerequisite for isolation and culti- 
vation in pure cultures. Although the species of Beggiatoa identified are considered 
as belonging to the six species described in Bergey's manual according to the 
usual nomenclature, the occurrence of Beggiatoa of identical diameters both in 
freshwater and marine habitats ; and the differences in growth in rice straw 
medium with different waters of varying salt content suggest that width is not 
the sole feature to be taken into consideration for species differentiation, substan- 
tiating the views of Pringsheim (1964) and others. That physiological groups 
exist within the smaller forms of Beggiatoa is also confirmed, though based only 
on growth in crude enrichment culture media. However, there is every need for 
isolating and cultivating these forms in pure culture media for further detailed 
consideration of species differentiation, and their nutrition. 



Acknowledgements 

The award of a fellowship by the Council of Scientific and Industrial Research, 
New Delhi, to one of the authors (MRRM) is duly acknowledged. 



References 

Buchanan R E and Gibbons N E 1974 Sergey's manual of determinative bacteriology (Baltimore : 

William and Wilkins) pp. 1416 
Cataldi M S 1940 Aislamiento de Beggiatoa alba en cultivo puro ; Rev. Inst. Bacteriol. (D.N.ff.) 

9 393-423 

Ellis D 1932 Sulfur bacteria (New York : Longmans, Green) p. 21 
Faust L and Wolfe R S 1961 Enrichment and cultivation of Beggiatoa alba ; J. Bacteriol. 81 

99-106 
Jorgensen B B 1977 Distribution of sulfur bacteria (Beggiatoa spp.) in coastal marine sediment ; 

Mar. Biol (Berlin) 41 19-28 



174 M R R Mohan and A Narayana Rao 

Joshi M M and Hollis J P 1977 Interaction of Beggiatoa and rice plant : Detoxification of 

hydrogen sulfide in the rice rhizosphere ; Science 195 179-180 
Keil F 1912 Beitrage Zur Physiologic der forblosen Schwefelbakterien ; Beitr. Biol. Pflanzen. 

11 335-372 
Kowallik U and Prmgsheirn E G 1966 The oxidation of hydrogen sulfide by Beggiatoa ; 

Am.J. Bot. 53801-806 
Lackey J B 1961 Occurrence of Beggiatoa species relative to pollution ; Water and Sewage 

Works 729-31 
Pitts G, Allarn A I and Hollis J P 1972 Beggiatoa : Occurrence in the rice Rhizosphere ; 

Science 178 990 

Pringsheim E G 1946 Pure cultures of algae (London : Cambridge University Press) 
Pringsheim E G 1964 Heterorrophism and species concepts in Beggiatoa ; Am. J. Bot. 51 

898-913 
Scotten H L and Stakes J L 1962 Isolation and properties of Beggiatoa ; Arch. Mikrobiol 42 

353-368 

Winogradsky S 1887 Uber Schwefelbakterien ; Bot. Z. 45 489 tT. (Nos 31-37) 
Wlnogradsky S 1888 Beitrage Zur Morphologic Und Physiologic der Bakterien Heft. 1. 

Schwefelbakterien. Leipzig : Felix 
Winogradsky S 1889 Recherches physiologiques surles sulfobacteries ; Ann. Inst. Pasteur. 

349-60 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 3, June 1982, pp. 175-181. 
Printed in India. 



Photoperiodic control of extension growth, bud dormancy and 
flowering of Nerium indicum Mill, and Thevetia peruviana Schum. 



KUSHAL SINGH, SURINDER KUMAR and K K NANDA 

Department of Botany, Panjab University, Chandigarh 160 014, India 

MS received 12 November 1981 

Abstract. Plants of N. indicnm and T. peruviana grew taller and produced more leaves 
under LD than under ND condition. While T. peruviana plants were taller and had 
more leaves under ND than under SD, those of N. indicum did not differ under the two 
photoperiods. In both cases LDS delayed the onset of bud dormancy but hastened the 
initiation of floral buds. While in T. peruviana floral buds were not formed under SD 
condition, in N. indicum floral buds were formed but they did not develop into 
flowers. While in N. indicum more flowers were produced under LD than under 
ND condition, in T. peruviana the number produced was higher under ND than under LD 
condition. 

Keywords. Photoperiod; extension growth; bud dormancy; flowering; Nerium indicum; 
Thevetia peruviana. 



1. Introduction 

Studies on the effect of photoperiod on extension growth, bud dormancy and 
flowering of woody species have shown that in general while long days prolong the 
period of extension growth and therefore delay the onset of dormancy, short days 
hasten the onset of rest (Nanda 1963; Whalley and Cockshull 1976; Bhatnagar and 
Talwar 1978; Singh and Nanda 1981). The floral response, however, varies with 
the plant species. Thus, the young seedlings of Cojfea arabica produce floral buds 
under short day condition (Piringer and Borthwick 1955) while in Hydrangea 
(Piringer and Stuart 1955) and Mains hupehensis (Zimmeraann 1971) floral buds 
are induced under long day conditions. Davidson and Hamner (1957) have reported 
that although long days induce floral buds in Rhododendron catawbiense, short days 
are needed for their development into flowers. In contrast to this, Mirov (1956) 
has reported that photoperiod does not affect the flowering response of 35 exotic 
pines. This paper deals with the effect of photoperiod on extension growth, bud 
dormancy and flowering of two garden plants, Nerium indicum and Thevetia 
peruviana. 



176 Kushal Singh, Surinder Kumar and K K Nanda 

2. Materials and methods 

Plants of Nerlum indicum Mill, were raised by planting one-year old stem cuttings 
(15 cm each), while those of Thevetia peruviana Schum. were raised from the seed 
collected locally from a healthy tree growing in the Panjab University Campus. 
The seed was sown on February 1, 1980 in 3:1 mixture of field soil and sand in 
earthenware pots (25 cm dia) under three photoperiodic regimes namely; long day 
(LD) - consisting of continuous illumination which was provided by supplementing 
the normal day-length by unfiltered 200 watt light from incandescent lamps which 
provided light intensity of about 3000 lux at the level of the plants; normal day 
ND - consisting of natural day-length conditions ; prevailing at Chandigarh 
(figure 1) and short day (SD) - consisting of 8 hr daily light alternating with 16 hr 
dark which was provided by covering the plants with thick tarpaulin sheets daily 
from 1700 hr to 900 hr which led to the rise in temperature in the range of 3-6C 
throughout the course of experimentation. To ensure healthy growth, Hoagland's 
nutrient solution (Hoagjand and Arnon 1939) was supplied to the plants twice a 
week during the course of experimentation from April 1980 to June 1981. 



20- 



o. 

01 

I 

to- 



MAXIMUM TEMPERATURE 

MI'MMUM TEMPERATURE 




ISi 

I* 



10 



I i I I ! i I 



APRJMAYjJUNjjTlTlAUG^ 

1980* ' ' 1981 ' 

TIME (MONTH) 



Photoperiodic control of extension growth ... 



177 



Observations of extension growth were recorded at 14-day intervals whereas the 
number of leaves was counted daily. Records were also maintained of the dates of 
initiation of floral buds. The number of floral buds produced and the time taken 
for them to open into flowers were also recorded. The 'period of dormancy' in 
this paper refers to the period during which the shoot apex remained apparently 
inactive and no new leaves were produced on the plant. 
3. Observations 
The results are presented diagrammatically in figures 2 and 3 and table 1 . 

150i 



130- 



120- 



110- 



100- 



T 
4- 

T 



o 

S! 80- 



en 
1 



: 70- 


50- 

40- 
30- 

20- 
10- 



N. indicum 



i 



I I HEIGHT 

NUMBER OF LEAVES 






10 NO SO 



CO NO SO 



Figure 2. Effect of photoperiod on height of the main axis and number of leaves 
produced on N. indicum and T. peruviana. 



178 



Kushal Singh, Surinder Kumar and K KNanda 



glS23 PERIOD OF FLORAL euo INITIATION 
I 1 PERIOD OF ACTIVE GROWTH 
E23 PERIOD OF BUD DORMANCY 



T. peruviano 



SD 



ND 



^^ 



LD 



?^^vj 



N- indicum 



SD 



HiMWWW^^ 



ND 



*~vmw^^ 



LD 



o : so: hoo j 150 : 200; ?so 

APR jMAYljUNlJUL jAUGjSEPjOCT JNOVJOEC 



19*0 
TIME 



360 ; 3$o i A%o i 4io 

JANjFEBjMARiAPRiMAY; JUN 



1981 



NUMBER OF DAYS 



Figure 3. Effect of photoperiod on periods of active growth and bud dormancy in 
N. indicum and T. pemviana. 



Table 1. Effect of photoperiod on flowering of N. indicum and T. peruviana. 



Photoperiod Days to floral Number of floral Days taken to Number of Number of plants 
bud initiation buds flower opening flowers out of 10 that pro- 
duced floral buds 








N.vindicum 






LD 


190.52.50 


70.82.81 


314.4+2.81 


17.51.71 


10 


ND 


315.23.40 


35.63.10 


356.74.31 


10.53,81 


10 


SD 


340,04.21 


3.01.05 








10 








T. peruviana 






LD 


340.53.42 


6.1 2.40 


360.84.21 


4.52.85 


10 


ND 


320.75.16 


18.73.08 


348.93.91 


10.61.31 


10 


SD 


















SE at 95% level of significance 



3.1 Extension growth and number of leaves 

Figure 2 shows that plants of both the species grew taller and produced more 
leaves under LD than under ND and SD conditions. While in N. indicum the height 



Photoperiodic control of extension growth .... 179 

and number of leaves produced under SD condition did not differ from that 
produced under ND condition, in T. peruviana plants remained significantly shorter 
and produced fewer leaves under SD than under ND condition. 



3,2. Periods of growth and bud dormancy 

In N. indicum, plants continued growth till mid November under LD as compared 
to that till early October and mid September under ND and SD conditions, 
respectively. The period of rest that followed also lasted till early February under 
LD but till mid February and early March under ND and SD conditions, respecti- 
vely (figure 3). 

In T. peruviana, growth continued till early February ui^der LD condition but lasted 
till the end of December and October under ND and SD conditions, respectively. 
The period of rest that followed and which lasted till mid-February and early 
February under ND and LD conditions, respectively, continued till early March 
under SD condition. 

It may be noted that in both the species the period of rest was shorter under LD 
but longer under SD than under ND condition (figure 3). 



3.3. Days to floral bud initiation 

In TV. indicum, floral bud initiation in plants under LD condition started in early 
October prior to the onset of dormant phase. In contrast to this under ND 
condition floral buds were initiated in mid February. Floral bud formation under 
both these conditions continued till the end of June when the experiment was termi- 
nated. In contrast to this, the formation of floral buds under SD condition was 
delayed to early June. Floral bud initiation, thus, occurred earlier under LD but 
later under SD than under ND condition (figure 3). 

In T. peruviana floral bud initiation was observed immediately after the period of 
rest in plants exposed to LD or ND conditions so that it occurred earlier under ND 
than under LD condition (table 1). Plants maintained under SD condition did not 
produce floral buds. It may be noted that the hastening effect of LDs was 
markedly more in N. indicum than in T. peruviana. 



3 .4. Days to floral bud opening 

Although LDs hastened the initiation of floral buds, the initiated buds in N. indicum 
took longer to develop into flowers under LD than under ND condition. Floral 
buds produced under SD condition in this species did not develop into flowers at 
all. In contrast to this in T. peruviana while the initiation of floral buds occurred 
earlier under ND condition, their development was hastened under LD condition 
(table 1). As stated earlier, plants of this species did not produce floral buds 
under SD condition. 



180 Kushal Singh, Surinder Kumar andK K Nanda 

3.5. Number of floral buds and flowers 

Table 1 shows that while in N. indicum the number of floral buds and flowers was 
higher under LD than under ND condition, in T. peruviana it was higher under ND 
than under LD condition. As stated earlier, in T. peruviana no floral buds were 
produced under SD condition and in N. indicum a few floral buds which were 
produced did not develop into flowers. 

4, Discussion 

The early onset of dormant phase under SD condition in plants of both the species 
reported in this paper is in accord with the results reported earlier in some other 
plants (Nanda 1963; Whalley and Cockshull 1976; Bhatnagar and Talwar 1978; 
Singh and Nanda 1981). Hastened onset of rest period in plants under SD 
condition may be due to accumulation of some growth inhibitory substance (s) 
(Wareing and Saunders 1971) or to a decrease in photosynthates due to reduced 
daily light period. 

The fact that in N. indicum floral buds are produced even under SD condition, 
while in T. peruviana they are not produced under this photoperiod shows that 
while the former is quantitative, the latter is qualitative long day in its response to 
photoperiod. 

But the more interesting point is that in N. indicum although floral buds, are 
produced, they fail to develop into flowers showing that the photoperiodic require- 
ment for the completion of these two phases i. e. 9 (i) induction of floral buds and 
(ii) their development into flowers, may not be the same. That the requirement of 
these two phases may vary is shown in soybean (Jindal and Nanda 1978) and 
Bauhinia acuminata (Singh and Nanda 1981). It cannot, however, be ruled out that 
failure of buds of this species to develop into flowers may be due to the limitation 
of photosynthates under SD condition. This is particularly in the light of work of 
Ramina et al (1979) and Even-Chen and Sachs (1980) who reported that photo- 
synthetic availability influences the flowering intensity in Bougainvillea. 

Acknowledgement 

This work was supported by a grant (HCS/DST/2/76) from the Department of 
Science and Technology, Government of India and the Council of Scientific and 
Industrial Research, New Delhi. 

References 

Bhatnagar H P and Talwar K K 1978 Photoperiodic response of growth of Pinus caribaea 
seedlings. I. Effect on stem height and diameter and tracheid characters. Indian For. 
104 212-226 

Davidson H and Hamner C L 1957 Photoperiodic responses of selected woody ornamental 
shrubs. Mich. Exp. Stat. Quart. Bull. 40 327-343 



Phot aperiodic control of extension growth .... 181 

Even-Chen Z and Sachs R M 1980 Photosynthesis as a function of short day induction and 

gibberellic acid treatment in Bougainvillea "San Diego Red". Plant Physio! . 65 65-68 
Hoagland D R and Arnon D I 1939 The water culture method for growing plants without soil. 

Calif. Agric. Exp. Stat. Cir. 347 
Jindal R K and Nanda K K 1978 Effect of time of sowing on flowering of some varieties of 

soybean (Glycine max) under varying photoperiods; in Physiology of Sexual Reproduction in 

Flowering Plants, (eds) C P Malik, N C Bhattacharya, A K Srivastava and Rattan Singh 

(New Delhi: Kalyani Publishers) pp. 148-151 

Mirov N T 1956 Photoperiod and flowering in pines. For. Sci. 2 328-332 
Nanda K K 1963 Studies on growth and development of forest trees. I. Effect of photoperiod 

on annual growth cycle of seedlings of some Indian species. Indian J. Plant PhysioL 6 14-33 
Piringer A A and Borthwick H A' 1955 Photoperiodic responses of coffee Turrialba 5 72-77 
Piringer A A and Stuart N 1955 Responses of Hydrangea to photoperiod. Proc. Am. Soc. 

Hortic. Sci. 65 446-454 
Ramina A, Hackett W P and Sachs R M 1979 Flowering in Bougainvillea. A function of 

assimilate supply and nutrient diversion. Plant PhysioL 64 810-813 
Singh K and Nanda K K 1981 Effect of photoperiod on extension growth, rest period and 

flowering of Bauhinia acuminata L. seedlings. Part I - Analysis of the first year growth. 

Indian J. Exp. Biol. 19 337-340 
Wareing P F and Saunders P F 1971 Hormones and dormancy; Ann. Rev. Plant PhysioL 22 

261-288 
Whalley D N and Cockshull K E 1976 The photoperiodic control of rooting, growth and 

dormancy in Cornus alba L.; Sci. Hortic. 5 127-138 
Zimmermann R H 1971 Flowering in crabapple seedlings. Methods of shortening the juvenile 

phase. /. Am. Soc. Hortic. Sci. 96 404-411 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 3, June 1982, pp. 183-188. 
Printed in India 



Interaction of kinetin with B group vitamins on the seedling growth 
of green gram (Phaseolus radiatus L.) 



P GOPALA RAO and J KODANDARAMAIAH 

Department of Botany, Sri Venkateswara University, Tirupati 517 502, India 

MS received 17 November 1981 ; revised 13 May 1982 

Abstract. Kinetin (50 and 100 mg 1 inhibited both the shoot and the root growth. 
Inhibition of root growth by kinetin is considered to be mainly due to inhibition 
of protein synthesis. Vitamins of the B group v/z., riboflavin, thiamin, niacin and 
pantothenic acid are found to be antagonistic to kinetin in reversing the inhibition 
of protein synthesis of the root. Vitamins, probably by acting as inducers of protein 
synthesis, antagonized the action of kinetin. The response of kinetin to shoot protein 
content is different from that of the root. 

Keywords. Kinetin ; B-vitamins ; root protein ; green gram. 

1. Introduction 

Our knowledge of cytokinins and their possible functions in root growth 
is extremely limited. That cytokinins inhibit root growth was shown by Gaspar 
and Xhaufflaire (1967). But how they inhibit root growth was not categorically 
explained. Hussain et al (1980) working on nutsedge with vitamins of B group 
and kinetin have noticed that riboflavin and pyridoxin up to 100 mg I"" 1 promoted 
root and shoot growth of the plantlets, whereas kinetin produced short, thick 
shoots and inhibited root growth, with increasing concentration. 

The present study has been designed to understand the interaction of vitamins 
and cytokinins which might throw some light on the growth of root and shoot 
systems of intact seedlings. Only inhibitory concentrations (50 and 100 mg I"" 1 ) 
of kinetin were used in order to find out the efficacy of vitamins to counteract the 
influence of kinetin. Earlier report by Gopala Rao et al (1975) indicated that 
riboflavin can effectively reverse the chloramphenicol inhibited growth of green 
gram seedlings. It is a well-known fact that chloramphenicol is a potent inhibitor 
of protein synthesis. Interaction of auxins and riboflavin in growth reactions in 
plants was studied by Artamonov (1974). The present study is intended to 
find out the capacity of vitamins to counteract the effect of kinetin in shoot or root 
growth inhibition. 

2. Materials and Methods 

Seeds of green gram (variety G.G 525) were surface sterilized with 0.1% mercuric 
chloride for 3 min, washed thoroughly with distilled water and allowed to grow 

183 



184 



P Gopala Rao and J Kodandaramaiah 



in petridishes. The seeds were subjected to presowing soaking for 24 hr with 
kinetin (50 and 100 mg I- 1 ) and vitamins of the B group viz., riboflavin, 
thiamine, niacin and pantothenic acid, each at a concentration of 100 mg I"" 1 
since it was found to be effective in reversing the kinetin inhibited protein 
synthesis. Then the seeds were allowed to grow in distilled water in diffuse light 
in the laboratory up to 8 days. Growth in length of the seedlings was measured 
for shoot and root separately. Ten replications, each replications representing 
five seedlings were maintained for growth measurements. The protein content 
was estimated separately in the shoot and the root portions using the method 
of Lowry et al (1951) at 2 day intervals. Three replications were maintained 
for protein estimations. 

3. Observations 

3.1 Extension growth 

With kinetin (50 and 100 mg I- 1 ) treatment both the root and shoot growth were 
inhibited (table 1). The results discussed pertain to 100 rng I"" 1 concentration 
alone. On the eighth day, for example, the root growth of control seedlings was 
8.0 cm while that of kinetin treated';; seedlings was 35cm with 50 mg I"" 1 and 
1.03 cm with 100 mg I- 1 respectively. The shoot growth of control seedlings was 
17. 5 cm while that of kinetin treated seedlings was 16.1cm with 50 and 0.8cm 
with 100 rng I- 1 respectively (table 1). 

Table 1. Effect of kinetin on growth (cm) and protein content (mg/g. dry wt.) of the 
seedlings. 



Treatment Observation 


Part of 
the 
seedling 


Age of seedling 


2 days 


4 days 


*6 days 


8 days 




Root 


2.55 


6.46 


760 


8.00 


Growth 




0.05 


091 


0.66 


0.65 




Shoot 


1.30 


9.25 


9.75 


17.50 


Control 




0.03 


0.27 


0.81 


0.41 




Root 


109.5 


124.5 


117.0 


82.5 


Protein 




7.6 


7.3 


9.1 


1.8 




Shoot 


124.5 


172.5 


127.0 


90.0 






1.9 


3.8 


1.8 


2.6 




Root 


0.55 


0.70 


1.13 


3.45 


Growth 




0,03 


0.02 


0.04 


0.25 




Shoot 


0.63 


253 


2.63 


16.15 


Kinetin 




0.03 


0.07 


0.03 


029 


(50 mg I-') 


Root 


153.0 


96.5 


90.0 


70.5 


Protein 




1.63 


1.51 


0.87 


0.88 




Shoot 


208.5 


182.0 


159.0 


102.0 






2.91 


4.15 


1.35 


2.01 




Root 


0.43 


0.6 


0.8 


1.03 


Growth 




0.06 


0.08 


0.09 


0.04 




Shoot 


0.6 


2.53 


430 


8.03 


Kinetin 




0.09 


0.07 


0.46 


0.21 


(100 mg l~i) 


Root 


109.5 


60.0 


51.0 


45.0 


Protein 




1.25 


0.62 


0.57 


0.69 




Shoot 


187.5 


185.5 


163.5 


79.5 






3.56 


2.25 


1.79 


1.27 



Seedling growth of green gram (Phaseolusradiatuis L.) 



185 



3.2 Protein content 

The root protein content was reduced with both concentrations of kinetin (50 and 
lOOmgl- 1 ). There was only an initial increase with 50 mg I- 1 kinetin on the 
second day of seedling growth. On the eighth day, for example, the root pro- 
tein content of control seedlings was 82.5 mg while that of kinetin treated seedlings 
was 70.5 mg with 50 mg l^ 1 and 45.0 mg with 100 mg I-" 1 respectively (table 1). 

It is tempting to note that the shoot protein content was enhanced with kinetiu 
(100 nig I- 1 ) except on the eighth day (table 1). 

3.3 Interaction of kinetin (100 mg 1~~*) with vitamins on protein content 

There was about 50% reduction in root protein content with kinetin treatment 
when compared to that of control seedlings (from 80 mg to 40 mg) on the eighth 
day. Although there was an initial raise with riboflavin treatment up to fourth 
day, it was followed by a steep fall during later stages. The interaction of 
kinetin with riboflavin enhanced protein content of the root from 40 mg to about 
140 mg on the eighth day (250%) or a 3 fold increase. Thiamine, niacin and 
pantothenic acid could raise the protein level from 40 mg to 80 mg not exceeding 
the control level when they interacted with kinetin (figure 2) . 



5240 

>s 

C. 

Q 



160 



z 

Ul 
H- 

o 
o 



o 

(X 
Q. 



80 



40 



SHOOT 

O CONTROL 
A RIBOFLAVIN 
KINETIN 

RlBOFtAVtN-*- 
KINETIN 




O 
A 



A 



SHOOT 

CONTROL 
TH/AMINE 
KfNETlN 
THIAMJNE-f 




68 
AGE OF THE 



024 
SEEDUNOS IN OAY$ 



8 



Figure 1A. 



186 



P Gopala Rao and J Kodandaramaiah 



$240 



200 

t 

JTI60 

z 

LU 

g 120 

U 



80 



o 
cr 
CL 



40 



SHOOT 

O CONTROL 

A NIACIN 

O KINETIN 

A NIACIN 4- KIN&TIN 




SHOOT 

CONTROL 

NJACIN 

KINETIN 

PANTOTHENIC AGIO + 
KINETIN 




JL 



AGE 



68 024 

OF THE SEEDLINGS IN DAYS 



Figure 



la the case of shoot, riboflavin (figure 1A) increased the protein content from 
a value of 124.5 mg to 172.5 mg (38%) on the second day and from "a value of 
90 mg to 105 mg (16%) on the eighth day. Thiamine also increased the protein 
content of the shoot. Niacin and pantothenic acid (figure IB) could not increase 
the protein as effectively as the former two vitamins except on the second day. 
As opposed to the response of the root, shoot protein content was not reduced by 
kinetin, but instead, increased up to the sixth day at least. The interaction of 
riboflavin with Jkinetin caused a significant increase in protein content of the shoot. 
Thiamine interaction with kinetin caused an initial increase only, followed by a 
gradual reduction. Interaction of pantothenic acid with kinetin was quite similar 
to that <5f thiamine. Interaction of niacin with kinetin was almost similar to that 
of riboflavin in that it caused an additive effect in increasing the protein content of 
the shoot (figure IB). The behaviour of riboflavin and niacin on the one hand 
and that of thiamine and pantothenic acid on the other hand were found to be 
similar in their interaction with kinetin. 



4. Discussion" 

Kinetin at 50 and 100 mg I- 1 is "inhibitory to both the shoot and the root growth. 
One of the main causes of root growth inhibition (table 1) by kinetin is the reduc- 
tion in protein content. Since all the B vitamins used in the present study were 
able to effectively reverse the inhibition of protein synthesis by kinetin, it might be 



Seedling growth of green gram (Phaseolus radfatus L) 



187 



O 
cr* 



Ul 
I 

z 
o 
(J 

z 

UJ 
I 

o 
a 
a 



200 

160 

120 

80 

40J 

o! 



160 
120 
80 
40 



ROOT 

CONTROL 

& KINETIN 
A RIBOFLAVIN 
- O RIBOFLAVI.M 4 KINETIN 




CONTROL 



A NIACIN 

O WIACIN + KINETIN 




ROOT 

CONTROL 

4 KINETIN 

A THIAMINE 

O THIAMINE + KINETIN 




CONTROL 
A KINETIN 

A PANTOTHENIC ACID 
O PANTOTHENIC ACID + 
K'lNETIN 




2 4 6 8 24 6 
AGE OF THE SEEDLINGS IN DAYS 



Figure 2. 



assumed that vitamins increase protein synthesis by operating either at transcription 
or translation level. This assumption can be corroborated by the observation made 
by Srivastava (1967) that cytokinins can also inhibit RNA synthesis. A consequence 
of the inhibition of RNA synthesis is inhibition of protein synthesis. Gopala Rao 
et al (1975) earlier reported enhanced protein synthesis by B vitamins In green 
gram. It is a well-known fact that chlorarnphenicol is an eflfective inhibitor 
of protein synthesis. Gopala Rao et al (1976) observed that riboflavin can effec- 
tively reverse the chloramphenicol inhibited growth of green gram. Basing on 
these observations it can be assumed that a probable site of action of vitamins of 
the B group could be at transcription or translation level. 

The present study also reveals that the response of kinetin to root protein might 
be different from that of shoot protein since the root protein content was signifi- 
cantly reduced and that of the shoot was significantly enhanced. The present 
study forms the basis for future work on the role of vitamins at molecular level. 



1&8 P Gopala Rao and J Kodandaramaiah 

Acknowledgements 

The authors are grateful to Prof. V S Rama Das for his encouragement and 
suggestions. One of the authors (JK) is highly thankful to the UGC for providing 
financial assistance. 

References 

ArtamonovV 1 1974 Interact ion of auxins and riboflavin on growth reactions of plants Dokl 

AkadNauk SSSR Ser Biol 210 978-981, Biol.Abst. 57 68993 
Gaspar T and Xhaufflair A 1967 Effect of kinetin on growth, auxin catabolism, peroxidase and 

catalasc activities Planta. 72 252-257 
Gopala Rao P, Nagi Reddy A and Raja Kumar N 1975 Reversal of chloramphenicol inhibited 

growth by riboflavin in green gram Curr. Sci. 44 399-400 
Gopala Rao P, Nagi Reddy A and Raja Kumar N 1976 U C incorporation into amino acids in 

chloramphenicol inhibited growth and its reversal by riboflavin in green gram 

Z. Pflanzenphysiol. 80 279-282 
Lowry O H, Rosenbrough N J, FarrAL and Randall R J 1951 Protein measurement with 

folin phenol reagent ; Biol Chem. 193 265-275 
Srivastava BIS 1967 Effect of kinetin on nucleic acid synthesis in barley leaf segments ; 

Biochim. Biophys. Ada 145 166-169 



Proc. Indian Acad. Sci, (Plant Sci,) Vol. 91, Number 3, June 1982, pp. 189-20C). 
(8) Printed in. India. 



Leaf architecture of Apocynaceae 



J S S MOHAN and J A INAMDAR 

Department of Biosciences, Sardar Patel University, Vallabli Vidyanagar 388 120, 
Gujarat, India. 

MS received 16 May 1981 ; revised 12 April 1982 

Abstract. Leaf architecture including venation pattern has been studied in 19 genera 
and 29 species of the Apocynaceae. The leaves are simple, alternate, opposite or 
\vhorled with entire margin and a simple midrib. The major venation pattern 
conforms to pinnate camptodromous type with festooned brochidodromous seconda- 
ries. The qualitative and quantitative features are charted. The leaf size, areolc 
size, number of vein endings entering the areoles and number of vein terminations 
entering the areoles vary from species to species even within the same species. The 
highest degree of vein order is observed up to 7. Isolated tracheids, isolated vein 
endings, isolated free vein endings and tracheoidal elements are noticed. Bundle 
sheath cells ensheaths all category of veins. 

Keywords. Anatomy ; leaf architecture ; venation pattern ; Apocynaceae. 



1. Introduction 

Only sporadic information is available on the venation in some members of the 
Apocynaceae (Chandra et al 1969, 1972; Kapoor et al 1969; Sharma et al 1970). 
The leaf architecture of the Apocynaceae has not been studied in detail. The 
present investigation has been undertaken to give a comprehensive and detailed 
account of leaf architecture of the Apocynaceae. 



2. Materials and methods 

The material of the 19 genera and 29 species of Apocynaceae was collected from 
different places in Gujarat, Karnataka (Lalbagh, Bangalore) and Kerala States. 
The mature leaves were cleared following the procedure of Rao et al (19 80). 
Photomicrographs were taken with a Carl-Zeiss photomicroscope 1 using yellow 
filter and OR WO NP 15 film. Leaf size was measured using graph paper. The 
areole size, the number of veinlets entering in areole and the number of vein 
endings entering in areole were taken in five different fields of different leaves, and 
the average value was recorded. Terminologies to describe leaf architecture 
were adopted from Hickey (1973), Hickey and Wolfe (1975) and Melville (1976). 

189 



190 / S S Mohan and J A Inamdar 

Table 1. Showing the qualitative features and numerical data on the venation pattern 
of some Apocynaceae. 



SI. No. Name of the taxa 
1 2 


Locality 
3 


Shape 

4 


Apex 
5 


Base 
6 


Margin Texture 
7 8 


1. 


Aganosma caryo- 


local 


ovate 


acute 


obtuse 


entire charta- 




phyllata G. Don. 










ceous 


2. 


Allamanda cathartica 


Bulsar, 


ovate 


acumi- 


obtuse 


entire coria- 




Linn. 


Gujarat 




nate 




ceous 


3. 


A. nerifolia Hook 


Lalbagh 


oblong 


39 


> 


chart a- 






Bangalore 








ceous 


4. 


A. violacea Garden 


,, 


ovate 


acute 


acute 


,, coria- 




& Field 










ceous 


5. 


Alstonia scholaris 


Dangs, 


99 


99 


99 


99 99 




R. Br. 


Gujarat 










6. 


Alyxia pubescens 


99 





99 


obtuse 


chart a- 




Turril 










ceous 


7. 


Carissa carandas 


Kerala 


9) 


,, 


acute 


coria- 




Linn. 










ceous 


8. 


C. congesta Wight 


Dangs, 


,, 


obtuse 


obtuse 


charta- 






Gujarat 








ceous 


9. 


C. spinarum Linn. 


9t 


99 





99 


99 


10. 


Catharanthes major 


Kerala 


> 


19 


cordate 


" 99 




Linn. 












11. 


C.pusillus GDon. 


Bulsar, 





acute 


obtuse 


99 






Gujarat 










12. 


C. rosem G. Don. 


Local 





obtuse 


9> 


,, membra- 














naceous 


13. 


C. variegata 


99 


J 


99 


J9 


charta- 














ceous 


14. 


Cerbera manghas Linn. 


Lalbagh, 


oblong 


acute 


acute 


coria- 






Bangalore 








ceous 


15. 


C. odollum Gaertn & 


99 





obtuse 


99 


99 ff 




Bunt 












16. 


Chonemorpha macro- 


Lalbagh, 


wide 


obtuse 


obtuse 


entire coria- 




phylla G. Don. 


Bangalore 


ovate 






ceous 


17. 


Holanhena antidy- 


Dangs, 


ovate 


acute 


obtuse 


charta- 




sent erica G. Don. 


Gujarat 








ceous 


18. 


Kopsia fruticosa 


Kerala 


elliptic 


acumi- 


acute 


9> 




A. DC. 






nate 






19. 


Nerium indicum 


Local 


narrow 


acute 





coria- 




Mill. 




ovate 






ceous 


20. 


Parsonsia spiralis 


Kerala 


ovate 


acute 


obtuse 


charta- 




Vidal 










ceous 


21. 


Plumeria rubra Linn. 


Local 


ob ovate 


obtuse 


acute 


i, coria- 














ceous 


22. 


Rauwolfia serpentina 


Lalbagh, 


ovate 


acute 


obtuse 


, t charta- 




Benthe & Kurz 


Bangalore 








ceous 


23. 


R. tetraphylla Linn. 


99 


99 


99 


>9 


" 99 


24. 


Tabernamantana 


Local 


99 


acumi- 








divaricata R. Br. 






nate 




" 


25. 


Thevetia peruviana 


99 


needle- 


acute 


acute 


,, coria- 




(Pers.) K. Schum. 




shaped 






ceous 


26. 


Trachelosp ermum 
jasminoides Lem. 


Lalbagh, 
Bangalore 


ovate 





obtuse 


charta- 
ceous 


27. 


Vallaris solanacea 


Dangs, 


oblong 


acumi- 








(Roth.) O. K. 


Gujarat 




nate 




9t 


28. 


Wrtghtia tinctoria 


Lalbagh, 


ovate 


9) 








R. Br. 


Bangalore 








ft ft 


29. 


W. tomentosa 


Dangs, 


99 


acute 








Roem & Schutt 


Gujarat 






_C 


> 



Leaf architecture of Apocynaceae 



191 



Predomi- Marginal 
nant ultimate 
tertiary venation 
vein 
origin 
angle 
9 10 


Leaf 
area 
in mm 2 

11 


No. of 
2 veins 
along 
one side 
midrib 

12 


Range of 
angle 
between 
1&2 
veins 

13 


No. of No. of vein 
areoles/ veinlets ending 
mm 2 entering termina* 
in areole/ t ion/mm 1 
mm 2 

14 15 16 


RR, RA incomplete 


3870 


8-12 


50-65 


1 


11 


21 


AR, RO incomplete 


3635 


15-50 


60-85 


1 


8 


32 


RA, OR, 


2830 


12- 15 


50-75 


1 


8 


27 


00 














RR, OR, 


5290 


12- 16 


40-65 


1 


8 


56 


RA 














absent ,, 


3775 


35- 38 


65-70 


absent 








RA, RO, Firnbriate 


2565 


15- 18 


45-60 a 


1 


11 


18 


OR 














RO, AR, incomplete 


125 


8- 10 


25-40 


2 


8 


13 


RR 














AA, AR, 


1170 


7- 10 


70-85 


1 


22 


49 


RA 














AR, RA, 


965 


8- 10 


60-80 


1 


20 


37 


AO 














RR, RO, 


735 


8- 10 


45-75 


1 


9 


13 


RA 














RO, RA, 


815 


9- 12 


3S-55* 


1 


15 


22 


RR 














RR, RO 


935 


7-10 


250.450 











RR, RO 


760 


8 - 10 


50-75 


1 


10 


9 


RR, RA, 


4210 


15- 18 


45-60 


1 


10 


25 


RO 














RA, RO, 


4490 


16-20 


40~55 


1 


9 


22 


RR 














RR, RA incomplete 


12875 


20-25 


70-85 


2 


9 


19 


AR, RR, 


3180 


12- 16 


45~65 


2 


6 


19 


RA 














RA, AA, fimbriate 


8850 


18-22 


550.750 


3 


11 


45 


OA 














RR, RO, 


2460 


110- 125 


70-85 


6 


3 


1 


RA 














RR, OR incomplete 


5135 


14-16 


65-80 


1 


6 


31 


RO, RR, 


12015 


30-35 


35-55 


1 


9 


29 


RA 














OR, RR, 


2615 


12-15 


45-70 


1 


10 


40 


AR 














JR. A, jxibv i 


765 


9-12 


65-75 


1 


12 


41 


RR, AR, 


2135 


10-14 


35-65 


1 


14 


42 


RO 














AR, AO 


775 


15 - V 20 


30-45 


2 


14 


10 


RA, OR, 


715 


6-9 


60-80 


2 


5 


26 


RR 














RO, RR, 


2850 


12-15 


70-80 


1 


9 


33 


RA 














OR, RA, 


5365 


12- 15 


70-85 


2 


8 


17 


RR 














OR, RR, 


5530 


11-15 


60-80 


1 


9 


21 


RA 















192 J S S Mohan and J A Inamdar 

3. Observations 

3. 1 Morphological description 

Leaves simple, alternate, opposite or whorled. Shape ovate, narrow to w; 
ovate, obovate, oblong (figure 1 A), elliptic. Margin entire (figures 1 A, B, I 
Apex acute obtuse (figure 1 A) or rounded. Base acute, obtuse (figure 1 A) 
cordate. Texture chartaceous, membranaceous or coriaceous. The qualitative li 
features of the species studied are given in table 1. 

3 . 2 Major venation pattern 

The venation pattern conforms to pinnate camptodromous type with festoor 
brochidodrornous secondaries in which secondaries do not merge at the marj 
but upturn and join together in a series of prominent arches forming brochic 
dromous secondaries having a set of secondary loops outside the main brochic 
dromous and form "festooned brochidodromous" type of Hickey and Wolfe (19' 
or multiarcuate wherein secondary veins form a coarcuate infra-marginal v 
and breaking up into a series of small arching loops forming a zone between 1 
infra- marginal vein and the margin (Melville 1976) in all the species, except 
Catharanthus roseus where the venation pattern seems to be eucamptodromo 
In eucamptodromous venation pattern, secondaries upturned and gradua 
diminishing apically inside the margin, connected to the super adjacent secom 
ries by a series of cross veins without forming prominent marginal loc 
(Hickey 1973) or simple curvipinnate. Here, secondaries curve gradually towa; 
the margin and often form marginal or submarginal veins (Melville 1976). 1 
primary vein or midrib is the thickest vein of the leaf and after its departure fr< 
petiole it traverses straight or markedly curved. The thickness of the primi 
vein gradually decreases towards the apex. The next smaller size class of ve 
are the secondary veins (2 veins) whose origin may be on either side of 1 
primary vein in an alternate or sub-opposite fashion. The number of 2 veins 
either side of the primary vein varies from species to species irrespective of 1 
leaf size. The secondary veins do not merge into the margin but turn upwaj 
and form arches with super adjacent secondaries with acute, right angle or obti 
angle. Composite intersecondary veins are observed in all species. Intramargii 
vein is observed in Nerium indicum. Intramarginal vein closely paralleling 1 
leaf margins and into which the secondary veins are fused; probably the result 
the fusion and straightening of the exmedial brochidodromous secondary ai 
segments into what appears to be am independent vein (Hickey 1973) or margit 
vein simple and linear which is situated close to the leaf edge and without a 
other veins extending beyond it formed by linking the ends of all of the excurr< 
veins at the margin (Melville 1976) (figure 1 B). 

The tertiary veins arise from the secondaries having no definite patterns of an 
of origin. Predominant tertiary vein angles of origin are right angle right an, 
(RR), right angle acute (RA), right angle obtuse (RO), acute acute (A A), aci 
right angle (AR), obtuse right angle (OR), acute obtuse (AO) or obtuse obtuse (0 
in all the species studied except in Alstmia scholaris, where the predoratu* 
tertiary vein origin angle is absent. Species-wise details of angles of origin i 



Leaf architecture of Apocynaceae 



193 




Figure 1. (A) direct photograph of cleared leaf showing venation pattern in Carissa 
congesta ; (B) infra-marginal vein in Nerium indicum ; (C) incomplete marginal 
ultimate venation in Rauwolfia serpentina ; (D) looped marginal ultimate venation 
in Allamanda nerifolia ; (E) areolation and loop formation in Trachelospermum 
jasminoides ; (F) loop formation and veinlets in Trochelospermum jasminoides ; 
(G) free vein ending and bundle sheath in Wrightia tinctoria 
A 2.1 X ; B 55 X ; C 149 X ; 

D 115 X ; E 43 X ; F 270 x ; 

G 335 X 

L Loop T tracheid 



194 J S S Mohan and J A Inamdar 




Figure 2. (A) vessel element at the terminal position of the veinlet in Catharanthus 
major ; (B) undifferentiated tracheary element (extension cell) in multiseriate vein 
in Catharanthus major ; (C, D, E, F) uniseriate or biseriate tracheids in Rauwolfia 
serpentina and Vallaris solanacea ; (G) uniseriate elongated tracheid in Catharanthus 
major ; (H, I) tracheoidal elements in Rauwolfia serpentina and JR.. tetraphylla. 
A ' 617 x ; B 435 X ; C - 402 X ; 

D - 335 X ; E 371 X ; F 672 X ; 

G 385 X ; H 471 X ; I 335 X. 



Leaf architecture of Apocynaceae 1 95 

given in table 1 . The pattern of the tertiary veins is either random or orthogonal 
reticulate (Hickey 1973) or scalariform where inter-coastal areas are bridged at 
regular intervals by transverse veins either at right angles or with a regular 
orientation and having the appearance of rungs on a ladder; transverse ramified 
(Hickey 1973) or dendroid - regularly or irregularly dichotomous veins occupying 
an areole and attached to the areolar veins at one point (Melville 1976) 
(figure 1 E). But in Alstonia scholaris admedial ramification of tertiary veins 
branching into higher orders without rejoining the secondary veins and oriented 
towards the leaf axis (Hickey 1973) or pendulous type with branching veins lying 
free in an intercostal area or an areolus, attached at their distal ends and appear- 
ing to be pendulous from a submarginal vein or costal vein. 

.3 Minor venation pattern 

Ihe highest order of veins is identified up to 7, but in Catharanthusroseus'u.p to 
3 or 4, in some cases up to 5 or 6. The numerical data on the venation pattern 
are charted in table 1 . Marginal ultimate venation is incomplete (Hickey 1973) 
3r marginal vein simple and incomplete i.e. marginal vein broken, linking some 
of the excurrent veins but leaving others free (Melville 1976) (figure 1 C), 
fimbriate (Hickey 1973) or marginal vein simple and arcuate i.e. marginal vein 
formed of arching veins linking the ends of the excurrent veins (Melville 1976), 
looped (Hickey 1973) or marginal vein simple and irregular (Melville 1976) 
(figure 1 D). 

The areoles are the smallest areas of the leaf tissue surrounded by the major 
veins which taken together form a contiguous field over most of the area of the 
leaf. The areoles in most of the cases are either well developed or imperfect. 
But in Alstonia scholaris the areolation is lacking. The arrangement of the 
areoles is either random or oriented. The shape of the areoles may be quadran- 
gular, pentagonal, polygonal or irregular. The size of the areole is not constant, 
but varies in different species and even in the same species. Venation characters 
show variations in areole size, number of veinlets entering per areole and the 
organizations of terminal vein endings in different species. 

3.4 Veinlets 

The ultimate veins of the leaf are either simple or branched. Simple vein 
endings may be linear or curved (figure 1 F) . The branched ones divide dichoto- 
mously.once or twice symmetrically or asymmetrically (figure 1 F). The veinlets 
may be uniseriate (figure 1 C), biseriate or multiseriate (figure 2 B). They may 
be thin and long or thick and short. The veinlet number vary irrespective of 
ireole size. The veinlets whether uni-, bi- or multiseriate without terminal 
tracheids are known as free vein endings (figure 1 G). Occasionally a vessel 
with a single scalariform perforation plate is present at the vein tip alongwith 
the tracheids in Catharanthus major (figure 2 A at arrow). In most of the cases 
where areoles are devoid of vein endings a loop-like structure is seen 
(figure 1 A, E, F) which is formed either due to the union of tracheids, veins or 
tracheids and veins. Rarely, in a multiseriate veinlet some of the elements fail 
to differentiate into tracheary elements (figure 2 B at arrow). 



196 J S S Mohan and J A Inamdar 

3.5 Tracheids 

The tracheids manifest extraordinary variation in size, shape and nature and 
situated at the terminal position of the veinlets. Tracheids may be uniseriate 
(figure 2 C, G) or biseriate (figure 2 F). They may be juxtaposed (figure 2 F) or 
superimposed having V, *T J or club-shape (figures 2 C, D, E, F; 3 F). They 
may be isodiametric grouped or elongated. 

3.6 Tracheotdal elements 

Tracheoidal elements are observed in most of the species. They lie lateral and 
parallel to the veins and veinlets (figures 2 H, I; 3 A). The tracheoidal elements 
may be isodiametric or elongated and arranged either scattered or in rov/s. 
Probably the function of tracheoidal elements is to provide mechanical support 
and also aid in retention of water for the leaf. 

3.7 Isolated tracheids 

Uniseriate or biseriate tracheids that lie free and disjunct in the mesophyll and 
those connected with veinlets by extension cells are called isolated tracheids 
(figure 3 B, C). 

3.8 Isolated vein endings 

Vein endings with terminal tracheids either uni- or biseriate lying free and 
disjunct in the areole are known as isolated vein ending (figure 3 D, E). These 
are common in most of the cases. 

3.9 Isolated free vein endings 

These are uniseriate or biseriate vein ending without terminal tracheids lying free 
and disjunct in the areole. These vein endings are seen in Catharanthus roseus 
(figure 3 I). 

3.10 Extension cells 

These are parenchymatous cells which either adjoin isolated tracheid with a vein 
(figure 3 G) or a vein with another vein (figure 3 H). Extension cells have 
failed to differentiate either into sieve or tracheary elements. 

3.11 Bundle sheath 

All the major and higher order veins are ensheathed by parenchymatous bundle 
sheath ceils. The thickness of bundle sheath varies from primary to higher order 
veins. The shape of the bundle sheath cells may be either round, isodiametric 
or rectangular (figure 1 G). 

4. Discussion 

According to Hickey (1973) and Melville (1976) the Apocynaceae leaves fall 
under the pinnate camptodromous with festooned brochidodromous secondaries. 



Leaf architecture of Apocynaceae 



197 




Figure 3. (A) iracheoidal element in Catharanthus major ; (B) isolated tracheid in 
Rauwolfiu serpentina ; (C) grouped isolated tracheid in JRaitwolfia tctraphylla ; 
(D, E) isolated vein endings in Rauwolfia serpentina and Allamanda neri folia ; 
(F) isodiamctric tracheids in Rauwolfia letraphylla ; (G) extension cell between 
tracheid and vein in Rauwolfia serpentina ; (H) extension cell between vein and 
vein in Catharanthus roseus ; (I) isolated free vein ending in Catharanthus roseus. 



A 252 X 



D 
G 



- 292 X 

471 X 
EC 
IFVe 
It 
IVe ~ 



B 289 x ; 

E 303 X ; 

H 435 X : 
extension cell 
isolated free vein ending 
isolated tracheid 
isolated vein ending 



C 335 X ; 
F 429 x ; 
I 271 X. 



Leaf architecture of Apocynaceae 1 99 

rarely pinnate eucamptodromous or curvipinnate venation pattern with multi- 
arcuate type, rarely simple curvi-pinnate respectively. Sehgal and Paliwal (1974) 
classified the euphorbiaceous leaves as imi-, bi- or triveined on the basis of the 
number of strands entering the base of the leaf. Apocynaceae leaves fall under 
the univeined category. 

Reports on the significant variation in the size, shape and number of vein 
endings entering the areole are contradictory (see Nicely 1965; Sehgal and 
Paliwal 1974). Sehgal and Paliwal (1974), Singh era/ (1976), Jain (1978) and 
Inamdar and Murthy (1978) concluded that there is no direct relationship 
between size of an areole and the number of vein endings and vein termination 
in different species as well as in the same leaf. 

Hickey (1973) classified the marginal ultimate venation into looped, fimbriate 
and incomplete. In Nerium indicum the intramarginal vein is formed by secondaries 
only and there are no higher order veins beyond it. Therefore, it becomes 
extremely difficult to classify the marginal ultimate venation according to Hickey 
(1973), however, it can well be classified into marginal and linear type of 
Melville (1976). 

Tracheids or tracheoidai idioblasts (Foster 1956) also regarded as peculiar 
cells (Giibort 1881; Bierhorst and Zamura 1965), storage tracheids ~ speichert- 
racheiden (Solereder and Meyer 1930), mechanical cells (Mangin 1882), water 
storage cells (Kny and Zinimermann 1885), water cells (Pirwitz 1931). Tucker 
(1974) referred to these elements as hybrid cells. According to Oltunji and 
Nengim (1980) the occurrence of the tracheoidai elements in many unrelated 
plants may be due to convergent adaptation to xerophytic habit. The occurrence 
of tracheoidai elements along the lateral side walls of veinlets and rarely on the 
4 and 5* veins are noticed in this family. These tracheoidai elements differ 
from tracheids in form, arrangement and thickening. 

Kasapligil (1951), Foster and Arnott (1960), Herbst (1972) reported the 
occurrence of isolated veins in dicotyledonous leaves. The tracheidal elements 
tfhich lie free in the areole are designated as "free vein ending'* by Sehgal and 
Paliwal (1974). The isolated tracheids, isolated vein endings and free veins are 
)bserved in most of the species. Terminologies such as vein endings, free vein 
mdings, isolated vein endings and isolated tracheids as defined by Inamdar and 
Vlurthy (1981) are used here. Sehgal and Paliwal (1974) termed the parenchy- 
natous sheath cells which surrounds the veins, as 'ornamented'. Bundle sheath 
s present in all the cases around the veins, vein ending and even tracheids. 

Acknowledgements 

Ve thank Dr (Sr) Avita for kindly supplying some material. Mr J S S Mohan 
hanks the CSIR for awarding a junior research fellowship. 



References 

Jierhorst D W and Zamura P M 1965 Primary xylem elements and element association of 
angiosperms ; Am. J. Bot. 52 567-710 



200 / S S Mohan and J A Inamdar 

Chandra V, Kapoor S L, Sharma P C and Kapoor L D 1969 Epidermal and venation studies in 

Apocynaceae 1 ; Bull. Bot. Surv. India 11 286-289 
Chandra V, Mitra R, Kapoor S L and Kapoor L D 1972 Epidermal and venation studies in 

Apocynaceae IV ; Bull. Bot. Surv. India 14 76-82 
Foster A S 1956 Plant idioblasts, remarkable examples of cell specialisation ; Protoplasma 46 

184-193 
Foster A S and Arnott H J 1960 Morphology and dichotomous vasculature of the leaf of 

Kingdonia uniflora ; Am. J. Bot. 47 684-698 
Gilbortt W H 1881 Notes on the histology of picher plants ; /. Quekett Microscopical Club 6 

151-164 

Herbst D 1972 Ontogeny of foliar venation in Euphorbia forbesii ; Am. J. Bot. 59 843-850 
HickeyLJ1973 Classification of the architecture of dicotyledonous leaves ; Am. J. Bot. 60 

17-35 
HickeyL land Wolfe J A 1975 The bases ofangiosperm phylogeny vegetative morphology; 

Ann. Misso. Bot. Gard. 62 538-589 

Inamdar J A and Murthy GSR 1978 Leaf architecture in some Solanaceae ; Flora 167 265-272 
Inamdar J A and Murthy GSR 1981 Vein endings in some Solanaceae ; Proc. Indian Acad. ScL 

(Plant Sci.) 90 53-58 

Jain D K 1978 Studies in Bignoniaceae III Leaf architecture ; /. Indian Bot. Soc. 57 369-386 
Kapoor S L, Sharma P C, Chandra V and Kapoor L D 1969 Epidermal and venation studies in 

Apocynaceae II ; Bull. Bot. Sury. India 11 372-376 
Kasapligil B 1951 Morphological and ontogenetic studies of Umbellularia calif arnica Nutt. an 

Laurus nobilis L. 
Kny L and Zimmermann A 1885 Die Bendentung der spiralzelien von Nepenthes ; Ber. Dtsch. 

Bot. Ges. 3 123-128 
Mangin L 1882 Sur le development des cellules spiralis ; Ann des Sciences Naturelles Paris 

(Botanique) 13 208 

Melville R 1976 The terminology of leaf architecture ; Taxon 25 549-561 
Nicely K A 1965 A monographic study of calycanthaceae ; Castanea 30 38-81 
Olatunji O A andNengimRO 1980 Occurrence and distribution of tracheoidal elements in 

the Orchidaceae ; Bot. J. Linn. Soc. 80 357-370 
Pirwitz K 1931 Physiologische und anatomische untersuchengen an spechertracheiden und 

velamen ; Planta 14 19-76 

Rao VS, ShenoyK N and Inamdar J A 1980 Clearing and staining technique for leaf archi- 
tectural studies ; Microsc. Acta 83 307-311 
Seagal L and Paliwal G S 1974 Studies on the leaf anatomy of Euphorbia. II Venation patterns; 

Bot. J. Linn. Soc. 68 173-208 
Sharma P C, Chandra V, Kapoor S L and Kapoor L D 1970 Epidermal and venation studies in 

Apocynaceae III, Bot. J. Linn. Soc. 12 92-96 
Singh V, Jain D K and Meena Sharma 1976 Leaf architecture in Salix; J. Indian Bot. Soc. 55 

140-150 

Solereder H and Meyer F 1930 Systematische anatomic der Monocotyledonen Stuttgart 6 92-242 
Tucker S C 1974 DedirTerentiation of guard cells in Magnoliaceous leaves ; Science 185 445-447 



roc. Indian Acad.Sci. (Plant Sci.), Vol. 91, Number 3 t June 1982, pp. 201-209, 
*\ Printed in India. 



mpact of extension growth and flowering on the cambial 
ictivity of Delonix regia Rafin. 



A K M GHOUSE and SHAMIMA HASHMI 

Department of Botany, Aligarh Muslim University, Aligarh 202 001, India 

MS received 3 July 1981; revised 18 May 1982 

Abstract. Shoot growth in Delonix regia takes place in three distinct flushes, the 
first commencing in mid-March, the second in late May and the third in October. 
The cambial reactivation starts in April after the initiation of the first flush of shoot 
growth, but the addition of new vascular derivatives does not take place until the 
second flush of shoot growth has occurred. Heavy flowering which ensues following 
first flush of shoot growth seems to delay cell divisions in cambial initials in April. 
Once the formation of cambial derivatives starts, it continues till mid-November 
whence the cambium enters the dormant phase. Xylogenesis begins from July and the 
formation of phloem in October, while the precursor phloem differentiates in early 
April. 

Keywords. Tree growth; extension growth; flowering cambial activity; vascular deri- 
vatives; Delonix regia. 

.. Introduction 

Foster as early as 1927-28, considered that bud bursting and new leaf emergence 
re highly significant for the initiation of cambial activity. The later works on 
everal tropical and temperate trees have supported Coster's contention (Chow- 
ihury 1958, 1969; Chowdhury and Tandon 1950; Wareing et al 1964; Waisel and 
<ahn 1965; Waisel et al 1966; Fahn et al 1968; Ghouse and Hashmi 1979a). It 
5 believed that an intricate relationship exists between bud burst, leaf emergence 
nd initiation of cambial activity, the first two preceding the last. The present 
eport deals with the relationship between shoot growth, flowering and the cambial 
ctivity in Delonix regia, a flowering tree which originated in West Africa and is 
presently grown in many parts of India. It has a deciduous habit and diffuse 
torous wood. The study has been undertaken at Aligarh which is located at 
;7 53' N lattitude and 78 4' longitude in the monsoon belt of the great gangetic 
>lain of North India. 

1. Materials and methods 

"hirtysix trees of Delonix regia Rafin. planted 30 years earlier at the University 
Uampus of Aligarh were used for the present investigation. To study the phenology 

201 



202 



A K M Ghouse and Shamima Hashmi 



of the selected trees, 40 current year branches were tagged with aluminium labe! 
in each tree, at the rate of 10 branches facing east west, north and south side; 
Observations were recorded on leaf fall, bud burst, leaf emergence, flowering 
fruit setting, fruit persistence and bud burn for 3 consecutive calenda 
years commencing from 1974. 

The daily data on atmospheric temperature, relative humidity and rainfa 
were collected from the local meteorological unit maintained at the Universit 
Campus. The monthly average of physical factors was calculated and represents 
in figure 1 . 

Samples of cambial strips of 2 cm sq, together with the inner bark and som 
sapwood were collected at fortnightly intervals for 3 consecutive calen 



* Re!. hum,(% 
Temp* ( C) 
Rain fall (cm) 




JFMAMJ JASO NO 



I 



J'F'M'A'M'J'J'A'S'O'N'D 

ZZ Bud bursting 

m Pre- cursor phloem 

El Flowering 

S Cambial activity 

ED Leaf fall 

^ Phloem production 

S3 Extension growth. 

ID Xylem production 

Figure 1. Graphs showing the relationship between the atmospheric temperature, 
relative humidity, rainfall, phenology and cambial activity in Delonix regta 
during a calendar year. 



Extension growth and flowering on the cambial activity 203 

dar years starting from 1974, Chisel and hammer were employed to take out the 
blocks from the main trunks at chest height (1.5 in from ground level). Three 
trees were sampled on each turn and from each tree four blocks were obtained, 
one each from east, west north and south side of the tree. The excised blocks 
were fixed on the spot in FAA (formalin-acetic acid and ethanol mixture) and 
aspirated after an hour. After fixation, they were softened in an alco-glycerol 
1:1 mixture of 50% ethanol and 50% glycerol for 4 weeks. Sections were pre- 
pared using a sliding microtome in transverse, tangential and radial longitudinal 
planes at a thickness of 10 /"m. The sections were stained with tannic acid and 
ferric chloride (Foster 1934) and with lacmoid combination (Cheadle et al 1953) 
and mounted in Canada balsam, after dehydrating in ethanol-xylol series. 



3. Observations 

3.1 Extension growth 

Dehnix regia, being a deciduous tree, starts shedding leaves from December and 
becomes completely bare by early January and remains so till mid-March (figure 1). 
In March- April, the weather becomes a little warm in day and large number of 
buds emerge from the naked branches of the previous year in the axils of fallen 
leaves. The bud grows rapidly and produces two or three bi-paripinnate leaves 
within a fortnight. By mid-April buds of floral axes come up in large quantity 
from the axils of new leaves on current year shoots. These buds develop into a 
large corymbose inflorescence, each having a large number of conspicuously coloured 
showy flowers. The trees bloom heavily in late April and early May and as 
a result, the green crown appears as flaming red or orange. 

The first flush of extension growth which begins in late March ends by late 
April when reproductive growth becomes established. As a result of heavy 
flowering, the vegetative growth of the shoots stop completely. In late Mayor 
a little earlier, flowering declines and the fruits start setting. It may be noted 
that the fruits remain on the tree for about 14-15 months. 

The second flush of shoot growth starts in the second half of May and continues 
up to August. It occurs at a rapid rate and accounts for the major part of the 
year's growth product. 

The third flush of shoot growth occurs in October in the same year, although 
for a short duration because the low night temperature of November burns down 
the apices of the branches. Leaf fall ensues in early December and the events 
repeat once more (figure 1). 



3 . 2 Cambial activity 

The microscopic analysis of the cambial samples, collected at fortnightly intervals, 
indicates that cambial reactivation commences in the first half of April soon after 
bud burst and leaf emergence in first flush of extension growth. To start with 



204 A KM Ghouse and Shamima Hashmi 

the protoplasmic contents of the cambial cells stain lighter than before. Concur- 
rently, the nuclei also lose their chromaticity. In late May a few cells of the 
cambial zone enlarge in radial direction and later enter the active phase by under- 
going cell division in the first week of June. The radial growth thus resuming in 
early June continues up to the second week of November, stretching over a period 
of five and a half months against the eight month extension, growth occurring in 
three distinct flushes. 

After cell division, the size of cambial zone population swells up (figures 2 
C,D) touching its peak in August-September and declining later, as the derivatives 
continue to differentiate at a rapid rate. In October the activity slowly declines 
and stops by mid-November. The cambium thus enters its dormant phase in 
late November when the cells develop dark protoplasmic contents (figures 2 A, B). 
The walls of the cambial cells, especially the radial walls become thicker and 
develop beaded appearance on account of unthickened primary pit-fields. The 
cambium remains in this state till the following spring. 



3.3 Formation of xylem and phloem 

New xylem differentiates in trunks in July after the break of monsoon, although 
the cell divisions start in cambial cells one month earlier. Xylem formation takes 
place at a high rate throughout August and September and slows down in October 
and later stops by mid-October (figure 1). The phloem production, on the other 
hand, initiates in October, when the air temperature becomes somewhat moderate 
and lasts for about a month. 

Earlier to the initiation of cambial activity, but after the advent of summer in 
early April, a few layers of cells differentiate into a narrow strip of new phloem 
out of the overwintered mother cells. This precursor phloem measures about 75 
to 100 [im in depth in transections. 

In a growth year, as viewed in transections, about 800 /im of xylem and about 
500 /tm of phloem are produced by D. regia at Aligarh conditions of weather. 



4. Discussion 

The majority of dicotyledons and gymnosperms show a sharp periodicity of shoot 
growth including radial growth. However, in certain exceptional cases growth 
may occur throughout the year without break, particularly in the tropical envi- 
ronment (Alvim 1964; Fahn and Sarnat 1963; Fahn et al 1968). 

The pioneering work of Chowdhury (1958, 1968, 1969) has shown that the 
radial growth in certain Indian trees may extend up to 10 months in a calendar 
year. Other works also indicate that the tree growth in tropics takes place for a 
considerably longer duration than at the temperate regions (Fahn and Sarnat 1963; 
Lawton 1972; Rao 1972; Chau and Chiang 1973; Lu and Chiang 1975; Khan and 
Ghouse 1978, 1980; Ghouse and Hashmi 1979a). The present study on D. regia 
also indicates the same, as far as the extension growth is concerned. However, 



Intension growth and flowering on cainhlal activity 



205 




Figure 2. Photomicrographs showing the active (A and C) and dormant phases 
(B and D) of vascular camlium in tangential (A and B) and transverse 
(C and D) sections-A f B, C at X 372; D at X 160 



Extension growth and flowering on the cambial activity 207 

radial growth in D. regia takes place only for a period of five and a half months 
in a year and it, therefore, does not resemble other trees which grow in tropics. 
This may be due to the flowering habit of this species in which the cell division 
in cambial cells is seemingly delayed by heavy flowering till June. 

The direct relationship between extension growth and the cambial activity 
established by earlier workers like Chowdhury and Tandon (1950), Chowdhury 
(1958, 1969) and Ghouse and Hashmi (1979a) is further getting confirmed in the 
present study, since it demonstrates that the occurrence of extension growth 
acts as a prerequisite factor for the initiation of cambial reactivation in the inves- 
tigated species. In Polyalthia longifolia the authors noted that the reactivation 
of vascular cambium is invariably preceded by the swelling phenomenon in certain 
cambial cells and this incidence is initiated, in turn, by the emergence of leaves 
(Ghouse and Hashmi 1979a). The results obtained in the present study also indi- 
cate the same for D. regia. In an earlier communication the authors further 
brought to light that high temperature and high humidity accelerate the differentia- 
tion of xylem and low humidity favours the formation of phloem in P. longifolia 
(Ghouse and Hashmi 1978). A careful scrutiny of the data obtained in the 
present study reveals that the requirements for differentiation of phloem and xylem 
are different and they follow more or less the same trend in D. regia and P. longi- 
folia (Ghouse and Hashmi 1978). 

The decrease in chromaticity of the~protoplasmic contents, tanniferous substan- 
ces and the cell wall characteristics have also been noticed in the past (Derr and 
Evert 1967; Tucker and Evert 1969; Ghouse and Hashmi 1979a). 

Differentiation of phloem preceding that of xylem has been recorded in a number 
of tropical trees including those that grow in India (Lawton 1972; Ghouse and 
Hashmi 1978, 1979b). In the present study xylem has been noticed to differentiate 
first during a growth year. However, the precursor phloem differentiation 
as noted in D. regia is more commonly observed in the Indian trees (Ghouse and 
Hashmi 1979b, 1979c, 1980) than reported so far in the temperate trees (Evert 
1960, 1963; Derr and Evert 1967; Davis and Evert 1968). 

The differentiation of phloem two times in a year first in early April 
and next in October, as found in D. regia invites special attention. The pheno- 
menon appears to be controlled more by the environmental conditions than by the 
internal make up of the species. A cursory look of the weather data provided 
in figure 1 indicates that the temperature during the periods of phloem differentia- 
tion happens to be almost the same in the present case. It appears that a slight 
rise in temperature above this level does not seem to favour phloem differentiation 
in this species. A situation of more or less similar nature has been observed in 
Polyalthia longifolia by the authors (Ghouse and Hashmi 1978). 



Acknowledgements 

We are thankful to the Council of Scientific and Industrial Research, New Delhi 
for financial assistance in the form of a Senior Fellowship awarded to 
Miss Shamima Hashmi. 



208 A KM Ghouse and Shamima Hashmi 

References 

Alvim P T 1964 Tree growth periodicity in tropical climate ; in The formation of wood in fores 

trees (ed) M H Zimmerman (New York: Academic Press). 
Chau T and Chiang S HT 1973 Seasonal changes of cambial activity in the young branch of 

Psidium guajava Linn. ; Taiwania 18 35-41 
Cheadle V I, Gifford E M and Esau K 1953 A staining combination for phloem and contiguous 

tissues ; Stain Techno I. 28 49-53 
Chowdhury K A 1958 Extension and radial growth in tropical perennial plants; in Modern deve 

lopments in plant physiology (ed.) P Maheshwari (Delhi : Delhi Univ.) 
Chowdhury K A 1968 History of botanical researches in India, Burma and Ceylon X WoOi 

Anatomy (Aligarh : Muslim Univ. Press) 

Chowdhury K A 1969 Cambial activity in temperate and tropical tree XI Int. Bot. Congr. Seatth 
Chowdhury K A and Tandon K. N 1950 Extension and radial growth in trees ; Nature (London 

165 732-733 
Coster C 1927-28 Zur anatomic and physiologic der zuwachszonen and jahressingbildung in der 

tropen ; Ann. Jard.]Bot. Buitenz. 37 49-160 
Davis J D and Evert R F 1968 Seasonal development in the secondary phloem in Populu, 

tremuloides ; -Bot. Gaz 129 1-8 
Dcrr W F and Evert R F 1967 The cambium and seasonal development of the phloem in Robinit 

pseudoacacia ; Am. J. Bot. 54 147-153 
Evert R F 1960 Phloem structure in Pyrus communis L. and its seasonal changes ; Univ. Call) 

Publ. Bot. 32 127-194 
Evert R F 1963 The cambium and seasonal development of phloem in Pyrus malus; Am. J. Bot 

50 149-159 
Fahn A and Sarnat C 1963 Xylem structure and annual rhythm of development in trees an 

shrubs of the desert IV Shrubs ; Bull. Res. Counc. Israel 11 198-209 
Fahn A, Waisel Y and Benjamin L 1968 Cambial activity in Acacia raddiana SavL; Ann* Bot. 3^ 

677-686 
Foster AS 1934 The use of tannic acid and iron chloride for staining meristernatic tissues 

Stain TechnoL 9 91-92 
Ghouse A K M and Hashmi S 1978 Seasonal cycle of vascular differentiation in Polyalthh 

longifolia (Annonaceae); Beitr. Biol. Pflanzen 54 375-380 

Ghouse A KM and Hashmi S 1979a Cambium periodicity in Polyalthia longifolia Benth <S 

Hook; Phytomorpholo^y 29 61-67 
Gbouse A K M and Hashmi S 1979b Longevity of phloem in Polyalthia longifolia Benth <S 

Hook Bull. Torrey Bot. Club 106 182-184 
Ghouse A K M and Hashmi S 1979c Longevity of phloem in Delonix regia Rafin ; Proc. Indiai 

Acad. Sci. (Plant Sci.) 89 67-72 
Ghouse A K M and Hashmi S 1980 Seasonal production of secondary phloem and its longevit] 

in Mimusops elengi L. Flora 170 1 75-1 79 
Khan M I H and Ghouse A K M 1978 Occurrence of intermittent growth waves in the shoots o 

Psidium guajava L. ; in Environmental Physiology and Ecology of Plants (ed) D N Sen (Dehi 

Dun : B S M P Singh) 351-355 

Khan M I H and Ghouse A K M 1980 Studies on the impact of heavy flowering and fruiting 01 
the extension growth ofguava (Psidium guajava L.); Flora 169 453-455 

Lawton J R 1972 Seasonal variation in the secondary phloem of some forest trees from Nigeria 
II. Structure of the phloem ; New Phytol. 71 335-348 

Lu C Y and Chiang S H T 1975 Seasonal activity of the cambium in the young branches o 
Liquidamber formosana Hance ; Taiwania 20 32-47 

Rao A N 1972 Periodic changes in the cambial activity of Hevea brasiliensis ; /. Indian Bot. Soc 
51 13-17 



Extension growth and flowering on the cambial activity 209 

Fucker C M and Evert R F 1969 Seasonal development of the secondary phloem in Acer negun- 

do\ Am. J. Bot. 56 275-284 
iVaisel Y and Fahn A 1965 The effects of environment on the wood formation and cambial 

activity in Robinia pseudoacacia ; New Phytol. 64 436-442 
tVaisel Y, Noah I and Fahn A 1966 Cambial activity in Eucalyptus cameldulensis Dehn. I. The 

relation to extension growth in young sapling; La-Yaaran 16 103-108 
Wareing P F, Hanney C E A and Digby J 1964 The role of endogenous hormones in cambial 

activity and xylem differentiation; in The formation of wood in forest trees (ed) 

M H Zimmerman (New York : Academic Press) 323-344 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 3, June 1982, pp. 211-226. 
Printed in India. 



Pharmacognostic studies on the flower of Mesuaferrea L. 



USHA SHOME, SHANTA MEHROTRA and H P SHARMA 

Pharmacognosy Laboratory, National Botanical Research Institute, Lucknow 226 
India 

MS received 15 June 1981 

Abstract. Stamens of Mesua ferrea L. constitute the genuine 'Nagkeshan 
Ayurveda, a drug considered to be astringent, stomachic and expectorant. The pr 
communication deals with detailed pharmacognosy of the drug and includes mo 
logical, anatomical as well as certain phytochemical characters of the floral pai 
Mesuaferrea. Some of the distinguishing characters are : cortical fibres, num 
resin canals and calcium oxalate crystals in the cortex and pith of the pe< 
anamocytic, anisocytic or paracytic stomata on sepals and petals ; and 
zonocolporate pollen grains with reticulate exine surface. Fluorescence ans 
behaviour of the drug with certain chemical reagents and thin layer chromatog 
were also carried out. 

Keywords. Mesuaferrea ; flower ; pharmacognosy ; cluseaccae. 

1. Introduction 

Mesua ferrea Linn, belonging to family Cluseaceae is considered the gen 
'Nagkeshara' (Chunekar I960; Dymock ef #71885; Mudaliar 1957; Va 
1971; Waring 1868). It is known as Nagpushpa champeya in Sanskrit; I 
nagkeshara in Hindi ; Nageshwara in Bengali ; Nagchampa in Madhya Prad 
Pilu-nagkeshara in Gujrati ; Viluha champkan in Tamil ; Iron wood tree 
Cobras Saffron in English. 

Stamens of M. ferrea constitute the actual drug (Sharma 1978). It : 
mention in all the important ancient Ayurvedic literature (Bhavmishra li 
Charaka 1949 ; Sushruta 1952) to be used as an astringent stomachic 
expectorant. Flowers are given in bleeding piles, in the form of a paste n 
with butter and sugar. These are also applied for relief in burning sensatic 
the feet (with old and a hundred times washed 'Ghee') (Chakradatta, See Nadk 
1937; Kirtikar and Basu 1933). A syrup of the flower buds (1 in 10) is sa 
cure dysentery (Nadkarni 1937). The drug was also adopted by Arab 
Unani physicians into their Materia Medica (Anonymous 1970) accordinj 
which it has a depressent action on uterine muscles, is digestive, diaphor 
antidysenteric and an emmenagogue. It is used as a constituent of aboul 
compound Unani formulations (Anonymous 1970). 



N.B.R.L Research Publication No. 139 (New Series) 



212 Usha Shome, Shanta Mehrotra and H P Sharma 

Mesua ferrea L. is a large, evergreen tree with a short trunk, often buttressed 
at the base. It is found in the Himalayas from Nepal eastwards ascending to an 
altitude of 1500m. Jt is also cultivated in the gardens for its beautiful flowers 
and attractive foliage (Anonymous 1962). Detailed pharmacognostic work on 
the flower of Mesua has not been carried out so far and hence the preient study. 



1.1. Previous Work 

Several workers have carried out market surveys of Nagkeshara. Satkopan and 
Thomas (1967) surveyed Gujrat markets and found only one sample 
from Surat as genuine 'Nagkeshara'. These authors have also tried to identify 
other samples being sold as Nagkeshara (Satkopan and Thomas 1967a,b ; 
1968). All (1967) surveyed the South Indian Markets. However, he found 
none of the samples as Mesua ferrea L. 

The presence of an essential oil and two bitter substances has been reported 
in the flowers by Boorsma in 1904 (see Chakraborty et al 1959). Subramanyara 
el al (1975) have isolated mesuanic acid, a new carboxylic acid, from the 
acetone extract of Mesua ferrea stamens. Subramanyam and Subba Rao (1969) 
had earlier isolated mammeisin from the seeds of Mesua ferrea L. Bala and 
Seshadri (1971) isolated mammeigin and mesuol as the main phenolic components 
from two different samples of seed oil. Dutt et al (1940), Chakraborty and Bose 
(1960) and Chakraborty and Das (1966) isolated two crystalline antibiotic 
principles mesuol and mesuone from the seed kernel oil. Chakraborty et al 
(1959) investigated anti-bacterial activity of mesuol and mesuone. None of the 
constituents, however, was found to be antifungal. Bhattacharya et al (1979) 
have synthesized an antibiotic mesuagin from Mesua ferrea. 



2. Material and methods 

Fresh material was collected from the Forest Research Institute, Dehra Dun 
in the month of May and preserved in form acetic-alcohol. Hand sections 
stained with safranin were used for the present study. Phloroglucinol, iodine 
and terric chloride were used to test lignin, starch and tannin respectively. 
For whole mounts petals were treated with a dilute solution of nitric acid 
followed by clearing in chloral hydrate. Physicochemical studies were 
performed with the powdered, shade dried material. 



3. External characters of the flower 

The flowers are fragrant ; cream coloured ; ^ebracteate ; pedicellate ; pedicel 
short, axillary or terminal ; solitary or in pairs and 2.5-7.5 cms in diameter 
(figure 1). The buds are subglobose. 

Sepals 4, large persistent, boat shaped, 2 outer slightly shorter than the 
inner ones and depressed at the base. Petals 4, large, cream coloured, spreading, 
cuneate, margins undulating, caducous ; stamens indefinite, golden-yellow, united 



Pharmacognostic studies of Mesuaferrea L. 



213 




Figure 1. Mesuaferrea Linn : Two tuigs with flowers x 0.425. 



214 



Usha Shorn e, Shanta Mehrotra and H P Sharma 




Figures 2-12. 2, 3. T. S. pedicel (Diagrammatic) 4. A portion of transverse section through 
the pedicel region showing details. 5. Upper epidermis of a sepal showing stomata and 
striations. 6. Upper epidermis of a sepal showing elongated narrow cells over the vein. 
7. Lower epidermal cells of the sepal showing trichome bases and stomata. 8. Lower 
epidermal cells showing trichomes only. 9 and 10. uni and multicellular uniseriate 
trichomes. 11. T. S. sepal (Diagrammatic). 12. A portion of the same showing details. 
Abbreviations: ASP, airspace; BS, bundle sheath; COL, collenchyma ; COR, 
cortex; CR, crystal ; CU, cuticle ; EP, epidermis ; GT, ground 
tissue ; HYP, hypodermis ; LEP, lower epidermis ; MXY, 
metaxylem ; PH, phloem ; PI, pith ; PXY, protoxylem ; RC, 
resin canal ; S, starch grains ; SCL, sclerenchyma, ST, stomata ; 
STC, stone cells ; TR, trichome ; TRB, trichome base ; UEP, upper 
epidermis ; YB, Vascular bundle ; XY, Xylern. 



Pharmacognostic studies of Mesuaferrea L. 215 

at the base and forming a fleshy basal sheath, filaments small, anthers oblong ; 
ovary superior, bicarpellary, syncarpous, style twice as long as stamens, stigma 
capitate, style and stigma persistent in young fruit but are shed away later on. 

Fruit conical when young, ovoid to almost round with a prominent bea? 
when mature, pericarp hard, warty, 2-valved after dehiscence. Seeds 1-4, 
angular, smooth and chestnut brown. 



4. Histology 

4.1 Pedicel 

A transverse section of the pedicel is almost circular in outline. The epidermis 
is highly cuticularised and is formed by narrow elongated cells bearing one- to 
five- celled, uniseriate hairs. The cortex is 19 or 20 layered and is composed 
of rounded, collenchymatous cells. Cells of the outer cortical layers have 
characteristic semi-lunar, lignified thickenings on their walls. The inner cortical 
cells have fairly large starch grains as eragastic inclusions. As the pedicel 
matures sclerenchymatous fibres develop in the form of radially elongated 
patches outside the phloem. The phloem is well developed and forms a 
concentric cylinder. It consists of sieve tubes and phloem parenchyma. The 
xylem is endarch and comprises of vessels, fibres and xylem parenchyma. 

The pith is well developed and collenchymatous like the cortex. Resin 
canals (which are lined by numerous elongated secretory cells) and Ca-oxalate 
crystals of rosette and prismatic types are abundant in both cortex and 
pith (figures 2-4) . 

4.2 Sepals 

In surface view cells of the upper epidermis are larger as compared to those of 
the lower one (figures 5-8). Stomata are anisocytic or paracytic (figures 5-7) and 
those on the upper surface are sparse and larger in size. The guard cells have 
parallel striations radiating out from the stomata (figures 5 and 6). 

Cells over the veins are narrow and elongated (figure 6). Uni- to multicellular, 
uniseriate trichomes (figures 9 and 10) are present on both the surfaces but are 
more abundant on the lower one. 

A transverse section of the sepal is semi-lunar in outline. Cells of the 
upper epidermis have a thick cuticle on the outside ; are larger, and broader 
than long and have lignified walls and sunken stomata (figures 11 and 12). The 
epidermis is followed by a well demarcated hypodermis, again with lignified 
large rounded cells. In contrast the lower epidermis comprises of small, narrow, 
vertically elongated and unlignified cells. 

The ground tissue is many layered and has rounded collenchymatous cells. 
Up to four layers of these have greatly thickened cells similar to those in the pedicel 
cortex. The vascular bundles are scattered in the middle of the ground tissue. 
These are usually amphicribral and are surrounded by a bundle sheath formed 
of two to three layered sclerenchymatous fibres. Resin canals, lined by a layer 
of narrow epithelial cells are present throughout the ground tissue. The 



216 



Usha Shome, Shanta Mehrotra and H P Sharma 




Figures 13-21. 13. Upper epidermal cells of a petal. 14. A portion from figure 13 
enlarged to show the details. 15-17. Cells of the lower epidermis of a petal from apex, 
middle and base respectively. 18. Epidermal cells of a petal showing stomata arranged at 
right angle to the cells. 19 and 20. T. S. of petal from lower and middle portions. (Dia- 
grammatic). 21. A portion from figure 19 magnified to show the details. 
Abbreviations : CC, cell contents ; CU, cuticle ; GT, ground-tissue ; LEP, lower 

epidermis ; PH, phloem ; RC, resin canal ; ST, stomata ; UEP, 

upper epidermis ; XY, xylem. 



diameter of the canals is variable, being sometimes even larger than the 
vascular bundles. Disc shaped Ca-oxalate crystals are also present in 
abundance. 



Pharmacognostic studies of Mesua ferrea L. 217 

4.3 Petals 

The cells of the upper epidermis are uniform, laterally elongated, have 
anisocytic or paracytic stomata and are larger than those of the lower side 
(figures 13 and 14). The lower epidermal cells, on the other hand, vary 
considerably in shape and size from base to apex (figures 15-18). Epidermal 
cells in the apical region are squarish with wavy anticlinal walls (figure 15). 
In the lower region, however, these are narrow elongated and thick walled 
with comparatively smaller stomata (figures 16 and 17). The stomata are either 
oriented in the same plane or may be arranged at right angles to the epidermal 
cells (figures 17 and 18). 

The transverse section of a petal near the basal region shows a thick 
central portion and narrow curved margins (figure 19). The upper epidermis 
is undulating and has a thick cuticle on the outside. Sometimes even the 
anticlinal inner walls may also be thickened. A little below the epidermis 
one or two layers of cells show conspicously brown tannin containing cells. This 
layer is absent at higher levels (figure 20). The lower epidermis is smooth 
but cuticularised and is formed of comparatively smaller cells. The ground 
tissue is multilayered and is formed of parenchymatous cells with irregular 
outlines (figure 21). Resin canals are very few at lower levels but at higher 
levels these are quite abundant and are larger than the vascular bundles (figure 
20). The vascular bundles are arranged almost in a row, some large and some 
small. They are collateral and formed of a few xylem and phloem elements. 



4.4 Androecium 

The stamens are numerous, have short filaments and somewhat thick, elongated 
anther lobes. These are tetra-sporangiate (figures 22 and 23). Some fused stamens 
(figures 24 and 25), alongwith their respective vascular supplies, have also been 
observed. Occasionally the fusion may involve the anther lobes as well. In such 
cases, however, fusion of the anther lobes is not complete (figure 24). Another 
stamen in which only connectives are fused in the middle can be seen in a 
transverse section (figure 25). 

A semi diagrammatic representation of a transverse section of dehisced anther 
lobe can be seen in figure 26 and details of the vascular bundle in figure 27. 
The cells of the connective are elongated and papillate (figure 28). They are 
lignified and are positive to phloroglucinol cone, hydrochloric acid stain. 
The epidermis of the anther lobe is formed of large colourless highly cuticularised 
cells followed by a fibrous endothecium (figures 26, 29). 



4.5 Pollen grains 

Pollen grains are 3-(4-) zono-colporate with average size 35.89 X 48.71 /"m (range 
30.77 48.71 X 35.89 58.97^m) ; shape oblate to suboblate ; exine surface 
reticulate ; thickness 1 2 /*m, muriduplibaculate, colpus measures 13 X 33 /"m 
(figures 30-32). 



218 Usha Shome, Shanta Mehrotra and H P Sharma 

Table 1. Mesuaferrea Linn. : Behaviour of powder with different chemical reagents. 



SI. No. Treatment 


Observations 


Flower 


Stamen 


L 


Powder -f IN NaOH in methanol 


Blackish brown 


Brown with 








yellow tinge 


'2. 


Powder -f Picric acid (saturated) 


Yellowish 


Yellowish 






orange 


orange 


3. 


Powder -f Acetic acid 


Brown 


Orange yellow 


4. 


Powder -f Cone. HC1 


Brown 


Brown 


5. 


Powder + Cone. HNOs 


Reddish orange 


Reddish orange 


6. 


Powder -f Iodine (5%) 


Blackish brown 


Blackish brown 


7. 


Powder -f- SeliwanoflPs reagent 


Yellowish brown 


Yellowish brown 


8. 


Powder + Ferric chloride (5%) 


Dark blackish- 


Dark brown 






brown with 


with green 






green tinge 


tinge 


9. 


Powder + 40% NaOH + a few drops of 


Blackish- 


Light brown 




10% lead acetate 


brown with 


with yellowish 






yellowish 


white ppt. 






white ppt. 




10. 


Powder + Sudan III (alcoholic) 


Dark reddish 


Reddish orange 






orange 




11. 


Powder + Cone. HNOs 4- ammonia. 


Orange with 


Orange with 






ye] low ppt. 


whitish 








yellow ppt 



4.6 Gynaecium 

The ovary is superior, bicarpellary and syncarpous (figures 33 and 34). Epidermal 
cells of the ovary are small, hexagonal in surface view and have stomata measuring 
21.09 X 11.59 (range 19.00 26.60 X 9.50 14.25 f^m (figure 35). 

In transection the ovary is circular in outline (figures 36-38). The placentae 
are swollen and united at the base (figure 36). But at higher levels they separate 
(figures 36-39). The ovules arise one from each margin in each of the locales 
(figure 36). The placentation is thus axile tending to become parietal at higher 
levels. A transverse section passing through the style shows a stylar canal in the 
centre and three bundles, a dorsal flanked by two laterals on either side (figure 
40). Resin canals are abundant even at this level. 



5. Powder study 

The powder of the whole flower is greyish brown in colour, pleasant smelling and 
slightly astringent in taste. The powder of stamens is golden brown in colour 
pleasant smelling and astringent to taste. The powder of whole flower was sieved 
through No. 40 mesh, cleared in chloral hydrate and mounted in glycerine. A 
microscopic examination revealed the following elements, (i) Pieces of trichomes 
(figures 41 A-F). (ii) Pieces of tissues showing stomata (figure 41 G), simple pits 
(figure 41 H-I). (iii) abundant pollen grains in different planes (figures 41 J and K). 
The behaviour of the powdered drug with different chemical reagents was also 
studied and is presented in table 1 . 



Pharmac agnostic studies of Mesua ferrea L. 



219 




Figures 22-41. 22. A stamen showing one antherlobe. 23. T. S. of the antherlobe showing 
tetra sporangiate condition (Diagrammatic). 24. A stamen showing two anther lobes 
partially fused. 25. T. S. of the fused antherlobe. (Diagrammatic). 26. T. S. of dehisced 
amherlobe-(Semi-diaprammatic). 27. Vascular bundle from figure 26 magnified to show 
the details. 28. Cells from the connective (surface view). 29, A portion of antherlobe 
wall magnified showing cuticularised epidermis and endothecium. 30. A pollen grain 
showing wall stratification and ornamentation. 31. A palynogram of pollen. 32. L O 
pattern of pollen ornamentation. 33 and 34. Diagrammatic representation of very young 
and slightly mature ovaries. 35. Epidermal cells from ovary showing stomata in surface 
view. 36-38. Serial T. S. of ovary from base to apex (Diagrammatic) 39. L. S. of ovary 
(Diagrammatic). 40. T. S. of style (Diagrammatic). 41. A- K Different tissues from the 
drug powder. For details refer to the text. 
Abbreviations : AL, antherlobe ; C, connective ; CO, Colpus ; CU, cuticle ; 

epidermis ; EX, exine ; END, endothecium, 

ovary ; OV, ovule ; OW, ovary wall ; PG, 

phloem ; PL, placentum ; RC, resin canal ; 

ST, stomata ; STI, stigma ; VB, vascular bundle ; VS, Vascular 

supply ; XY, Xylem. 



EP, 

FI, filament ; O, 
pollen grain, PH, 
SC, stylar cavity ; 



Pharmacognostic studies of Mesua ferrea L. 
Table 2. Mesua ferrea L. : Fluorescence analysis 



221 



SI. No. 



Treatment 



Fluorescence under UV light 



Stamen 



Flower 



1. 


Drug as such 


Purplish brown 


Blackish brown 


2. 


Powder + Nitrocellulose in a myl acetate 


Olive green 


Purplish brown 


3. 


Powder + IN HC1 


Reddish brown 


Blackish brown 


4. 


Powder + IN HC1 -f Nitro-cellulose 


Dirty green 


Dark brown 




in amyl acetate 




with purple tinge 


5. 


Powder + aq. IN NaOH 


Dark brown 


Dark brown 


6. 


Powder + aq. IN NaOH + Nitro- 


Blcckish brown 


Blackish brown 




cellulose in amylacetate 






7. 


Powder + 1 N NaOH in Methanol 


Blackish brown 


Blackish brown 


8. 


Powder + IN NaOH in Methanol -f 


Greenish brown 


Greenish brown 




Nitrocellulose in amylacetate 






9. 


Powder + 50% Nitric acid 


Reddish brown 


Dark brown 


10. 


Powder + 50% Sulphuric acid 


Greenish brown 


Reddish brown 






with red tinge 





The fluorescence analysis was carried out according to the method described by 
Chase and Pratt (1949) and Kokoski et <2/(l958). The observations are recorded 
in table 2. 



6. Phytochemical studies 

Phytochemical characters of the drug were studied separately for the whole flower 
and stamens respectively. The determination of ash values, sugar, tannins, 
alcohol and water soluble extractives were made from air dried material. The 
procedures recommended by Anonymous (1966) were followed for calculating 
total ash, acid insoluble ash, alcohol and water soluble extractive percentages, 
whereas for calculating tannins, total and reducing sugars Fohlin-Denis reagent 
and Shaffer's Somogyi micromethods prescribed by AOAC (Anonymous 1965) were 
followed. 
(1) Ash Flowers Stamens 

(a) Total ash 9.297% 2.370% 

(b) Acid insoluble ash 3.730% 0.230% 



(2) Tannins 

(3) Sugars 

(a) Total sugars 

(b) Reducing sugars 

(c) Non-reducing sugars 



(4) 
(5) 

(6) 



Volatile oil 

Alcohol soluble extractive 
(I.P. Method) 

Water soluble extractive 
(I.P. Method) 



4.4% 

1.244% 
0.960% 
0.350% 

0.25% 
21.57% 

14.86% 



3.76% 

1.852% 
1.160% 
0.692% 

1.75% 
25.31% 

1 5.40% 



222 



Usha Shome, Shanta Mekrotra and HP Sharma 





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Pharmacognostic studies of Mesua ferrea L. 



223 













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47 



Figures 42-47. A Stamen extractive 
B Flower extractive 

BR, brown ; BY, bright yellow ; D.BR, dark brown ; GY, greenish yellow ; LBR, 
light brown ; LP, light purple ; LRY, light reddish yellow ; LY, light yellow ; P, 
purple ; PY, purplish yellow; RO, reddish orange ; Y, yellow ; YB,Yellowish brown. 



Beside the above 15 gms of air dried powdered flowers and stamens were 
sxtracted separately in a Soxhlet apparatus with hexane, benzene, chloroform, 
ilcohol and water successively and percentage of each extractive calculated after 
evaporation of respective solvents. These were further screened for steroids, 
and triterpenoids (L.B. test - Peach and Tracy 1955) ; flavonoids (Shinoda's test - 
Loc.Cit.); alkaloids (Mayer's reagent - Loc.Cit.) ; and tannins (ferric chloride 
test-Z0c.CzY.)- The results obtained are presented in table 3. 



224 



Usha Shome, Shanta Mehrotra and H P Sharma 



The hexane and benzene extractives of both the parts show presence of 
triterpenoids and resins. Percentage of reducing sugars in alcoholic extractive, 
in the case of flower, was found to be quite high. 

Water soluble portion of both the parts show presence of tannins and saponins. 
Concentration of sapcnins, however, was quite high in the case of flower. 

Thin layer chrornatography of the above extractives was also carried out 
(figures 42-47). The hexane soluble extractive of stamens shows a larger number 
of discrete spots as compared to that of the flower (figure 42). With hexane-ethyl 
acetate (72 : 29) as solvent system the hexane extractive separates into spots with 
shades of yellow and brown in the case of flower (figure 43b). Those of the 
stamens are, however, purple (figure 43a). 

The benzene extractives (figure 44) are quite comparable. Again, chloroform 
extractive resolved into 4 spots each with toluene-acetone (35 : 15) as the solvent 
system. Of these the two upper most and the lowermost on Rf 0.96, 0.82 and 
0.35 are comparable. However, an oval yellow patch at Rf 0.43 and a brown spot 
at Rf 0.65 in stamen and flower extracts respectively are diiferent. The same 
extractive resolved into 5 spots in flower and 6 spots in stamens with solvent 
system chloroform : acetone : methanol, 32.5 : 15 : : 2.5 (figure 46). It is interesting 
to note that the alcoholic portion of the stamens gave 8 spots while the flower 
portion gave only 2 spots (figure 47) (Solvent system : formic acid-ethyl formate- 
toluene (1 : 4 : 5), The Rf values of different extractives have been recorded in a 
tabular form (table 4). 



Table 4. Mesuaferrea Linn : TLC Rf. Values 



Extractive 


Solvent system 


Rf Values 


Flower 


Stamen 


Hexane 
soluble 
extractive 


Hexane : Benzene 
(30 : 70) 


0.067,0.12,0.19,0.82, 
0.93 


0.04,0.10,0.17,0.25, 
0.64,0.89,0.95 


Hexane 
soluble 
extractive 


Hexane : Ethyl- 
acetate (72 : 29) 


0.76,0.83,0,91 


0.79,0.83,0.91,0.99 


Benzene 
soluble 
extractive 


Benzene : Chloroform 
Acetone 
(3:1: 0.05 cc.) 


: 0.46,0.63.0.80,0.95 


0.50,0.63,0.79,0.99 


Chloroform 
soluble 
extractive 


Toluene : Acetone 
(35 : 15) 


0.35,0.65,0.83,0.96 


0.35,0.43,0.81,0.97 


Chloroform 
soluble 
extractive 


Chloroform : Acetone 
Methanol 
(32.5 : 15 : 2.5) 


0.28,0.43,0.59, 

0.78,0.90 


0.28,0.41,0.68,0.76, 
0.00,0.93 


Alcohol 
soluble 
extractive 


Formic acid : 
Ethyl formate : 
Toluene 
(1:4:5) 


0.19,0.36,1.0 


0.03.0.06.0.08, 
0.23,0.29,0.35 
0.4,0.48,0.99 


Spraying reagent 2% H2$O4 
Underlined Rfs denote spots which 


are distinctive. 





Pharmacognostic studies of Mesua ferrea L. 225 

acknowledgements 

"he authors are extremely grateful to Dr T N Khoshoo, Director, National 
Jotanical Research Institute, for his continued and keen interest in the progress 
>f this work. The late Mr K M Vaid was greatly helpful in procuring plant 
naterial for us for which we are sincerely thankful to him. We are also thankful 
o Dr Mithilesh Chaturvedi for her helpful suggestions regarding the pollen 
tudy. Our thanks are also extended to Messrs A Jha, R S Ojha and M K Tandon 
or technical assistance. 



References 

Vnonymous 1962 The wealth of India. A dictionary of Indian Raw Materials and Industrial Product 

L-M (New Delhi : CSIR) 
Anonymous 1965 Official methods of analysis of the AOAC (Benjamin Franklin Stn : Washington 

DC 20044) 

Vnonymous 1966 Indian pharmacopoeia 2nd ed (Delhi : Government of India) 
Vnonymous 1970 Hamdard pharmacopoeia of Eastern Medicine (Karachi: The Times) 
Ui U S 1967 Nagkeshara as known in South India : J. Res. Indian Med. 2 57-65 
3ala K R and Seshadri T R 1971 Isolation and Synthesis of some coumarin components of Mesua 

ferrea seed oil. Phy to chemistry 10 1131-1134 
Bhattacharya P, Chatterjee D, Chakrabarti A and Chakrabarti D P 1979 Synthesis of mesuagin a 

plant antibiotic from Mesua ferrea Linn. Indian J. Chem. B17 11 1-112 
ihavmisra 1949 Sri Bhavprakash (Varanasi : The Chowkhamba) 
rhakraborty O P, Purkayastha M and Bose P K 1959 On the antibiotic properties of some 

constituents of Mesua ferrea L. Proc. NatL Inst. Sci. India B25 8-11 
3hakraborty D P and Bose P K 1960 On the constitution of raesuol, the bitter antibiotic principle 

of M. ferrea L. Proc. NatL Inst. Sci. India Pt. A26 1-11 
^hakraborty D P and Das B C 1966 The structure of mesuol (an antibiotic from Mesua ferrea 

L.) Tetrahedron Lett. 46 5727-5730 

Hharaka 1949 The Charaka Samhita (Jamnagar : Gulab Kunverba Ayurvedic Society) 
3iase C R and Pratt R J 1949 Fluorescence of powdered vegetable drugs with particular reference 

to development of a system of identification ; /. Am. Pharrn. Assoc. 38 324-331 
rhunekar K C 1960 Commentary on Bhavprakash Nighantu of Shri Bhavmishra (Varanasi : The 

Chowkhamba) 
Dutt P, Deb N C and Bose P K 1940 A preliminary note on mesuol, the bitter principle of Mesua 

ferrea ; /. Indian Chem. Soc. 17 277-279 
>ymock W, Warden C J H and Hooper D 1885 (reprinted in 1972) Pharmacographia indica vol. 

History of the principle drugs of vegetable origin met within British India. (Delhi: Vivek 

Vihar D-42) 
Urlikar K P and Basil B D 1933 Indian Medicinal Plants 2nd ed (Allahabad : L N Basu and 

Co,) 
Cokoski J, Kokoski R and Slama F J 1958 Fluorescence of powdered vegetable drugs under ultra 

violet radiation /. Am. Pharm. Assoc. 47 715 
""Mudaliar Murugesa C S 1957 Gunapadam-vegetable, 1st ed. Section (in Tamil) (Government 

Press) 318-320 

^adkarni K M 1937 Indian Materia M edica (Bombay : Karnataka Printing Press) Vol. 7 
Peach K and Tracy M V 1955 Modern methods of plant analysis (Heidelberg : Springer Verlag) 

3rd and 4th Vol 
Satkopan S and Thomas P J 1967a The identity of Ayurvedic Market Drugs. I-A. Nagkeshara 

Mesua ferrea L. Nagarjun 10 461-467 
Satkopan S and Thomas PJ 1967 The identity of Ayurvedic Market Drugs 1-B Nagkeshara 

(Ochrocarpus longifolius} B and H, Nagarjun 11 67-72 



226 Usha Shome, Shanta Mehrotraand H P Sharma 

SatkopanS and Thomas P J 1968 The identity of Ayurvcdic Market Drugs Pt|l-C, Nagkeshara 

(Cinnamomum sps. Nagarjun) 474-477 

Sharma P V 1978 Dravyaguna vigyana 4th ed. (Varanasi : The Chowkhamba) 
Subrarnanyam R M and Subba Rao N V 1969 Isolation of mammeisin from the seeds of Mesua 

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Subramanyam R M, Srimannarayana G and Subba Rao N V 1975 Structure of mesuanic acid ; 

Indian J. Chem. 12 884-886 

Sushruta 1952 Sushmta Samhita (Kanpur : Sri Saraswati Pustakalaya) 

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Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 3, June 1982, pp. 227-234. 
Printed in India 



Effect of COz in ovecroming self-incompatibility barriers in Brassica 
carnpestris L. van toria 



A S DHALIWAL* and C P MALIK 

Department of Botany, Punjab Agricultural University, Ludhiana 141004, India 
*Seed Technology Section, Department of Plant Breeding, Punjab Agricultural 
University, Ludhiana 141 004, India 

MS received 1 May 1981 ; revised 14 May 1982 

Abstract. CO2 mediated induction of pollen adhesion, germination, tube pene- 
tration, tube growth through the stylar tissue and seed set was studied in Brassica 
campestris L. var. toria following incompatible pollination. This report stresses the 
effect of COa not at a single step but at various levels which normally prevent and/or 
hinder successful fertilization and seed set. The possible role of CO2 in overcoming 
these barriers is discussed. 

Keywords. Brassica campestris L. var. toria; incompatibility; COa; seed set. 



1. Introduction 

The incompatibility reaction in Brassica is sporophytically controlled with multiple 
S-alleles (Bateman 1955 ; Thompson 1957). Double cross hybrids raised from 
self-incompatible inbred lines would give low yielding crop because of high level of 
cross-incompatibility with each other. In recent years, two developments have 
taken place to overcome such problems. One is based on the manipulation of 
genetic attributes (Thompson 1 978) and the other involves putting the self-incom- 
patible plants in an atmosphere of 3 to 5% CO 2 after selfing the flowers (Nakanishi 
et at 1969, 1975). The latter technique is now successfully employed in obtaining 
self-seeds. 

Following incompatible pollination various check points exist at the level of 
pollen germination, adhesion, germination, tube penetration, tube growth through 
the stylar tissue and ultimately seed set. The present study describes the effect of 
CC>2 on the aforementioned levels following illegitimate pollination. 



2. Material and methods 

Seeds of Brassica campestris L. var. toria were obtained from the Department of 
Plant Breeding, Punjab Agricultural University, Ludhiana and the plants were 

227 



228 A S Dhaliwal and C P Malik 

raised in the Botanical Garden. Experiments were performed in full flowering 
season to avoid early or late season effect. 

Excised pistil technique was employed to study the effect of CO 2 on pollen 
adhesion, germination, pollen tube penetration and growth through the stylar 
tissue. Flower buds were emasculated in the evening before opening. Next 
morning the flowers were removed and the pedicels with pistils were embedded in 
1% agar medium in small glass vials. Self- and cross-pollinations were done with 
a fine brush to obtain a thin and even coating of pollen on the stigmatic surface. 
The vials containing self- and cross-pollinated pistils were put in a glass container 
which was sealed with parafilm. CO 2 was injected with the help of an air tight 
syringe to obtain 4% concentration. After incubation for 6 hr at 222C pollen 
adhesion, germination and tube penetration into stigmatic papillae were studied by 
staining pistils with cotton blue (0.1%) in lactophenol. 

Growth of pollen tubes in cross-pollinated pistils without CO 2 and self-pollinated 
pistils with 4% CO 2 was followed at times ranging between 6 to 30 hr after polli- 
nation. The number of pollen tubes in 0.5 mm region alongwith the length of the 
pistil were counted in the two situations (three replicates of six flowers each). 

The emasculated flowers were pollinated with self- and cross-pollen and enclosed 
within known volume of polythene chamber and 4% CO 2 was injected with an air 
tight gas syringe. After exposing the flowers for 6 hr the polythene chamber was 
replaced by emasculating bags. The number of seeds formed per siliqua was deter- 
mined 50-60 days after pollination. 



3. Results 

Due to full operation of self-incompatibility, adhesion of self pollen to the stigmas 
was low. Those which made contact with the papillae showed poor germination. 
Figure l(a, b, c) depicts the adhesion of pollen grains in three situations. Figure 
la shows incompatible pollination with very few pollen grains adhering to the 
stigmatic surface and without any germination. Figure Ic is that of compatible 
pollination where most of the pollen grains are adhering to the papillae. Pollen 
grains show germination and pollen tubes grow in the stigmatic and stylar tissues. 
The firm attachment of pollen grains, following self-pollination, was observed when 
they were enclosed in an atmosphere of CO 2 (4%) (Figure Ib). 

The data given in table 1 show that no pollen tube was recorded in the pistil 
following self-pollination. In majority of the styles, the longest pollen tube 
reached almost the same position in response to both self-pollination + COa 
and cross-pollination styles. The data on percentage of pollen tubes present at 
different levels of the pistil after 24 hrs of pollination are set in figure 2. A 
gradual reduction in the number of pollen tubes from the top of the pistil down 
into the ovary was noticed. A decrease in cross pollen tubes as well as self 
pollen tubes (in the presence of CO 2 ) is evident. This decrease was more 
pronounced in the cross pollen tubes as compared with the self pollen tubes with 
CO 2 . 

Table 2 shows the number of seeds produced per siliqua after different pollination 
treatments. The number of seeds per siliqua following bud pollination was nearly 



Self -in compatibility barriers in Brassica campestris L. 



229 





Figure 1. Pollen adhesion, germination and tube penetration after 6 hr pollination. The 
stigmas alongwith styles were stained with 0.1% cotton blue in lactophenol. (a) Noticed 
very few pollen grains adhering to the stigmadc surface following self-pollination 
(b) The number of pollen grains sticking to the stigmatic surface is more as compared 
with figure (a) following CO2 treatment. Some pollen tubes are penetrating the 
stigmatic papillae, (c) Most of the pollen grains germinate and penetrate following 
cross-pollination (pollen tubes arrowed). 



Self -incompatibility barriers in Brassica campestris L. 



231 



Table 1. Growth of pollen tubes in the pistil at different times after pollination in cross-, 
self- and self 4- CO2 pollinations. Data are the mean of six flowers. 



Time after Pollination 
pollination treatment 
(hr) 


Maximum 
pollen tube 
length (mm) 


Rate of pollen tube growth 
mm/br 


Max. rate 


Mean rate 


6 CP 




0.66 


0.11 


0.05 


SP + 


C0 2 


0.45 


0.08 


0.04 


SP 




0.03 


0.01 


0.00 


11 CP 




1 .40 


0.13 


0.07 


SP 4- 


C0 a 


1.35 


0.12 


005 


18 CP 




3.00 


0.17 


0.09 


SP -4 


C0 2 


2.70 


0.15 


0.05 


24 CP 




3.50 


0.15 


0.08 


SP-h 


CO* 


3.00 


0.13 


0.07 


30 CP 




4.00 


013 


0.07 


SP + 


CO* 


3.60 


0.12 


0.05 



Table 2. Effect 



(4%) on seed production following self- and cross-pollination. 



Pollination 
treatment 




Self seeds 


Cross seeds 




Number of 
fruits 


Number of 
seeds per 
fruit 


Number of 
fruits 


Number of 
seeds per 
fruit 


Bud 
Open flower 
CO 2 
-hCOa 


36 



34 


17.3 

0.0 
8.2 


30 

38 
30 


18.2 

19.6 
18.8 



same in the two situations i. e, self- and cross-pollinations. No seed set was 
recorded when the freshly opened flowers were self-pollinated. However, about 8 
seeds per siliqua were formed when self-pollinated flowers were kept in 4% CO 2 
atmosphere for about 6 hr. CO 2 did not affect seed set after cross-pollination. 



4. Discussion 

The strong self-incompatibility barrier exhibited by Brassica campestris var. toria 
depends on the recognition and the rejection reaction. The events grouped in these 
two stages constitute the various check points as mentioned earlier. The rejection 
response is characterized by callose deposition. 

The adhesion of the pollen grain to thestigmatic surface seems to be the initial 
but most important event to ensure pollen germination and tube penetration. CC>2 
plays an important role in the adhesion of the pollen grains to the self stigma 
surface as is evident from the presence of more pollen grains on the self stigma 
following CO 2 treatment than control (without COa) even 6 hr after pollination. 



232 



A S Dhaliwal and C P Malik 



JOO 
in 



A 

5 80 

c 

I 60 



.Cross 




0.5 i 2 3 

Oistanc* from top of stigma(mm) 

Figure 2. Percentage of pollen tubes at different levels from top of the stigma following 
24 hr of pollination. 



If pollination is compatible the tryphine is induced to 'gel' and promotes both 
adhesion and hydration of the pollen grain (Dickinson et al 1980). The number 
of pollen grains adhering to the stigma surface following cross-pollination is higher 
than self-pollination. Stead et al (1980) proposed that differences in the degree/ 
extent of adhesion may result from physical or chemical alteration(s) taking place 
in the components that bind the pollen to the stigma surface. They suggested the 
involvement of stigma surface proteins in the pollen grain adhesion which have a 
rapid turnover rate. CO 2 has been shown to affect changes in the kinetic 
behaviour of allosteric proteins (Mitz 1979). CO 2 may be affecting pollen adhesion 
through the above mentioned processes. CC>2 has been reported to affect protein 
synthesis in maize roots (Splittstoesser 1966), Chlorella (Miyachi and Hogestu 
1970) and germinating Amaryllis vittata pollen grains (Sharma et al 1981). 

Dhaliwal and Malik (1980) and Dhaliwal et ah (1981) have suggested the role 
of COa in pollen hydration and recognition rejection response which are essential 
for the activation of enzymes and acceptance of pollen grain, respectively. Non- 
specific esterases have been suggested to be involved in the active cutinase complex 
essential for the breakdown of cuticle. C0 2 stimulates the leaching of these 
esterases on to the stigma surface following illegitimate pollination (Dhaliwal and 
Malik 1982). 

COs increased both pollen germination and tube penetration following incom- 
patible pollination. CO 2 action was correlated with the metabolic changes of 
pollen tubes and/or papilla cells at the time of their attachment (Nakanishi et al 
1969). The growth rates of pollen tubes in self-pollination + COs and cross- 
pollination styles indicate that the fastest self pollen tube (in presence of COs) grew 
nearly as fast as the fastest cross pollen tube. The gradual reduction in the number 
of pollen tubes from the top of the pistil down into the ovary indicate much 
variation in the rate of pollen tube growth. Thus, the most rapidly growing tubes 
registered two to three times faster rate than the average tubes. This decrease in 
the number is more pronounced in the cross pollen tubes as compared to self pollen 
tubes in the presence of CO 2 . The variation in the growth rate may be attributed 
to competition between the pollen tubes for the nutrients. Clearly, single pollen 



Self-incompatibility barriers in Brassica campestris L, 233 



Pollen lands on the stigma 

Adhesion 

Hydratjon 

Germination 

Cutinase activation 

Ptrwtration *-* I 

Orowth through 
the stylar 





Figure 3. Effect of COs (4%) on various check points that lead to self-incompatibility in 
Brassica campestris L. 

tube growing through the style will have less competition with each other than 
several of them growing at the top of the stigma. The seed set increased when 
the freshly opened flowers were self-pollinated and were kept in 4% COg atmos- 
phere for about 6 hrs. CO 2 did not affect seed set after cross pollination. 

Talcing the present discussion in conjunction with our previous inferences on the 
role of CO 2 on various check points is indicated (figure 3). We believe that CO 2 
act at several stages promoting successful fertilization and seed set in incompatible 
pollination. 



Acknowledgements 

Financial assistance in the form of Junior Research Fellowship to ASD from 
Government of India, Department of Atomic Energy is gratefully acknowledged. 



References 

Bateman A J 1955 Self-incompatibility systems in angiosperms. III. Heredity 9 53-58 

Dhaliwal A S and Malik C P 1980 Effect of relative humidity and COa on the shape, volume and 

fresh weight of Brassica campestris L. pollen in vitro ; Indian J. Exp. Biol. 18 1522-1523 
Dhaliwal A S and Malik C P 1982 Release of esterases from incompatible Brassica 

campestris L. pollen in vitro and in vivo ; Indian J. Exp. Biol. 20 95-96 
Dhaliwal A S, Malik C P and Singh M B 1981 Overcoming incompatibility in Brassica campestris 

L. by carbon dioxide and dark fixation of the gas by self- and cross-pollinated pistils; Ann. Bot. 

48 227-233 
Dickinson H G, Roberts I N and Stead A D 1980 The role of exine held substances in the 

development and behaviour of the pollen tube in Brassica : 5th International Palyrrological 

Conference, Cambridge, Abs. 
Mitz M A 1979 COa biodynamics: a new concept of cellular control; J. Theor. Biol. 80 537-551 



234 A S Dhaliwal and C P Malik 

Miyachi S and Hogestu O 1970 Effect of pre-illumination with light of different wavelengths or> 

subsequent CO 2 fixation in Chlorella cells ; Can. J. JBot. 48 1203-1207 
Nakanishi T, Esashi Y and Hinata K 1969 Control of self- incompatibility by CO 2 gas in 

Brassica: Plant Cell Physiol. 10925-927 
Nakanishi T and Hinata K. 1975 Self seed production by CO-2 gas treatment in self-incompatible 

cabbage; Euphytlca 24117-120 
Sharma S, Singh M B and Malik C P 1981 Dark CO2 fixation during germination of Amaryllis 

vittata pollen in suspension cultures ; Indian. /. Exp. Biol 19 710-714 
Splittstoesser W E 1966 Dark COa fixation and its role in the growth of plant tissue; Plant PhysioL 

41 755-759 
Stead A D, Roberts I N and Dickinson H G 1980 Pollen-pistil interaction in Brassica oleraceai 

Events prior to pollen germination; Planta 146 21 1-216 
Thompson K. F 1957 Self-incompatibility in narrow-stem kale Brassica oleracea var acephala L 

Demonstration of a sporophytic system; /. Genet. 55 45-60 
Thompson K 1978 Application of recessive self-incompatibility to production of hybrid 

rapeseed ; 5th International Rapeseed conference. Abstract 12.6-16.6 1978 Malmo, Sweden* 



oc. Indian Acad. Sci., (Plant ScL), Vol. 91, Number 3, June 1982, pp. 235-240. 
Printed in India 



harmacognosy of the stems of Portulaca quadrifida L. and 
ortulaca oleracea L. 



J LAL* and A M KHAN 

Pharmacognosy Research Unit, Central Council for Research in Unani Medicine > 
Department of Botany, Aligarh Muslim University, Aligarh 202 001, India 
*Dmg Standardization Research Circuit, Central Council for Research in Unani 
Medicine, National Botanical Research Institute, Lucknow 226 001, India 

MS received 3 December 1980 ; revised 5 September 1981 

Abstract. Pharmaco gnostic details of the stems of P. quadrifida L, and P. oleracea L , 
are reported to distinguish one from the other. 

Keywords. Pharmacognosy; Portulaca quadrifida L.; comparison with P. oleracea JL. 



Introduction 

wtulaca quadrifida and Portulaca oleracea, commonly known as Chhota Luniya 
d Bara Luniya respectively, are succulent annual herbs and grow abundantly in 
Id state throughout warmer regions of India (Anon 1969). It has been suggested that 
)m the therapeutic point of view they are quite similar and one can be used as 
mbstitute for the other by the drug dealers (Dymock et al 1980 ; Kirtikar and 
LSU 1975). Detailed chemical analysis of leaf and stem of P. oleracea was 
>rked out by Sadaaa and Ahmed (1947). Recently Lai (1980) described the 
.armacogaostic features of the leaf of P. quadrifida. The pharmacognostic 
tails of the stems of both species are presented in this paper. 



Materials and methods 

esh plants of P. quadrifida and P. oleracea collected from the Botanical Garden 
Aligarh Muslim University were fixed in FAA. After usual processing, free 
nd and microtome sections were cut and stained in safranin and fast green. 
uorescerice analysis and extractive and ash values of the powdered mass of stems 
:re carried out by Chase and Pratt (1949) and Indian Pharmacopoeia (Anon. 1966) 
sthods respectively. . 



236 



/ Lai and A M Khan 



2.1 Macroscopic characters 

The stem of P. quadrifida is succulent, diffuse, filiform, purple in colour at 
maturity, less than a milimeter in diameter; on crushing mucilaginous; mucilage 
slimy; rooting at the nodes; nodal appendages many, pilose, white; internodes 
1.5 to 3 cm long; without any smell and taste acidic. The stem of P. oleracea 
(figure 8) on the other hand, is about 2 mm in diameter; internodes 1.5 to 3.5 cm 
long; nodal appendages less in number, minute, scarious. The other morphological 
characters are more or less similar to P. quadrifida. 



2.2 Microscopic characters 

The cross-section of the stems of both the species are almost circular (figures 2 
and 9). The epidermal cells are polygonal in shape in both species and are 
surrounded externally by thick striated cuticle. The outer wall of some of the 




Figures 1-7. 1. A twig of the plant Portulaca quadrifida L. (X 2.5). 2. T.S. stem 
(diagrammatic) (X35). 3A, B. Anatomical details of a portion of figure 2 (X2973). 
4. Epidermal cell in T.S. containing acicular crystals (X2973). 5A, B, C D 
and E. Prismatic crystals and druses (X2973). 6 and 7. Macerated xylem 
vessels (X2973). 



Pharmacognosy of P. quadrifida L. and P. oleracea L. 



237 



epidermal cells of P. quadrifida slightly bulge out. Acicular crystals which appear 
as crystals and in cross section are present m some of the epidermal cells of 
P. quadrifida (figure 4). Epidermis is, followed by 2-3 layers of collenchyma cells 
in the stem of P. oleracea (figures 9 and 10A), whereas it is parenchymatoiis in 
P. quadrifida (figure 3A). The parenchyma in both species consists of thin-walled, 
more or less isodiametric cells with large intercellular spaces. These cells are 
loaded with starch grains, simple as well as compound. The compound starch 
grains are usually 2 or 3 celled (figures 5F and 11). Druses, prismatics, acicular 
crystals and colourless mucilage cells are commonly present in both the species. 
The endodermis in both species is not well defined. Collateral vascular bundles 
are arranged in a ring in both but the number of bundles in P. oleracea is almost 
double or even more than those in P. quadrifida (figures 2 and 9). Pith consists of 
thin-walled isodiametric cells some of which contain calcium oxalate crystals 
(figure 3B). The macerated xylem consists mostly of helical and scalariform 
vessel elements with simple perforation (figures 6, 7A> B, C and 12) and fibres 
with intrusive growth. 
The measurement of different tissues and cells is given in table 1. 



Table 1. Measurement of different tissues and cells in microns. 



P. quadrifida 



P*oleracea 



Cuticle M = 3.33 6.66 thickness 
Epidermis M = 23.31 x 6.66 39.96 

x 19.93 79.92 x 49.94 

Parenchyma M = 6.66 13.32 93.24 diameter 
Collenchyma Absent 

Vessels M = 15.65 19.98 33.30 diameter 
T = 79.92 X 23.31 123.21 
x 63.27 404.0 X 31.10 
Fibres M = 15.55 23.32 31.10 diameter 
T = 155.50 X 23.32 233.25 

X 31.10 

Pith M = 16.65 23.31 66.60 diameter 
Druse M = 33.30 39.96 49.96 diameter 
Starch grains M = 3.33 9.99 13.32 diameter 



3.66 6.89 thickness 
39.6 x 26.4 42.9 x 29.7 

49.6 x 36.4 

46.8 124.8 156.0 diameter 
M = 33.3 66.6 diameter 
9.99 _ 26.24 29.97 diameter 
90.0 x 28.54 223.31 x 68.27 

532.98 X 35.30 



46.8 124.8 153.50 diameter 
46.8 78.0 124.8 diameter 
1.66 3.33 16.50 diameter 



M = measurement in cross-section; T = measurement of macerate 



2.3 Macerate 

Macerate consists of cuticle, parenchyma cells, xylem vessel elements, fibres, starch 
grains and druses (figures 5, 6, 7, 11 and 12). 



2.4 Extractive and ash values 

Extractive and ash values were determined according tcf Anoja (1966) and the 
results are given in table 2. 



238 



J Lai and A M Khan 




Figures 8-12. 8. A twig of the plant Portulaca oleracea L. (Xl). 9. T.S. 
stem (diagrammatic) (X20). 10A, B and C. Anatomical details of a portion 
of figure 9 (X2973). 11. Starch grains and druses (x2973). 12. Macerates 
(X2973). 



Table 2. Extractive and ash values of the sterns of P. quadrifida and P. oleracea. 



Extractive and ash values 


P. quadrifida 


P. oleracea 


Water soluble extractive 


19.73% 


25.00% 


(chloroform water) 






Alcohol soluble extractive 


10.32% 


18.50% 


Total ash 


9.09% 


25.18% 


Acid insoluble ash 


0.63% 


3.18% 



2.5 Fluorescence analysis of the powdered drugs 

The stem powders prepared by drying fresh specimens at 60C # were chemically 
treated and exposed to ultraviolet light. The fluorescence observed is recorded 
in table 3. 



Pharmacognosy of P. quadrifida L. and P. oleracea L. 239 

Table 3. Fluorescence analysis of the stem powders of P. quadrifida and P. oleracea. 



Chemical treatments 


Fluorescence 




P. quadrifida 


P. oleracea 


>owder as such 


Light green 


Dark green 


>owder mounted in IN NaOH 






in methanol 


Yellowish green 


Green 


*owder mounted 4n nitrocellulose 


Orange 


Brownish green 


owder treated with IN NaOH 






in methanol and mounted in 






nitrocellulose 


Brown 


Dark brown 



1.6 Chromatographic studies 

Vlcohol extracts of the stems of P. quadrifida and P. oleracea were subjected to 
bin layer chromatography with the solvent system methanol : chloroform (3:7). 
rhe plates were developed by iodine vapours. They showed the presence of four 
pots (figure 13) with Rf Values 0.05, 0.65, 0.73, 0.90 and 0.05, 0.65, 0.76, 0.88 
espectively. This indicates thai the two species have more or less the same 
jhemica 1 . constituents. 



Conclusion 

two species differ considerably in their measurements of cells and tissues 
[table 1), extractive and ash values (table 2) and fluorescence analysis of the 
)owdered drugs under uv light (table 3). Little or no differences were obtained 
n TLC studies as shown in the chromatogram (figure 13), Undoubtedly, the 



Chromotogram 






o o 

o o 

t-f 



A - Alcoholic extract 

of portulaca oleracea linn 

B- Alcoholic extract of 
portulaca quadrifida linn 



Figure 13. Chromatogram. 



240 / Lai and A M Khan 

stems of P. quadrifida and P. oleracea differ morphologically and anatomically. 
They also differ in extractive and ash values as well as in fluorescence analysis 
under UV light; yet in view of the similarities in therapeutic properties the stem 
of P. quadrifida can be used as a substitute for that of P. oleracea. 



Acknowledgements 

The authors are thankful to the Director, Central Council for Research in Unani 
Medicine for his unceasing encouragement and for providing financial assistance. 



References 

Anon 1969 Portulaca Linn. (Portulacaceae); The Wealth of India (New Delhi: CSIR), 

pp. 219-.221 
Anon 1966 Determination of ash Pharmacopoeia of India (New Delhi: Govt. of India) 

pp. 947-949 
Chase C R and Pratt R 1949 Fluorescence of powdered vegetable drugs with particular 

reference to development of system of identification; /. Am. Pharm. Assoc. (Sci. ed). 

38 pp. 324-331 
Dyrnock W, Warden C H J and Hooper D 1980 Portulacaceae; Pharmacographia Indica 

(Dehra Dun : B S M P Singh), 1 pp. 158-159 
Kirtikar K R and Basu B D 1975 Portulacaceae; Indian medicinal plants (Reprint Edition) 

(Dehra Dun: B S M P Singh) 1 pp. 240-245 
Lai J 1980 Foliar Pharmacognosy of Portulaca quadrifida Linn. Bull. Med. Ethnobot. Res. 

I pp. 46-54 
Sadana J C and Ahmad B 1947 Observations on the Carotenoid pigments of common Indian 

vegetables; /. Sci. Ind. Res. B6 pp. 47-52 



Abbreviations : CA : Cambium; ACO : Angular collenchyma; CR : Druses; 
cs : Crystal sand; csx : Compound starch grains; cu : Cuticle; EP : Epidermis; 
INV : Involucre; LF : Leaf; MA : Nodal appendages; PAR : Parenchyma; 
PHI : Phloem; PIT : Pith; PR : Prismatic crystal; ST : Starch grains; 
VB : Vascular bundle; XY : Xylem. 



Proc. Indian Acad. Sci. (PJant ScL), Vol. 91, Number 3, June 1982, pp. 241-253. 
O Printed in India. 



Structure and function of a sub-tropical humid forest of 
Meghalaya L Vegetation, biomass and its nutrients 



JASBIR SINGH and P S RAMAKRISHNAN 

Department of Botany, School of Life Sciences, North-Eastern Hill University, 
Shillong 793 014, India 

MS received 10 January 1981; revised I May 1982 

Abstract. The peripheral disturbed zone of a 50-year old stand of the 
forest at Lai lad was dominated by D emir o calamus hanriltonii, an early successional 
bamboo characteristic of a secondary successional fallow of not more than 
20-25 years while the central undisturbed zone had Schima wallichii, Castanopsis 
indica and Shorea robusta as important components. The biomass of the forest 
was computed as 137 X 10" kg/ha of which 64.7% was in the central zone. The 
contribution by different species both in the central and peripheral zone of the 
forest was worked out. Linear relationship between dbhfd^h and biomass was 
worked out for different species. The standing crop had : N, 953 ; P, 284; K, 600; 
Ca, 2281 ; and Mg, 450 kg/ha, of which 60% was in the central zone of the forest. 

Keywords. Biomass ; nutrients; sub-tropical forest. 



1 . Introduction 

Understanding of the structural and functional attributes of a given forest 
ecosystem is important for proper management of the environment and utilization 
of the resource potential (Grantham and Ellis 1974). The present study on a 
50-year old humid tropical forest ecosystem of north-eastern India is important 
as it represents a stage in secondary succession after slash and burn agriculture 
(locally known as jhum) which is a wide spread land use practice in this region 
(Ramakrishnan et al 1981). The present series of three papers deal with forest 
ecosystem from the point of view of its organization, biomass and nutrient flow 
through litter and through water. 

The structure of a vegetational unit depends upon the species composition and 
their relative number (Gleason 1926). Biomass data form an important component 
of the structure of any ecosystem (New Bould 1967). Though much infor- 
mation on this and inventory of nutrients is available on different forest 
types from the parts of the world, little is known on Indian forest types except for a 
few studies (Dadhichi 1979; Agrawal 1980; Vyas et al 1980). This paper is related 

241 



242 



Jasbir Singh and P S Ramakrishnan 



to phytosociology and biomass analysis alongwith nutrient inventory of the forest 
type mentioned above. 



Max. temperature 
o Min. temperature 
Q Rainfall 



500 
400 



~ 300 



* 200 



TOO 





n 



10 1 



J FMAMJ J ASONO 
Months 



Figure 1. Climate of Lailad based on the average of two years - 1977 and 1978 (data 
obtained from Department of Silviculture, Government of Meghalaya). 



2. Study area, geology and climate 

The study area located at Lailad (2545"~ 260" N Latitude and 9145" - 92OE 
Longitude) at an altitude of 296 m, is tropozoidal shaped and is a part of a reserve 
forest known as Nonghyllem reserve under the Meghalaya Forest Department 
since 1910. The peripheral zone is subjected to biotic disturbances, 
such as cutting of trees for firewood and removal of bamboo for fencing and house 
building by the local tribal population. Therefore, only the central zone of the 
forest is representative of a 50-year old stand. The soil is red, sandy loam and 
is of laterite origin. ThepH ranged from 5.8 to 6.3. The climate is typically 
monsoonic with most of the rainfall (84%) during May to September. The mon- 
soon season is followed by a mild winter (mid November to mid February). March 
and early April represent a brief dry summer period (figure 1). 



2.1 Methods of study 

Phytosociological studies were done in July 1976, at the peak of the growing 
season both in the outer peripheral zone of about three hectare. The density, 
frequency, basal area and importance value index were based on 20 quadrats of 
10 X 10m for trees, 5 X 5m quadrats for shrubs and 1 X 1m quadrats 
for herbs along a transect running from the periphery to the centre of the forest 



Vegetation, biomass and its nutrients 243 

(Misra 1968; Kershaw 1973). Species diversity was calculated using the formula 
iven by Shannon and Weaver (1949) 



where H = Shannon index of general diversity, ni = importance value index of 
ipecies /, N = Importance \alue index in the community, 

The index of dominance of the community was calculated according to 
Simpson (1949). 



as C = 

where C = concentration of dominance ; ni importance value ofspscies /, 
N = total importance value for the community. 

Biomass estimation of tree species was done in a specified area during August at 
:he time when the leaves were fully mature. Three different girth classes with 3 repli- 
cates of 11 major tree species, namely, Shorea robusta, Schlma wallichii, Castono- 
?sis indica, Artocarpus chaplasha, Gmelfne arbor ea, Garcinia cowa, MiUiusa roxbur- 
%hiana 9 Sterculia villosa, Dillenia indica, Vitex pedimcularls and Dendro calamus 
hamiltonii were harvested. Various parameters like diameter of the bole at base, 
niddle and top, total height of the tree, diameter and length of the branches and 
;otal number of leaves were recorded after harvest. The fresh weight of all the 
branches and leaves were determined in the field and sub-samples of branches, 
small twigs and leaves were brought to the laboratory in polythene bags. Small 
liscs (2-3 cm thick) from the base and top of the bole were taken for cornpula- 
:ion of dry weight. All the sub-samples were oven dried at 80 - 85 C to cons- 
ent weight. The regression equations were obtained relating the biomass 
parameters to combinations of diameter at breast height (dbh) and diameter 2 X 
leight (d*h) . Regression equations were used to estimate the biomass of the 
itanding crop. 

Plant samples were ground and passed through a 20-mesh screen and chemically 
maiyzed for nitrogen, phosphorus, potassium, calcium and magnesium using 
rtandard methods as described by Peach and Tracy (1956) and Jackson (1958). 
rhus, nitrogen was determined by micro-kjaldahl method and phosphorus was 
estimated colorimetrically by molybdenum blue method. After dry ashing the 
;amples, calcium and magnesium were analyzed by EOTA-filtration method aad 
Dotassiurn by flame emission method. 



I. Results and discussion 

l.l Vegetation structure 

Some of the most important component of the peripheral disturbed zone were 
Dendrocalamus hamiltonii, Mesuaferrea, Millusa roxburghiana, Vitex peduncularis> 
Schima wallichii, Castonopsis indica and Shorea robusta. It may be noted that 
9. hamiltonii which is more dominant in the peripheral zone is an early successional 



244 



Jasbir Singh and P S Ramakrishnan 



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Vegetation, biomass and its nutrients 245 

species and reaches its peak in a 20-year old community after which it dec- 
lines (Ramakrishnan and Toky 1978). On the other hand species like Schima 
wallichii, Castonopsis indica and Shorea robusta were more dominant towards the 
central zone of the forest with higher IVi values. In forest community as a 
whole, dominant tree species were Dendrocalamut hamlltonii, Schima waUichii, 
Castonopsis indica, Shorea robust a, Milusa roxburghiana and Artocarpus chaplasha 
(table 1). M. roxburghiana had high density and frequency but low IVI values 
due to low basal area whereas Artocarpus chaplasha and Vitex peduncular is had 
high IVI values in spite of low density and frequency because of their greater 
basal area. 

Croton oblongifoliwn, Litsaea khasyana, Leea samhucina, Annona wallichli, 
Randia demi flora and Micromelum pubescence were the important of the shrub 
layer of the forest as a whole. It may be noted that species like Croton oblvngi- 
folium had high IVI values towards the peripheral zone of the forest along with 
species like Annona wallichi, Combretum decundrurn, Litsaea khusyana, etc. In the 
forest as a whole, Panicum khasianum, Cyperus elegans, Hedychhim gracile, etc. are 
predominant amongst herbaceous species (table 1). However, a number of these 
species possess greater IVI in the peripheral zone in comparison to the central 
zone of the forest. This may be due to lesser number of tree species with reduced 
canopy cover which permitted greater light penetration and less competition in the 
peripheral zone. 



3.2 Biomass 

The pattern of biomass distribution in the forest for the important species may be 
related to the species diversity. Of the total biomass 64.7% was along the central 
zone ducto greater species diversity whereas peripheral zone contributed about 
35.3%. Schima wallichii, Castonopsis indica and Shorea robusta contributed maxi- 
mum in the peripheral zone as well as central zone. Howqver, these three species 
contributed more than 2-fold in the central zone of the forest compared to the 
peripheral zone (table 2). 

Leea stambucina, Annona wallichii, Sterculia cacclnia, Litsaea khasyana and 
Croton oblongi folium account for the largest biomass contributed by the shrubs in 
the forest. Of the total biomass contributed by shrubs, 54.3% was along the 
peripheral zone and the remaining 45.7% was in the central zone of the forest. 
Of the total plant biomass in the forest as a whole 64.5% was in the central zone 
and only 35.5% along the peripheral zone. Along the periphery, shrubs and 
herbs contributed larger share of the total biomass whereas trees contributed 
mostly towards the central zone of the forest (table 3). 

The average biomass on an unit area basis i'n the forest was 137 X 10 3 leg/ha, 
the value being lower than those reported by other workers for the tropical 
forests (Jordan and Kline 1972). Whittaker and Likens (1973 a, b) reported a 
mean value of 35-45 X 10 4 kg/ha biomass for some tropical and seasonal forests. 
However, the present value lies between 60~350m*lia reported by Whittaker 
(1975) for some temperate evergreen and tropical seasonal forests and much higher 
than that for temperate coniferous forest (Akai et al 1968; Smith et al 1971). 



246 Jasbir Singh and P S Ramakrishnan 

Table 2. Biomass contribution by major tree, and shrub species in the 
central zones of the forest at Lailad (values in parentheses represents 
of the total amount). 



peripheral and 
the percentage 



Species 



Biomass (kg/ha) 



Peripheral zone 



Central zone 



Trees 








Artocarpus chaplasha 


3796.2 


(1.4) 


9124.2 (3.3) 


Castonopsis indica 


22484.1 


(8.2) 


45199.9 (16.4) 


Dendro calamus hamiltonii 


6914.8 


(2.5) 


3638.2 (1.3) 


Dillenia indica 


4594.6 


(1.7) 


2938.4 (J.7) 


Grnelina arborea 


2033.8 


(0.7) 


4589.2 (1.7) 


Garcinia cowa 


1967.3 


(0-7) 


51187 (1.9) 


Miliusa roxburghiana 


61857 


(2.2) 


4744.5 (1.7) 


Schima wallichii 


35113.9 


(12.8) 


74900.4 (27.2 


Shorea robusta 


8416.3 


(3.1) 


21395.9 (7.8) 


Sterculia villosa 


4674.8 


(1-7) 


2614.4 (0.9) 


Vitex peduncularis 


1057.1 


(0.4) 


1698.7 (0.6) 


Shrubs 








Anona wallfchti 


196.2 


(6.2) 


65.4 (2,1) 


Actinodaphne angustifolia 


141.2 


(4.5) 


94.4 (30) 


Croton oblongi 'folium 


184.0 


(5.8) 


147.2 (4.6) 


Combretum decandrum 


127.4 


(4.0) 


72.8 (2.3) 


Litsaea khasyana 


186.0 


(5.9) 


248.0 (7.8) 


Lfea sambiicina 


265.0 


(8.3) 


371.0 (11.7) 


Morinda umbel/ ata 


151.2 


(4.8) 


108.0 (34) 


Phlogacanthus thyrsiflorus 


2030 


(6.5) 


124.8 (3.9) 


Randia densiflora 


73.6 


(2.3) 


92.0 (2.9) 


Others 


193.0 


(6.1) 


131.2 (4.1) 



Table 3. Biomass contribution by major tree species, shrub and herbaceous layers 
in the peripheral and central zones of the forest at Lailad (values in parentheses 
represents the percentage of the total amount). 



Different layers 



Biomass (t/ha) 



Peripheral zone 



Central zone 



Tree 
Shrub 
Herbaceous 


97.234 (35.3) 
0.172 (0.01) 
0.008 (0.03) 


177.984 (64.6) 
0.146 (0.05) 
0.004 (0.01) 



Relationship between morphological growth parameters viz., diameter (dbh), 
and diameter 2 X height (d 2 h) on the one hand and biomass of individual tree 
species as well as fractional plant parts on the other was found to be highly 
significant for all the tree species except D. hamiltonii where significant correla- 
tions were found with dbh (tables 4,5). 



Vegetation, biomass and its nutrients 



247 



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Vegetation, biomass and its nutrients . 249 

.3 Nutrient content in biomass 

ji analysis of the concentration of M,P, K, Ca and Mg in different plant 
omponents (bole, branches and leaves) showed that the leaf had higher pcrcen- 
ige of N, P, K, and Mg while bole alongwith bark contained higher percentage 
f Ca in all the species except Dendro calamus hamihonii where the concentration 
f Ca was more in leaves. Next to the leaves, the branches had higher levels 
f N, P, K and Mg and the bole had the least concentration. On the other hand, Ca 
Dncentration was the least in branches with intermediate values from leaves 
:able 6). 



Table 6. Concentration of different nutrients in different compartments of 
different tree species at Lai lad. 



Species 


Compart- 
ment 


N 


P 


K 


Ca 


Mg 




Bole 


0.41 


0.12 


0.31 


3.00 


0.14 


Artocarpus chaplasha 


Branches 


0.61 


0.23 


0.32 


0.82 


0.49 




Leaves 


2.10 


0.64 


0.59 


2.96 


0.77 




Bole 


0.64 


0.14 


0.58 


0.84 


0.47 


Dcndrocalamus hamiltonii 


Branches 


0.70 


0.28 


0.52 


0,69 


0.70 




Leaves 


1.76 


0.59 


0.83 


1.99 


0.88 




Bole 


0.85 


0.14 


0.26 


2.90 


0.18 


Castonopsis indica 


Branches 


0.90 


0.28 


0.40 


1.00 


0.34 




Leaves 


1.90 


0.49 


0.66 


2.40 


0.79 




Bole 


0.58 


0.15 


0.26 


2.80 


0.29 


Miliusa roxburghiana 


Branches 


0.75 


0.28 


0.65 


1.00 


0.83 




Leaves 


1.65 


0.47 


0.85 


3.20 


1.09 




Bole 


0.59 


0.16 


0.48 


3.36 


0.26 


Schima wallichii 


Branches 


0.62 


031 


063 


0.70 


0.45 




Leaves 


1.86 


066 


1.19 


260 


0.67 




Bole 


0.42 


0.11 


0.34 


2.40 


0.30 


Shorea robusta 


Branches 


0.82 


0.26 


0.44 


0.88 


0.68 




Leaves 


1.90 


0.76 


1.20 


1.78 


1.08 



On hectare basis, the total amount of different elements contributed by different 
pecies (table 7) showed that Schima wallichii, Castonopsis indlca and Shorea 
riusta contributed maximum with respect to N, P, K, Ca and Mg. This could 
e related to the large biornass in the standing crop of these tree species 
i the same order as given above. Among other species a great variation 
fas observed with regard to percentage contribution of different elements 
'hich may be partly due to the concentration of nutrients. Thus, Dendro- 
alamus hamiltonii stands next to Artocarpus chaplasha and Miliusa roxbur- 
hiana in biomass but contributed more in terms of P, K and Mg due to 
igher concentration of these three elements in the plant tissue. Because of low 
'a concentration in the tissue coupled with low biomass the contribution of Ca 
y Dendrocalamus hamiltonii was far less than that of Artocarpus chaplasha and 
diliusa roxburghiana. 



250 



Jasbir Singh and P S Ramakrishnan 



The distribution of nutrients in different tree compartments showed that bole 
contained maximum amount of all nutrients followed by branches and leaves. 
This is in spite of the higher concentration of N, P, K and Mg in the leaf tissue 
This compartmentalization of nutrient is highly exaggerated for Ca due to the fact 
that this nutrient also had the highest concentration in the bole (figure 2). 



Table 7. Amount of different elements contributed by different species alongwith 
percentage contribution of the total for the forest given in parentheses. 



Species 



Nutrient (kg/ ha) 





N 


P 


K 


Ca 


Mg 


Artocarpus chaplasha 


34.7 


11.3 


21.2 


135.8 


17.2 




(3-8) 


(4.0) 


(3.6) 


(4.8) 


(3.9) 


Dendrocalamus hamiltcnil 


?2.4 


22.0 


31.7 


36.1 


29.2 




(3.5) 


(7.9) 


(5.4) 


(i-4) 


(>/) 


Dillenia indica 


23.7 


H.I 


21.1 


74.6 


10 3 




(2.6) 


(2.9) 


(3.6) 


(2.7) 


(23) 


Castanopsis indica 


291,7 


61.4 


102.5 


439.9 


79.3 




(32.0) 


(22.0) 


(17.4) 


(25.7) 


(17.9) 


Gmelina arborea 


18.8 


5.2 


16.6 


63.8 


12.7 




(2.1) 


(1.9) 


(2.8) 


(2.0) 


(2.9) 


Garcinia cowa 


18.1 


8.5 


10.5 


61.2 


12.8 




(2.0) 


(3.0) 


(18) 


(2.2) 


(2.9) 


Miliusa roxburghlana 


27,7 


11.0 


11.7 


113.1 


25.3 




(3.0) 


(3.9) 


(2.0) 


(4.7) 


(57) 


Schima wallichii 


346.7 


115.0 


294.0 


1086.6 


174.0 




(38.1) 


(41.2) 


(499) 


(45.5) 


(39.3) 


Shorea robusta 


83.7 


25.2 


58.1 


181.7 


61.0 




(9.2) 


(9.0) 


(9.9) 


(8.9) 


(13.8) 


Sterculia villosa 


23.1 


9.1 


14.6 


54.0 


16.8 




(2.5) 


(3.2) 


(2.5) 


(1.9) 


(3.6) 


Vitex peduncularis 


10.1 


2.5 


7.0 


22.4 


3.8 




(I.I) 


(0.1) 


(1.2) 


(0.9) 


(0.7) 



Figures indicates the percentage contribution (per hectare) by the different 
elements in the different compartments of the standing crop. The quantities of 
the different nutrients in the three compartments of the tree in a decreasing order 
are as follows : 

Ca>N>K>Mg>P. 

The pattern of distribution of nutrients by trees, shrubs and herbs along the peri- 
phery and the centre of the forest showed that about 60% of the total nutrient pool 
was in the undisturbed central zone and the rest along the disturbed peripheral zone 
(table 8), However, along the peripheral zone the contribution by shrub and 
herb species was more (0.9 and 3% respectively) in comparison with the central 
zone (0.7 and 0.2% respectively). Tree species contributed more towards the 
nutrient pool of the living biomass (59.9%) in the central zone than along the 
peripheral zone (37.9) of the forest. 



Vegetation, biomass and its nutrients 



251 







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252 



Jasbir Singh and P S Ramakrishnari 



2300 



2100 



1800 



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200' 



900 



600 



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Ca 



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00 



Figure 2. Pattern of compartmentalization of nutrients in trees at Lailad forest. 
Open column bole; hatched column branches; closed column leaves. 
Figure 3. Proportion of different nutrients in different components in living biomass of 
trees (percentage of the total amount in kg/ha). Closed column N; strippled column P; 
hatched column KL; open column Ca; cross hatched column Mg. 



The total standing crop of nutrients as worked out in the present studies was 
higher when compared with broad leaved temperate forests (Ovington 1958; 
Grier el al 1974); DIvigneud et al (1968) but the values are lower when compared 
with tropical forests of Ghana (Greenland and Kowa 1960) and some Indian 
forest type (Deshbandhu 1970 ; Faruqi 1972) 



Acknowledgements 

The help given by Mr Ashish K Das in the preparation of this manuscript 
is gratefully acknowledged. One of us (JS) was a recipient of a Fellowship of the 
North-Eastern Hill University. 



References 



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Vegetation., biomass and its nutrients 253 

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?aech K and Tracey M V (ed) 1956 Modern methods of plant analysis Vol. Ill (Berlin; Springer 

Verlag), pp. 542 

Ramakrishnan P S and Toky O P 1978 Preliminary observations on the impact of jhum (shift- 
ing agriculture) in the forested ecosystem. In : Resource development and environment in the 

Himalayan region', Dept. of Sci. and Tech. Govt. of India 343-354. 
Ramakrishnan P S and Toky O P 1981 Soil nutrient Status of hill agroecosystem and recovery 

pattern after slash and burn agriculture (Jhum) in north eastern India, Plant Soil 60 41-64 
Ramakrishnan P S, Toky O P, Mishra B K and Saxena K G 1981 Slash and burn agriculture 

in North-Eastern India. In: Fire regimes and ecosystem properties, (ed) H A Mooney, 

T M Bonnickson, N JL Christensen, J E Lotan and W A Reiners, USDA Forest Service 

general technical report WO-26 pp. 570-587 

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versity of Illinois Press), pp. 117 

Simpson E H 1949 Measurement of diversity; Nature (London) 163 688 
Smith W H, Nelson L E and Switzer G L 1971 Development of the shoot system of young 

loblolly pine II Dry matter and nitrogen accumulation; For. Sci. 17 55-62 
yyas L N, Garg R K, Vyas N L and Jindal K 1980 Community structure and above ground 

biornass in dry deciduous forests around Udaipur (Rajasthan) India; Acta EcoL 2 25-32 
Whittaker R H (ed) 1975 Communities and ecosystem. Second ed (London and New York: 

MacMillan) pp. 385 
Whittaker R H and Likens G E 1973a Carbon in the biota. In : Carbon in the biosphere (eds.) 

G M Wood Well and E V Pecan Brookhaxen Symp. Biol. Spring field. Virginia 
Whittaker R H and Likens G E 1973b Primary production the biosphere and man; Hum. EcoL 

1 357-369 



Proc. Indian Aca<L Sci. (Plant Sci.), Vol. 91, Number 3, June 1982, pp. 255-268. 
Printed in India. 



Structure and function of a sub-tropical humid forest of 
Meghalaya II- Litter dynamics and nutrient cycling 



JASBIR SINGH and P S RAMAKRISHNAN 

Department of Botany, School of Life Sciences, North-Eastern Hill University, 
Shillong793 014, India 

MS received 10 January 1981 ; revised 1 May 1982 

Abstract. The litter production in a 50 year old humid sub-tropical forest at Lailad 
was found to be 5.5 t/ha/yr, 77% of which was through leaf and the remainder 
through \vood. Litter production was more in. the peripheral disturbed zone 
compared to the undisturbed central zone which is related to the successional status 
of the community. Species differences in the rate of decomposition of litter was 
noted. Besides litter production and decomposition pattern were related to seasonal 
differences in temperature and humidity. The present results have been discussed in 
the light of the data from other studies done elsewhere. 

Keywords. Litter dynamics ; nutrient cycling. 



1. Introduction 

Studies on litter production and its decomposition are important for understanding 
of energy flow, nutrient cycling and primary production in the ecosystem. While 
much is known on these aspects for temperate forests (Olson 1963; Bray and 
Gorham 1964; Rodin and Bazilevich 1967; Gosz et al 1972) fewer studies 
are available from the tropics (Jenny et al 1949; Loudelot and Meyer 1954; 
Cornforth 1970 ; Singh and Gupta 1977 ; Edwards 1977). Little is known on the 
litter dynamics of forests in India (Seth et al 1963 ; Singh 1969 ; Singh and Gupta 
1977). Further, many of these studies pertain to leaf litter alone and that 
derived from wood is often ignored. 

In an earlier paper in this series (Part I), the phytosociology, biomass and 
nutrient inventory of a 50 year old forest developed after slash and burn 
agriculture (Ramalcrish.nan et al 1981) at Lailad in Meghalaya was considered. 
The present study deals with the estimation of leaf and wood litter, its decom- 
position and nutrient cycling in this forest stand. 

2. Methods of study 

For collection of litter, 20 permanent quadrats (1 X 1 m) made of wood (10 
cms high) were randomly laid out in each site. Litter was collected at monthly 

255 



256 Jasbir Singh and P S Ramakrishnan 

intervals, classified into leaves and wood and further sub-divided intc 
different 'important tree species and a miscellaneous category. The litter was 
oven-dried, weighed and passed through a 20-mesh screen for chemical analysis 
The peripheral part of the forest was considered separated from the centra 
zone (of Part I). 

Decomposition of leaf litter was studied by litter bag technique (Shanks anc 
Olson 1961 ; Singh and Gupta 1977). Freshly fallen leaves in April wen 
collected in bulk from the forest and sorted out into five important tree 
species, namely, Shorea robusta, Schima wallichn, Castonopsis indicci 
Dendrocalamus hamiltonii and Artocarpus chaplasha and the remainder wen 
treated as miscellaneous. The litter was brought to the laboratory and air 
dried at constant temperature. Litter bags of nylon (10 X 14 cm) with 1 mn 
mesh size containing 30 g samples were used. 50 bags of each species wen 
prepared and decomposition of litter samples were evaluated by placing th< 
bags on the surface of the soil. Three replicate samples of each categon 
were recovered from the fields at monthly intervals. The material from th< 
litter bags was washed with water using a 100^ mesh screen to remove all soi 
particles. It was dried at 80C, weighed and preserved for chemical analysis. 

The rate of decomposition of wood litter was estimated using the methoc 
given by Yoneda (1975). Freshly fallen wood pieces of moderate diamctc. 
(3-4 cm) of the above-mentioned species were collected in April, air-dried a 
constant temperature, cut into 12 cm long pieces and initial weight of end 
piece was determined. 50 pieces for each species were randomly placed on tin 
surface of the soil and three replicate of each species were picked at monthl; 
interval. These were processed in the laboratory using similar procedures a; 
used for leaf litter bags. 

Chemical analysis of the litter was done following standard procedure 
(Allen 1974). Thus, nitrogen was determined by the Kjeldahl method, anc 
phosphorus was estimated color imetrically by the molybdenum-blue method 
calcium and magnesium by EDTA method while potassium by flame ernissioi 
method after dry ashing the samples in a muffle furnace at 450C. 

Soil respiration was measured by the alkali absorption method (Colemai 
1973 ; Gupta and Singh 1977 ; Gupta and Singh 1981) using 16.6 cm diamete 
metallic cylinder with 50 ml of 1 N KOH solution. The alkali solution wa 
kept in the cylinders for 24 hours. Soil respiration was measxired in two type 
of situations, (i) On the mineral soil after removing the litter layer, and (ii) Oi 
the soil without removing the litter layer. A third set served as the blank for whicl 
the beaker was placed on a wooden platform lined by a layer of polythene sheet 
Three replicates were done for each treatment. 



3. Results and Discussion 

3.1 Litter production 

Total litter production was estimated to be 5 5 t/ha/yr of which 77% wa 
through leaves and the remaining through wood. Litter production was mor< 



Litter dynamics and nutrient cycling 257 

along the periphery than the central zone of the forest. Dendrocalamus hamiltonii 
(bamboo) a comparatively early successional species was an important compo- 
nent in the periphery due to frequent disturbances like felling of dicot trees for 
timber and fuel wood and occurrence of accidental fire ; this species alone 
contributed 19% of the total litter. This species has been shown to have a 
high rate of turnover of biomass through leaves in the younger fallows up to 20 
years (Ramakrishnan and Toky 1981). In the central zone of the forest contribu- 
tion of litter was chiefly through Shorea rohusta, Schima wallichii and Castonopsis 
indica with 13, 10 and 8% respectively of the total (table 1). 

The average litterfall of the forest as a whole (5.5 t/ha/yr) was slightly lower 
than the values obtained for other tropical forests. At one end of the range 
are the values recorded by Mitchell (cited by Bray and Gorham 1964) for 
Malaya (5.5-7.2), Klinge and Rodrigues (1968) for Amazonia (7.3) and 
Edwards (1977) for lower Montane Rainforest in New Guinea (7.5). The values 
reported on higher side are by Bernhard (1970) for Ivory Coast (8.3-13.4) 
and Ewel (1976) for Guatemala (9,0). Toky and. Ramakrishnan (1980) reported 
litterfall of 9.7 t/ha/yr in a successional forest of 20-year dominated by bamboo 
(D. hamiltonii) in the same area where this study was done. This may be due 
to the fast developing vegetation during the early successional stages and the 
consequent rapid turnover of biomass. Since an early successional community 
undergoes fast changes in species composition, often entire individual may con- 
tribute to litter production resulting in an over shoot of litter production in 
early successional phase (Toky and Ramakrishnan 1980 ; Ewel 1976). 

Table 1. Distribution of litter along the peripheral and central zones of the sub-tropical 
forest at Lailad (kg/ha/yr). (Figures in parentheses represent the percentage of the total 
litter in each zone). 

Species Peripheral zone Central zone 



Leaf litter :- 






Ar to car pus chap las ha 


102.2 (2.3) 


204.5 (3.1) 


Castanopsis indica 


245.8 (5.4) 


491.8 (7.5) 


Dendrocalamus hamiltonii 


846.6 (18.7) 


282.2 (4.3) 


Dillenia indica 


25.2 (0.6) 


33.1 (0.5) 


Garcinia cowa 


48.8(1.1) 


145.0 (2.2) 


Machillus khasiana 


4.0 (0.1) 


5.0 (0.1) 


Mesua ferrea 


40.2 (0.9) 


14.4 (0.2) 


Miliusa roxburghiana 


311.3(6.9) 


140.7 (2.2) 


Shorea rohusta 


261.9(5.8) 


849.5(13.0) 


Schima wallichi 


297.8 (6.6) 


643.6(9.9) 


Sterculia villosa 


64.7(1.4) 


26.3 (0.4) 


Vitex peduncular is 


36.6 (0.8) 


43.4(0.7) 


Other species (leaf) 


1530.5 (33.9) 


1796.0 (27.6) 


(miscellaneous) 






Total 


3815.6 


4675.5 


Wood litter :- 






Branches, twigs, barks. 


701.0(15.5) 


1833.8 (28.2) 


(all species) 






Total (leaf -f wood) litter 


4516.6 


6509.3 


Mean of peripheral and central zone 




5512.9 



258 



Jasbir Singh and P S Ramakrishnan 



Wood litter (1.3 t/ha/yr) formed 23% of the total litterfall and the value is 
comparable to 1.2 t/ha/yr (16% of the total) as reported by Edwards (1977) in 
Lower Montane Rain forest in New Guinea. For temperate forests a range 
of 22-78% of the total contribution through wood litter has been estimated 
(Carlisle etal 1966; Anderson 1970). 

The distribution of litterfall was markedly seasonal with a maximum leaf 
litterfall (57%) during the dry months of February, March and April. Wood 
litterfall showed a peak during April to July (figure 1). A similar seasonal trend 
in litterfall was also found in other tropical rain forests (Nye 1961),Klinge 
and Rodrigues (1968). Data of Laudelot and Meyer (1954) for young secondary 
forests showed two periods of litterfall that come at the end of the drier season. 
This may be related to the formation of abscission layer in leaves due to the 
severity of drought stress during dry periods. During the present study, the peak 
of the wood litterfall extended into the rainy season. This may be related to 
storms prevailing during the rainy season, as also reported elsewhere (Edwards 
1977). 



3.2 Litter decomposition 

Most of the workers who have considered the rate of decomposition of litter on 
the forest floor (Jenny etal 1949; Olson 1963; Edwards 1 977) have assumed 
that there is an exponential loss in weight as a result of decomposition, i.e. 

Xlx = exp ( kt) 

where x is the initial weight, x is the weight at time r, and k is the annual 
decomposition constant. This model expresses the loss as a negative exponen- 
tial function of the fraction and calculation of k remains the most convenient 
means of comparing forests. 

During the present study the rate of decomposition was rapid in 
D. hamfltonii, Schima wallichii and miscellaneous litter (high k values) than 
other types of litter (table 2). The overall rate of decomposition in this 
forest for wood and leaf litter was found to be lower than the values reported 
by Laudelot and Meyer (1954) for Zaire and Singh (1968) for deciduous forest 




Figure 1. Monthly litter production in a humid sub-tropical forest at Lailad 
(1977-78). 



Litter dynamics and nutrient cycling 259 

at Varanasl (India) and was comparable to the values reported by Edwards 
(1977) for New Guinea. Nitrogen content and the texture of the litter play a 
great role in decomposition (Ewel 1976; Singh and Gupta 1980), however, Singh 
(1968) correlated various chemical constituents and the rate of decomposition 
in tropical tree species and found that not only nitrogen but numerous 
chemicals interact to affect the rate of decomposition. The nitrogen content of 
leaf litter (table 3) in S. rohusta, C. indica and A. chaplasha was quite high 
but still a low rate of decomposition was observed in these species. This may 
be due to decay resistant petiole and mid-rib of leaves due to high content of 
lignin (Singh 1968). The lower decay rate may also partly be due to the 
smaller mesh size of litter bags which do not allow larger fauna to enter 
(Edwards and Heath 1963). 

After a period of one year, the highest loss of litter was observed for 
D. hamiltonii (78%) and least for S. rohusta (56%) (figure 2) 40-45% of litter 
was lost during May to August due to higher temperature and humidity. 
Subsequently the rate of decomposition slowed down (figure 3) due to low 
temperature and moisture levels. This pattern of decomposition was also evident 
from the evaluation of CO 2 from litter layer on the mineral soil (figure 4). 



Table 2. Decay constants and time required for the loss of one-half and 95% of the 
original leaf and wood dry weight in different species. 



Time parameter (years) 


Species 


Half-time 
(0.693) 


95% 
(3) 


K 


K 




Leaf 


Wood 


Leaf 


Wood 


Artocarpus chaplasha 


1.99 


0.79 


1.50 


3.78 


Castanopsis indica 


1.87 


1.10 


1.61 


2.78 


Dendro calamus hamiltonii 


2.17 


3.63 


1.38 


0.83 


Shorea robusta 


1.87 


0.76 


1.61 


3.97 


Schima wallichii 


2.18 


1.19 


1.38 


2.52 


Other species 


2.17 




1.38 




(miscellaneous) 











Table 3. Chemical composition of leaf litter collected in April 1977 from Lailad forest. 
Species N(%) P (%) K (%) Ca (%) Mg (%) 



Artocarpus chaplasha 


0.67 


0.35 


0.35 


2.50 


0.66 


Castanopsis indica 


0.82 


0.46 


0.36 


1.88 


0.59 


Dendro calamus hamiltonii 


0.68 


0.32 


0.22 


1.15 


.0.31 


Shorea robusta 


0.85 


0.63 


0.63 


1.12 


0.51 


Schima wallichii 


0.76 


0.54 


0.44 


1.93 


0.65 


Other species 


0.79 


0.56 


0.46 


2.08 


0.67 


(miscellaneous) 













260 Jasbir Singh and P S Ramakrishnan 




Months 



Figure 2." Rate'l of decomposition of leaf litter f expressed" as' percentage of the 
original dry weight of leaves remaining'after^various"periods"of decomposition. 




MJ JASONOJ F M A 



Figure 3. Rate of decomposition of wood litter expressed as percentage of the 
original dry weight remaining after various periods of decomposition. 




Figure 4. Monthly pattern of CO 2 evolution from the forest floor at Lailad. 



3.3 Nutrient content in litter 

The seasonal variations in concentration of nitrogen, phosphorus and potassium 
was well marked in both leaf and wood litter (figures 5, 6). This may be explained 



Litter dynamics and nutrient cycling 



261 



2-8- 




0-2 

M J S N J 

Months 

Figure 5. Monthly variation in concentration of different nutrients in leaf litter at 
Lailad forest. 



0-9 

i 0-7 
j 

io-5 



I 03 



0-1 




M J S N J M 
Months 

Figure 6. Monthly variation in concentration of different nutrients in wood litter 
at Lailad forest. 



as due to a number of factors like translocation of nutrients from leaves before 
senescence, leaching of nutrients particularly potassium through rainfall and 
the extent of decomposition of leaves and twigs before they fall (Nye 1961 ; 
Tukey 1970; Goszetal 1972). The concentration of N, P and K was higher 
in dry periods than during the rainy season ; this may be mainly due to leaching 
of nutrients through rainfall. 

Average concentration for the 12-months period, for various species showed 
great variations. Nitrogen and calcium content was highest in Mesua ferrea, 
Miliusa roxburghiana while phosphorus and magnesium concentration was found 



262 Jasbir Singh and P S Ramakrishnan 

Table 4. Nutrients content in leaf litter of different species and wood litter (Composite 
sample for all species). 



Species 


N(%) 


P(%) 


K (%) 


Ca (%) 


Mg(%) 


Leaf Utter :- 












Artocarpus chaplasha 


0.68 


0.35 


0.35 


2.92 


0.66 


Castanopsis indica 


0.85 


0.46 


0.36 


2.28 


0.59 


Dendro calamus hamiltonii 


0.74 


0,32 


0.22 


1.39 


0.36 


Dillenia indica 


0.64 


0.24 


0.54 


1.86 


0.28 


Garcinia cowa 


0.52 


0.31 


0.31 


1.65 


0.35 


Machillus khasiana 


0.85 


0.40 


0.30 


1.98 


0.65 


Mesua ferrea 


0.99 


0.27 


0.47 


3.23 


0.58 


Miliusa roxburghiana 


0.87 


0.22 


0.32 


3.16 


0.65 


Shorea robusta 


0.86 


0.63 


0.63 


1.35 


0.51 


Schima wallichii 


0.77 


0.54 


0.44 


2.31 


0.66 


Sterculia villosa 


0.66 


0.30 


0.40 


1.33 


0.42 


Vitex peduncular is 


0.57 


0.33 


0.23 


1.81 


0.35 


Other species 


0,79 


0.56 


0.46 


2.51 


0.67 


(miscellaneous) 












Wood litter :- 












Branches, twigs and barks 


0.60 


0.38 




0.73 


0.45 


(all species) 













to be more in Shorea robusta, Artocarpus chaplasha and Schima wallichii. For 
other tree species a great variety in concentration of different elements was 
observed. In general calcium content was more followed by nitrogen in leaf and 
wood litter (table 4). Ewel (1976) observed a reverse trend where nitrogen was 
the predominant element followed by calcium. 



3.4 Nutrient return through litter fall 

The percentage contribution by different species not only depends upon litter 
biomass contributed by them but also on the nutrient concentration in their 
litter. Thus, even though Dendrocalamus hamiltonii had greatest litter biornass 
in the forest as a whole, yet there were other species like S. robusta which 
contributed higher amount of P through litter. In contrast a species like 
M. ferrea which had high nutrient level in the leaf tissue for N and Ca, had 
very low total contribution of these elements through litter because of lesser 
litter production. Shorea .robusta, S. wallichii and C. indica in that order 
contributed high percentage of the nutrients both because of high litter production 
and high nutrient concentration in their litter. The total contribution of nutrients 
through litter was higher in the central zone of the forest than the periphery, 
though the total production of litter was higher in the periphery than the 
central zone (table 5), This maybe related to the difference in the species 
composition in the zones due to difference in maturity of the forest community. 
A comparison of nutrient returned through litterfall in the present study has 
' been compared with other tropical and temperate forest ecosystems (table 6). 
The values for all the elements were lower (except phosphorus) than the values 
reported by Laudelot and Meyer (1954) for Zaire, and Nye (1961) for Ghana, 



Litter dynamics and nutrient cycling 



263 



I 



ja 



d 
o 







i 



s 



d 
o 

1 
1 

o 
U 



Table 5 



I- 



01 

00 



S 



d d d o d 



3 S S ~ 8 8 a 3 8 R 2 



. 

" d rn d d d ^H d ^ ^ o o vo 



ON 

O\ 



q 

*H l> 



OS 

oi 



1 



I 



-a 






- 



tl 

.^ S s 






Jasbir Singh and P S Ramakrishnan 







oo , 






^ \o e 


*O 

1 


I 

o 




<D 


00 


nJ ^ 5 fc^ tt5 Cd ^ 

(M W 


& 






*- 




1 


*O 






CO 

1 




1 




,0 




*WJ 


m 


M-1 




a 


10 00 







{30 


? ^2 32 


a 




S 




S 








o 








a 




1 


n 

O 


cd 




2 








cd 




1 




u 


^ -i oo ir> 


O 


& 






(H 








JD 


5 


a 




P 


"bS 


^ 




CO 


jyj 


*co 


oo 


s" 

E 


1 


cd 


^ S S - 8 s 


s 








*-* 


S 






I 


* 







CO 




r? 




.s 




43 


^ c^ ^ oo 


_r 




to 


^ ^ (S V? 10 


S 




O 




5 




ft 




60 

1 




Nitrogen 


3 

<S Tt 

"'T OS \O Q\ Vfi 
*O ON O VO VJD 


(D 






S 






P 




cd 

a 


S 




cd jd 


CO 


c 
^o 


Q S pi 


s 

<U 


g"*"* 


<o <u ^ co 


"-i 

"3 

a 


3 


1 M 1 IS 


IS 






8 






a 




CO 


^o 




(D 


i I 






s 




S 


o 

c 




's 


1 




o 3 


. 




"S. ^ 


* 




.1 .1 ^ 


1 


1 


'a 'E _g "^ 

O cd " d> 

* * 1 -.73 5 




1 


*a 'd S 3 ^ 

-8 -M 1! 




> 


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s|lS-3 

G d *:r d ^ 



t 






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o ^. 

TH O 



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11 CO ^ 



S ^ g ^ 

2 s -a 53 



Litter dynamics and nutrient cycling 



265 



but higher than that for (except nitrogen) Amazonian tropical Rain-forest Klinge 
and Rodrigues (1968) and mixed deciduous forests (except calcium which is 
comparable) at Varanasi (Singh 1969). 



3.5 Nutrient release through decomposition 

During the first 4 months after the placement of litter bags in the field, there 
was a rapid fall in the concentration of all the elements ; potassium and nitrogen 
were lost more rapidly. Loss of calcium and magnesium was comparatively 
slower. A similar trend of decline in concentration of nutrients, was found 
during wood decomposition too (figures 7, 8, 9). These results are similar to 
the trends shown by Gupta and Singh (1977) and Ewel (1976). 




Figures 7 a-d. a. Loss of nitrogen from leaf litter through decomposition. b>Loss of 
nitrogen from wood litter through decomposition, c. Loss of phosphorus from leaf litter 
through decomposition, d. Loss of phosphorus from wood litter through decomposition. 




MJ J ASONOJ P M A 



Figures 8 a-d. a. Loss of potassium from leaf litter-through decomposition, b. Loss of 
potassium from wood litter through decomposition, c. Loss of calcium from leaf litter 
through decomposition. d Loss of calcium from wood litter through decomposition. 



MJJASONOJFMA 




MJJASONOJFMA 
Months 

Figures 9 a-b. a. Loss of magnesium from leaf litter through decomposition, b. Loss of 
magnesium from wood litter through decomposition. 



Litter dynamics and nutrient cycling 267 

It may be concluded that (i) the turnover of litter is more in the peripheral 
sturbed zone of the forest than in the central zone due to comparatively 
triy successional stage of the community, (ii) litter production and decomposi- 
3n was markedly seasonal related to the temperature and rainfall pattern. The 
suits are compared with data on litter production and decomposition of 
.her forest types. 



cknowledgements 

r e are grateful to Dr O P Toky and Shri Ashesh K Das for their help in the 
eparation of manuscript. One of us (JS) was a recipient of a Junior Research 
sllowship of the North - Eastern Hill University. 



eferences 

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333-346 



268 Jasbir Singh and P S Ramakrishnan 

Olson J B 1963 Energy storage and the balance of producers and decomposers in ecological 
systems Ecology 94 322-333 

Ovington J D 1959 The circulation of minerals in plantations of Pinus sylvestis L ; Ann. Bat. 
(NS) 23 229-239 

Ramakrishnan P S, Toky O P, Misra B K and Saxena KG 1981 Slash and burn agriculture in 
north-eastern India. In Fire regimes and ecosystem properties, (ed) H A Mooney, 
T M Bonnicksen, N L Christensen, J E Lotan and W A Reiners USDA Forest Service 
general technical report WO-26, pp. 570-587 

Ramakrishnan P S and Toky O P 1981 Soil nutrient status of hill agroecosystem and recovery 
pattern after slash and burn agriculture (jhum) in north-eastern India Plant Soil 60 41-64 

Rodin L E and Bazilevich N I 1967 (ed.) Production and mineral cycling in terrestrial vege- 
tation (London : Oliver and Boyd) pp. 288 

Seth S K., Kaul O N and Gupta A C 1963 Some observations on nutrition cycle and return of 
nutrients in plantations at New Forests : Indian For. 89 90-98 

Shanks R E and Olson J E 1961 First year breakdown of leaf litter in Southern appal achian 
forests Science 134 370-376 

Singh K P 1968 Litter production and nutrient turnover in deciduous forests of Varanasi. Proc. 
Symp. Recent Adv. Trop. Ecol. pp. 655-665 

Singh K P 1969 Nutrient concentration of leaf litter often important tree species of deciduous 
forests at Varanasi ; Trop. Ecol. 10 83-95 

Singh J S and Gupta S R 1977 Plant decomposition and Soil respiration in terrestrial eco- 
systems Bot. Rev. 43 pp. 449-528 

Tukey H B Jr 1970 The leaching of substances from plants. Ann. Rev. Plant Physiol. 21 305-324 

Yoneda T 1975 Studies on the rate of decay of wood litter on the forest floor I. Some physical 
properties of decaying wood ; Jpn. J. Ecol. 25 40-46 



Proc. Indian Acad. Sci. (Plant Scu), Number 3, June 1982, pp. 269-280. 
(P) Printed in India 



Structure and function of a sub-tropical humid forest of 
Meghalaya III. Nutrient flow through water 



JASBIR SINGH and P S RAMAKRISHNAN 

Department of Botany, School of Life Sciences* North-Eastern Hill University, 
Shillong 793 014, India 

MS received 10 January 1981; revised 1 May 1982 

Abstract. This paper deals with water and nutrient flow through incident rainfall, 
throughfall and stemflow through a 50- year old stand of forest at Lailad. A larger 
proportion of water was accounted as throughfall and stemflow in March -April and 
was related to canopy density and this was inversely related to interception loss which 
was maximum in December. Many of the nutrient concentrations like that of 
N, K, Ca, etc. increased with the maturation of the leaves during October-November. 
Though concentration of nutrients was higher in stemflow, the total quantity was 
more via throughfali because of larger quantity of water passing through this 
compartment. More of Ca ind K was lost through run-off and percolation due to 
greater release of these nutrients through litter. The significance of these results have 
been discussed in the context of secondary succession after slash and burn agriculture 
(jhum) of which this forms a later stage of community development. 



1. Introduction 

Atmosphere is a source of chemical inputs to terrestrial ecosystems which come 
through direct fall, throughfall and stemflow (Ovington 1959; Carlisle et al 
1965) Generalization of these aspects are chiefly based on temperate forests 
and studies on tropical forests are meagre (Jackson 1971). In India the few 
studies done on nutrient flow through water are on plantations (Debral and 
Subba Rao 1968 ; George 1978) and no work has been done on natural 
forests. 

The earlier two papers in this series (Part I and II) pertain to a 50 year old 
forest fallow at lailad in Meghalaya and deal with phytosociology, biomass and 
nutrient inventory and cycling through leaf and wood litter. The present study 
deals with the pattern of water and nutrient circulation through stemfiow and 
throughfall and quantity interception, run-off and percolation losses in this 
forested ecosystem. For the present study, only the central zone of the forest 
representing the undisturbed 50-year stand has been considered. 

269 



270 Jasbir Singh and P S Ramakrishnan 

2. Methods of study 

Steinflow was sampled using a spiral polythene gutter of 6 cm diameter fitted in 
each stem and sealed with paraffin wax. The gutter was fixed at a height 
of 1.5m above the soil surface on the tree trunk. A plastic funnel was 
attached to the two cut ends of the gutter and connected to a polythene container 
of 5 litre capacity. A nylon filter 1 'mm mesh size was placed in the mouth of the 
funnel to prevent entry of extraneous matter. Three replicates each for two girth 
classes namely 30 and 90 cm were selected for each of the following important 
species : (1) Shorea robusta (ii) Schima Wallichii, (iii) Castanopsis indica, 
(iv) Gmelina arborea and (v) Artocarpus chaplasha. 

Water of throughfall and incident rainfall was collected in polythene containers, 
the mouth of each being fitted with 20 cm diameter funnel which was provided 
with 1 mm mesh nylon filter to prevent entry of foreign matter. Three containers 
were kept outside the forest in open places to collect the water from incident rain. 
Twelve containers of the same size were kept under the forest canopy to measure 
the throughfall. In order to measure the atmospheric precipitation two standard 
rain gauges were kept in the open. All the containers were kept 50 cm above 
the surface on a wooden platform to avoid splashing of soil particles into the 
funnels. 2 ml toluene was added to the container to prevent microbial activity. 
Sampling was done at intervals ranging from 2 to 7 days depending upon the 
intensity and the frequency of rainfall during the monsoon. At the time of 
sampling, 500 ml of well homogenised water from stemflow/throughfall/incident 
rain was brought to the laboratory and the samples were filtered through a 
Whatman no. 44 filter paper and chemically analysed for N, P, K, Ca and Mg. 

For studies pertaining to run-off water and sedimentation, the loss from a 
confined area of 1x10 m along the slope was collected in drums of 200 litre 
capacity and periodically removed for analysis. Percolation studies were done 
using a simple lysimeter of the Russian type (Buckman and Brady 1980). The soil 
was cut out vertically to expose the profile. A small tunnel was excavated at a 
depth of 40 cm (this is the depth at which root density is high) and a collector of 
30 X 30 X 15 cm was placed inside the tunnel. The water percolating through 
soil was tapped out from time to time and chemically analysed for N, P, K, Ca 
and Mg using standard methods as described by Jackson (1958) and Allen (1974). 
Thus, nitrogen was determined by micro-kjeldahl method and phosphorus 
estimated colorimetrically by molybdenum-method. Calcium and magnesium 
were analysed by EDTA titration method and potassium by flame emission 
method. 



3. Results and discussion 

3,1 Throughfall 

A summary of the seasonal distribution (table 1) shows that the percentage 
of rainwater coming as throughfall is maximum in the month of March- April 
(over 70% of the total) while in other months it was *mich less with minimum 



Nutrient flow through water 



271 



percentage in December. The high proportion of throughfall in March-April 
was due to reduced crown density at this time when 41 % leaf fall occurred. The 
proportion of throughfall showed a decreasing trend with increase in canopy 
density in subsequent months. Similar relationship between crown density and 
throughfall was also reported by Szabo (1975). The average value of 52.4% 
throughfall during the course of this study was higher than the values obtained 
for other broad leaved forests (Debral and Subba Rao 1968 ; Aldridge and 
Jackson 1973) but lower than a few others (Ovington 1959; Leonard 1961; 
Szabo 1975). The quantity of throughfall is directly proportional to the gross 
rainfall and the relationship is indicated in figure la (P < *05). 



3.2 Stemflow 

The average stemflow during the period of study was measured at 8% of the 
precipitation (table 1). A low proportion of stemflow was measured in the month 
of November due to lesser intensity and frequency of rain. On the other hand, 




100 ?00 )00 

Gross rainfall I mm ' 



Figure 1. Relationship between gross rainfall and (a) throughfall, 
(b) stemflow and (c) interception. 



272 Jasbir Singh and P S Ramkrishnan 

reduced canopy density in the month of March and April allowed more water flow 
through stem. The gradual decline in percentage stem flow from May-October 
could be related to the high canopy density which in turn resulted in greater 
proportion of interception losses. The present value of stemflow (8%) was found 
to be higher than the values reported by Ovington (1959) and Szabo (1975) which 
ranged from 0.123.10% but lower than the values reported by a few others 
(Eidmam 1959 ; Aldridge and Jackson 1973). The relationship between gross 
rainfall and stemflow was found to be highly significant which is expressed by 
a linear regression line (figure Ib). 



3.3 Interception loss 

The proportion of the rainfall intercepted by the canopy was inversely related to 
the proportion of throughfall and stemflow with maximum percentage values 
recorded in December and minimum in March. This high percentage of 
water transmission from the canopy during October-December may be attributed 
to the fact that rainfall in these months was not regular in comparison to the 
monsoon period (April-September) and the vegetation often remained dry. Thus 
the greater quantity of water necessary to wet the vegetation accounted for the 
high interception loss. The low interception values during March and April were 
due to reduced crown density due to maximum leaf fall during this period. 
Similar observation with low interception losses during heavier leaf fall was 
made by Szabo (1975) in their study on Hungarian Oak forest ecosystem, 
The relationship between gross rainfall and interception loss which follow the 
same pattern as stemflow and throughfall is shown in figure Ic. 



3.4 Water balance of the ecosystem 

The total amount of water and percentage values given in table 1 are estimates 
based considering forest canopy as entirely closed. But in order to explain the 
total quantity of water in one hectare forest surface, the forest gaps are also to be 
considered. The total canopy coverage in the present forest was 90% and the 
rest 10% was accounted for gaps where the precipitation could reach the forest 
floor almost directly without any interruption. After making allowances (Szabo 
1975) for gaps in the forest canopy, the percentage distribution of the incident 
rainfall in a forest of one hectare is shown in figure 2a. However, it may be 
mentioned that the stemflow values would be on the higher side as only selected 
important tree species of same girth classess were considered and assumed 
contribute to the total cover of the forest. 



3.5 Loss through run-off and percolation 

Studies on surface run-off and percolation losses of rainwater at Lailad forest 
showed that they represented 19.6% and 6.78% respectively of the total annual 



Nutrient flow through water 



273 









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274 



Jasbir Singh and P S Ramakrishnan 




A S N 
Months 



Figures 2a-b. a. Distribution of precipitation in the forest (per hectare). Hatched 
columns, throughfall ; closed columns, stemtlow ; open column, intercepted 
loss. b. Monthly pattern of surface run-off and percolation loss of Lailad 
forest. 



rainfall, during the year. The monthly pattern of losses of water could be related 
to the rainfall pattern with maximum run-off and percolation losses occurring 
during May-September (with 87% and 37% of the total run-off and percolation 
losses respectively), with peak values in the months of July (figure 2b). Both, the 
high frequency and intensity of rainfall during monsoon contribute to heavy 
losses during this period. 



3.6 Nutrient return by throughfall, stemflow and incident rainfall 

The mean monthly concentration (mg/1) of the various elements throughout the 
study period in throughfall, stemflow and incident rainfalls are shown in 
figures 3a, b, c, d, e, The * concentration of total nitrogen in throughfall and 
stemflow was low during March-May, followed by a steady rise reaching a 
maximum towards October-November which could be related to the subsequent 
maturity of the leaves. ' Similar increase in N concentration of throughfall with 
increasing leaf maturity was shown by Tukey et al (1958) with subsequent 
decrease after the formation of abscission layer. The concentration of nitrogen 
in stemflow was more than that of throughfall which may be due to (i) release 



Nutrient flow through wafer 



275 




J A S N 
Months 



Figure3. Monthly variation in concentration of different elements, a, 
nitrogen ; b, phosphorus ; c, potassium ; d, calcium and e, magnesium 
in different water samples. 



of nutrient during bark decay during the rainy season (ii) the wash out of 
nutrients from leaves and . (iii) low quantity of water in stemflow and consequent 
high concentration of nutrients. Monthly variation in the concentration of N in 
incident rainwater could also be observed though the values were low compared 
to that of the throughfall and stemflow. 



276 Jasbir Singh and P S Ramakrishnan 

The concentration of phosphorus in incident rain was very low during the rainy 
season. A slightly higher level was noted during March-April which could be 
due to (i) high level of dust particles in the atmosphere during the preceding dry 
period, (ii) presence of partly burnt particles of organic matter in the air due to 
burning of slash in the neighbourhood during this period due to shifting agri- 
culture and (iii) low rainfall during this period with lesser dilution of this 
element. In throughfall., the level of P gradually increased reaching a maximum 
in August followed by a slow decrease in subsequent months. The gradual 
increase in concentration up to August may be attributed to the presence of high 
pollen grains of the tree species and also the production of new leaves which may 
have higher concentration of phosphorus as was reputed by Carlisle et al (1967) 
working on a sessile oak forest in England. A similar pattern of monthly 
concentration changes was also observed for stemflow except that the concentration 
of P on the average was two times higher than that for throughfall due to release 
of this nutrient from the decayed bark. 

The monthly pattern for potassium, calcium and magnesium concentration in 
throughfall, stemflow and incident rainfall was similar (figures 3c, d, e) . The 
gradual increase in concentration from the month of May to December is probably 
related to gradual maturation of new leaves produced in April and the consequent 
increase in release of some of these nutrients from more mature leaves, an 
observation also made by Tukey et al (1958) and Deneyear-DeSmet (1966). 
Amongst the three elements, the concentration of Ca was more in stemflow due 
to its high level in the bark. 

The total amount of nutrients (kg/ha/yr) contributed through stemflow, 
throughfall and incident rain is shown in table 2. Throughfall contributed 98% 
of all the nutrients. The low addition through stemflow is in spite of the high 
concentration of nutrients in the stemflow water, as the total quantity of stemflow 
is far less than that due to throughfall. Amongst the cations, calcium and potassium 
were highly leachable with heavy washout through stemflow and throughfall. 



Table 2. Nutrient return through stemflow, throughfall and rainwater at Lailad forests. 



Nutrients (kg/ha/yr) 





N 


P 


K 


Ca 


Mg 


Stemflow 


0.17 


0.02 


0.71 


0.84 


0.10 


Throughfall 


8.39 


0.89 


31.28 


35.19 


5.03 




Total 9.56 


0.91 


31.99 


35.93 


5.13 


Rainwater 


4.33 


0.43 


7.80 


9.96 


4.77 




Difference 5.23 


0.48 


24.19 


24.97 


0.63 



Nutrient flow through water 



277 



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J.7 Nutrient losses* through run-off and percolation of water 






berlateto greater release of nutrients! from freshly decomposing 



278 



Jasbir Singh and P S Ramakrishnan 







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Nutrient flow through water 279 

The concentration of nutrients in percolation water also followed a similar pattern 
as that in the run-off water, in that peak values were higher during July- 
September with a minor peak during March-April (figures 4a, b, c, d, e). 

Since the present forest represented a secondary successional fallow after jhum 
(Toky and Ramakrishnan unpublished) a comparison of the pattern of losses with 
a 0-year freshly burnt site would be interesting (table 3). The loss of Ca and K 
from a 50-year old fallow was higher compared to other nutrients may be due to 
greater release of these nutrients from the vegetation through litter decomposition 
(Timmons et al 1977). In fact even though the quantity of water lost from a 
deforested side througK run-off and percolation was not more than 2-3 fold 
compared to the Lailad forest, nutrient losses increased as much as 6-7 times 
compared to the forested site. This may be due to sudden release of nutrients 
through burnt and lack of vegetation cover to hold the same (Ramakrishnan 
et al 1980). 

4. Conclusion 

The three series of papers on the ecosystem structure and function of a 50-year 
old secondary successional fallow is significant as it represents a comparatively 
more stable forested ecosystem in the successional gradient. An early successi- 
onal weed stage which dominates up to about 5 years of fallow development after 
jhum is soon replaced by a bamboo dominated stand up to about 20 years beyond 
which dicot tree species gain importance (Ramakrishnan et al 1981). The 
peripheral zone of the Lailad forest (Part I in this series) represents such a stage 
due to frequent disturbances in this zone. The central zone, however, represents 
the 50-year old stand of a mixed broad leaved forest* Apart from the high species 
diversity and biomass and nutrient stored in the living compartment alongwith 
efficient cycling through litter (Ramakrishnan et al 1980), the loss of nutrients 
from the system is also minimal compared to younger fallows (Toky and 
Ramakrishnan 1981). In fact there is negligible loss from the system through 
both run-off and percolation as seen from comparison presented in Part III 
in this series. This presented in a dramatic way the damage done due to 
deforestation during shifting agriculture. With the climate and steep topographic 
conditions prevailing in the north-eastern hill region, the present study highlights 
the significance of maintaining a forested ecosystem for environmental 
stability. 



Acknowledgement 

We are grateful to Dr O P Toky and Dr Ashesh Kumar Das for their help in 
the preparation of manuscript. One of us (JS) was a recipient of Junior Research 
Fellowship of the North-Eastern Hill University. 

References 

Aldridge R and Jackson R J 1973 Interception of rainfall by bardbeach (Nothofagus truncata) at 

Taita, New Zealand. N.Z. J. ScL 16 185-198 
Allen S E 1974 (ed) Chemical analysis of ecological material. (Oxford: Blackwell Scientific Publ) 

pp 565 



280 Jasbir Singh and P S Ramakrishnan 

Buckman H O and Brady N C 1960 (eds) The nature and properties of Solids. (New York : 

The MacMillan) pp. 567 
Carlisle A, Brown A H F and White E J 1965 The interception of precipitation by oak (Quercus 

petraea) a high rainfall site ; Q. J. For. 59 140-143 
Carlisle A, Brown A H F and White E J 1967 The nutrient content of tree stemflow and ground 

flora litter and leachates in a sessile oak (Quercus petraea) Woodland. /. Ecol. 55 615-662 
Debral B C and Subba Rao B K 1968 Interception studies in chir and teak plantations New 

Forest. Indian For. 94 541-551 
Denaeyer-DeSmet S 1966 Bilan annuel des apports d'elements mineraux par les eaux dc 

precipitation lous convert, forestier dans la foret caducifoliee de blamont (Birelessachimay). 

Butt. Soc. R. Bot. Belg. 99 345-375 
Eidmann F E 1959 The interception in Buchan und Fichtebestenden, Ergelomis mohrajah riger 

untersuchengen in Rothaurgebirge (Samel and) Inst. Assn. Scientific Hydrology Publ. 

48 5-25 
George M 1978 Interception, stemflow and throughfal] in a Eucalyptus hybrid plantation. 

Indian For. 104 719-726 

Jackson M L (ed) 1958 Soil chemical analysis (New Jersey : Prentice Hall, fnc.) 
Jackson I J 1971 Problems of throughfall and interception assessment under tropical forests. 

/. Hydro!. 12 234-254 
Leonard R E 1961 Interception of Precipitation by northern hardwoods. North Eastern Forests 

Expl. Station Paper No. 159 1-16 
Ovington J D 1959 The circulation of minerals in plantations" of Pinus sylvestris L. Ann. Bot. 

(NS) 23 229-239 
Ramakrishnan P S, Toky O P, Mishra B K and Saxcna K G 1981 Slash and burn agriculture in 

North-Eastern India In : Fire regimes and ecosystem properties (ed) H Mooney, TM 

Bonnicksen, N L Christensen, J E Lotan and W A Reiners, USDA Forest Service general 

technical report WO 26, pp. 570-587 
Szabo M 1975 Net precipitation under a forest (Quercetum petraea Gerris.) Canopy in 1974 

Acta. Bot. Acad. Sci. Hung., 21 151-165 
Toky O P and Ramakrishnan PS 1981 Run-off and infiltration losses related to shifting 

agriculture (Jhum) in north-eastern India. Environmental conservation 8 313-321 
Timmons D R , Very N S, Burwell R E and Hold R F 1977 Nutrient transport in surface run-off 

and stemflow from an Aspen-Birch forest. Our Environ. Qual 6 188-192 
TukeyJBJr, Tukey H B and Wittwer SH 1958 Loss of nutrients by foliage leaching as 

determined by radioisotopes. Proc. Am. Soc. Hortic. Sci. 71 496-506 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 4, August 1982, pp. 281-237. 
Printed in India. 



Anatomy of the seedling of the Legnminosae 



UMAVATHI HEGDE and V D TILAK* 

Department of Botany, KG College, Maharshi Karve Road, Bombay 400 020, 

India 

*Department of Botany, The Institute of Science, Madam Cama Road, 

Bombay 400032, India 

MS received 10 February 1981 ; revised 22 January 1982 

Abstract. Anatomy of the juvenile nodes m the seedling of fourteen genera of 
Legumtnosae is described. The cotyledonary node exhibits a two-trace, urailacunar 
condition. The two traces show various degrees of approximation of the two traces 
leading to one-trace, unilacunar condition in some genera. At the second node 
level, only two genera show the one-trace, unilacunar pattern ; 11 of the remaining 
genera have a three-trace trilacunar supply and one genus exhibits an intermediate 
type. At the third node level all the genera show a three-trace, trilacunar pattern. 
The present data suggest that the three-trace, trilacunar condition is derived by the 
addition of a lateral trace on either side of the median trace and that the one- 
trace, unilacunar condition appears to be a result of the approximation of the two 
traces at the cotyledonary node. 

Keywords. Nodal anatomy ; seedlings ; Lcguminosae. 



1. Introduction 

Anatomy of the seedling in the Leguminosae has been studied by Compton (1912), 
Winter (1932), McMurray and Fisk (1936), Weaver (1960), Pillai and Sukumaran 
(1969), Pillai et al (1970, 1974), Bairathi and Nathawat (1974) and Narang (1978). 
Most of these studies are, however, restricted to the cotyledonary node and 
root-stem transition region. The evolutionary sequence followed by the diffe- 
rent types of nodal structures has always been of interest to plant anatomists. 
The present investigation was, therefore, undertaken to find out if the anatomical 
studies in the seedling of some dicots would be of help in this regard. For this 
purpose the anatomy of the juvenile nodes of the seedling of 14 species belonging 
to 14 genera of the Leguminosae were studied. 



2. Materials and methods 

Seedlings of the plants studied were raised in the garden of .the Institute of Science, 
Bombay, from seeds collected locally or purchased in the market. Portions of the 

281 



282 Umavathi Hegde and V D Tilak 

axis obtained by cutting a little below and above the first (cotyledonary), second 
and the third nodes were fixed in FAA. The usual paraffin method was followed. 
Sections of the paraffin infiltrated material were cut on a microtome at a thickness 
of 15-20 microns. The sewere stained with crystal/gentian violet using erythrosin/ 
orange G as counter strain 



3. Observations 

Crotalaria juncea Linn. The hypocotyledonary axis has a ring of about a dozen 
bundles (figure 1), Two traces depart from each of the two sides (figure 2). The 
two traces corne closer to each other in their upward course but retain their sepa- 
rate entity at the cotyledonary base (figure 3). Axillary buds are present in the 
axil of the cotyledons (figure 3). 

The epicotyledonary axis has a ring of about 15-20 bundles (figure 4). Three 
traces, one median and a lateral on either side of it, emerge from the ring (figure 5), 
The laterals in their upward course shift closer to the median and can hardly be 
differentiated from each other in the petiole (figure 6). 

The third node has, likewise, a three trace, trilacunar supply to. the leaf (figure 7). 

The laterals after giving off a branch to the stipule shift closer to the median and 
almost merge with it so as to become indistinguishable (figures 8-9). As a varia- 
tion, some series cut off at the second and third nodes exhibit a four-trace, four 
lacunar condition i.e. a median with two laterals on one side and one on the other 
(figures 10-11). 

The nodal structure at the first three nodes in Medicago sativa Linn., Tephorosia 
purpurea (Linn.) Pers., Cassia tor a Linn, and Mimosa pudica Linn, is similar to 
Crotalaria juncea except for minor differences regarding the degree of distinctiveness 
of the two traces to the cotyledons as they extend upwards and that of the lateral 
traces of the leaves of the second and third nodes from the median trace, as these 
extend upwards into the petiole. In Mimosa pudica the cotyledons separate out 
from the axis in the form of a ring which organizes into two cotyledons, and two 
non-vascularized stipular structures (figure 12). 

In Arachis hypogaea Linn., the cotyledonary node is two traced unilacunar with 
the two traces remaining distinct in the cotyledons (figures 13-14). The leaves at 
the second and the; third nodes are three-traced and trilacunar (figure 1 5) with the 
laterals as well. as the median bundles dividing in their upward course (figure 16). 
Some of the branches of the laterals extend into the stipules while the remaining 
ones alongwith the branches of the median and the median itself, extend into the 
petiole '(figure, 17). 

The cotyiedonary node in Pisum sativum Linn, is two-traced, xmilacunar, traces 
remaining distinct from one another in their upward course (figures 18-19). Tho 
leaves of the second and the third node, are three-traced and trilacunar. This 
plant is characterized by the precocious emergence of the lateral traces as compared 
to the median trace of the same leaf. Thus, the lateral tcaces of tb@, teaf of tbl? 
second node emerge a little above the cotyledonary node and those of the leaves 
of the third node emerge almost . alongwith the median bra.dk of the second ?Bodte 
(figure 20)* These precociously emerged latoai traces, extend outwards from the 



Anatomy of the seedling of the Legwninogae~I 283 

vascular ring (figures 21 and 22). The median bundle and the lateral traces divide 
in their upward course (figures 21-23). While some of the branches of the laterals 
extend into the stipules, the remaining ones alongwith the branches of the median 
and the median itself, extend into the petiole (figures 22,24). 

The cotyledonary node in Clitoria ternatea Linn, is two-traced, unilacunar. 
The two traces come together in their upward course. They ultimately lose their 
distincti veness. The leaves of the second node are opposite in phyllotaxy. Each 
leaf receives a single median trace and a common lateral only on one side of the 
axis (figure 25). The single common lateral splits into two (figure 26). The 
branches of the split lateral, after giving off branches to the stipule, shift closer 
to the median in their upward course and ultimately merge with it forming a con- 
centric bundle (figures 26-27). The stipules on the other side of the leaves receive 
their vascular supply from the median bundle itself (figure 26). The phyllotaxy 
at the third node is alternate. The leaves at this node receive a three-trace vas- 
cular supply. The lateral traces, as usual, in their upward course, at first, give off 
a branch to the stipule and later come closer to the median and merge with it. 

The cotyledonary node in Abrus precatorins Linn, and Cicer arietinum Linn, 
is two-traced, unilacunar. The two traces come closer to each other in their 
upward course. The leaves of the second node are one-traced, unilacunar (figures 
28,30). The single trace splits into many small strands, upwards (figure 29). The 
leaf of the third node, is as usual three-traced, trilacunar. 

The cotyledons in Leucaena glauca Linn, and Albizia kbbeck (Linn.) Benth 
receive two traces, which in their upward course merge into one and divide into 
three at the point of entry into the cotyledonary base (figures 31,32,33). The 
cotyledons in the Tamarindus indica Linn, and Pithecellobium dulce, (Roxb.) Benth 
are characterized by one-trace, unilacunar supply (figure 34). This single trace 
divides into three right at the point of emergence (figures 35-36). The vascular 
supply to the leaves of the second and the third nodes in the above mentioned plants 
is of the three trace, trilacunar type with the lateral traces in their upward course, 
at first giving off branches to the stipules and then shifting closer to median. 

4. Discussion 

Anatomists have treated different types of nodal structures in different ways, phylo- 
genetically. Thus, Sinnott and Bailey (1914) suggest that the three-trace, trila- 
cunar type is primitive as compared to unilacunar and multilacunar types. Ozenda 
(1949) on the other hand treats the multilacunar type as more primitive. Marsden 
and Bailey (1955) describe the so-called " fourth type " i.e., a two trace, unilacunar 
type as the primitive type of node. They, further, consider that the unilacunar 
node with one trace arose by the fusion of two traces. Bailey (1956) reexamined 
the earlier viewpoint which he shared with Sinnott (Sinnott and Bailey 1914) and 
reached a conclusion that the three-trace, trilacunar and multitrace, multilacunar 
types in angiosperms were derived at some stage in their evolution from the one- 
trace, unilacunar type. Canright (1955), Eames (1961), Esau (1960,1965) and 
Carlquist (1961) have more strongly emphasized the primitiveness of the unila- 
cunar node with two traces and consider it as basic in the evolution of angiosperm 
nodal structure. 



284 



Umavatki Hegde and V D Tilak 




Anatomy of the seedlin g of the Legummosae-l 



285 



.w Mfiil ! 




286 Umavathi Hegde and V D Tilak 

Pant and Mehra (1964) and Benzing (1967) do not treat the two-trace, unilacunar 
node as primitive. Instead, Benzing (1967) proposes that the three-traced condi- 
tion is likely to be more primitive in the angiosperms than the two-trace, unilacunar 
type. Takhtajan (1969) conceives of a hypothetical tc fifth type " of node with a 
central gap haying two traces. This type is supposed to have given rise to all 
other known types. 

All the plants studied show a basically two traced, unilacunar nodal pattern at 
the cotyledonary node. Some of these have a two-traced, unilacunar structure* 
with varying degrees of independence or approximation of the two traces from the 
point of emergence through their extention into the cotyledons. Other plants 
have a one-traced, unilacunar type. Thus, there is a trend towards the approxi- 
mation of the two traces leading to a one trace, unilacunar type of node. 

At the second node level Abrus precatorius and Cicer arietinum have a one-trace 
unilacunar condition. Clitoria ternatea presents an intermediate condition. It has 
a median trace, each, for the two leaves of the opposite phyllotaxy and a common 
lateral on one side of the axis. All the remaining plants have a three-trace, trila- 
cunar supply to the leaves of the second node. Further, all the plants studied have 
a three-trace, trilacunar structure at the third node. Thus, it is only in Abnis 
precatorius and Cicer arietinum that the three trace, trilacunar pattern is attained 
at the third node. Rest of the plants achieve this condition at the second node 
itself. 

The present data, therefore, shows that the three-trace, trilacunar condition 
is a characteristic feature of the more mature second and third nodes while the 
two-trace, unilacunar and one trace, unilacunar patterns are seen at the cotyledo- 
nary nodes. Similar observations have been made during the course of a study of 
- twentytwo genera of the Malvales (Rao 1980). Further, in no plant either of the 
Leguminosae or of the Malvales could a condition be recorded wherein a three- 
trace, trilaeunar condition of the cotyledonary or second node is succeeded by a 
one-trace, unilacunar type at the second and third nodes. Bailey (1956) records 
similar observations. These observations may be taken as an evidence in support 
of the? primitiveness of the one trace, unilacunar type as compared to the three-trace, 
trilacunar type. 

In Abrus precatorius the single trace at the second node divides into many 
small strands. Ifl Clitoria ternatea the branches of the split lateral give off a branch 
to the stipule and shift closer to the median and merge with it. The stipule on the 
other side receives its vascular supply from the median itself. In Arachis hypogaea 
and Pisum sativum the median as well as the laterals divide into smaller strands. 
While some of the branches of the laterals enter into the stipule, the remaining 
ones alongwith the .branches of the median and the median itself extend into the 
petiokf. In rest of the plants both at the second as well as the third nodes the 
laterals at first give off a branch to the stipule and their remaining portions shift 
closer to the median. These portions of the laterals may remain distinct from the 
median in plants such as Medicago sativa and Tephrosia purpurea or merge with the 
median as in Crotalaria juncea and Cassia tor a. Thus, the laterals, give vascular 
supply to the stipules, however, when absent, the stipules receive branches from 
the median itself. 



Anatomy of the seedling of the Legumino$ae-I 287 

Vascular bundles outside the main vascular ring have been recorded in the inter 
node ofPisum sativum by Kuptcha (1975). He designates these as cortical bundles 
but they appear to be precociously emerged lateral traces, 

Acknowledgements 

The authors sincerely thank Dr B C Haldar, Director, The Institute of Science 
for laboratory facilities and Prof. R M Pai, Professor and Head of the Botany 
Department, Marathwada University, Aurangabad for helpful suggestions and 
keen interest. They thank Shri A M Siddiqui for his help in the preparation 
of the illustrations. 



References 

Ba'ley I W 1956 Nodal anatomy in retrospect ; /. Arnold Arbor. Harv. Univ. 37 269-287 
Bairathi H K and Nathawat G S 1974 Morphology and anatomy of polycotylous and twin 

seedlings of Crotalaria juncea Linn ; /. Indian Bot. Soc. 53 196-201 
Benztng DM 1967 Developmental patterns in stem primary xylem of woody Ranales ; Am. J. 

Bot. 54 805-820 
Ca'inght J E 1955 The comparative morphology and relationships of the Magnoliaceac IV. Wood 

and nodal anatomy ; /. Arnold- Arbor. Harv. Univ. 36 1-50 

Carlquist S 1961 Comparative Plant Anatomy (New York: Holt Reinhart and Winston) 
Compton R H 1912 An investigation of the seedling structure in the Leguminosae ; /. Linn. 

Bot. 41 1-122 
Eamcs A J 1961 Morphology of Angiospenm (New York, Toronto and London : McGiaw-Hill 

Co. Inc.) 

Esau K 1960 Anatomy of the seed plants (New York and London : John Wiley and Sons, Inc.) 
Esau 1965 Plant anatomy (2nd ed.) (New York amd London : John Wiley and Sons. Inc.) 
Kupicha F K 1975 Observations on the vascular anatomy of the tribe Yicicae (Leguminosae) ; 

Bot. J. Linn. Soc. 74 131-162 
Marsden H P E and Bailey I W 1955 A fourth type of nodal anatomy in dicotyledons 

illustrated by Clerodendron trichotomum Thumb. ; /. Arnold Arbon. Harv. Univ. 30 1-50 
McMurray E B and Fisk E L 1936 Vascular anatomy of the seedling of Melilotus alba \ Bot. 

Gaz. 98 121-134 
Narang Arvind Kaur 1978 Seedling structure in Crotalaria and Tephrosia species ; J. Indian 

Bot. Soc. 57 52-57 
Ozenda P 1949 Recherches aur les Dicotyledones apocaspiques contribution aletude des Angiospermes 

dites primitives Paris Publ. Las. Ecde. Norm. Sap.ser biol. II 

Pant D D and Mehra B 1964 Nodal anatomy in Retrospect ; Phytomorphology. 14 384- 357 
Pillai S K and Sukumaran K 1969 Histogenesis, apical meristem and anatomy of Cyamopsis 

tetragonoloba (Linn) Taub, Phytomorphology ', 19 303-312 
Piilai S K, Trivcdi M L ard Abraham K 1970 Certain aspects of the anatomy of Crotolaria 

burhia Ham. ; /. Birla Inst. Technol. Sci. 2 153-164 
Pillai S K, Ramasita K and Ishwar Dutt 1974 Embryology, histogenesis, apical meristem, seed 

coat and seedling anatomy of Albizia lebbeck (Lirux) Benth. ; New Botany 1 23- 33 
Rao Gmja K 1980 Anatomical studies in the seedling of some dicotyledons II Ph.D. thesis 

University of Bombay 
Sinnott E W and Bailey I W 1914 Investigations on the phyjogeny of angiosperms I. The 

anatomy of the node as an aid in the classification of the ang'osperms ; Am. J. Bot. 1, 

303- 332 

Takktajan A 1969 Flowering plants origin and dispersal (Edinburgh: Oliver and Boyd) 
Weaver H L 1960 Vascular'zation of the root hypocotyl-cotyledon axis of Glydne max L ; 

Phytomorphology. 10 82- 86 
Winter C W 1932 Vascular system of young plants of Medicago sativa L.; Bot. Gaz. 94 156-16. 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 01, Number 4, Aufiust 1982, pp. 289-195. 
Printed in India. 



Cork-warts in Eucalyptus species 



PARVEEN FAROOQUI (nee KIDWAI*) * 
Department of Botany, The University, Allahabad. India 
* Regional Forest Research Centre, Jabalpur 4#2 020, India 

MS received 26 December 1980 ; revised 24 May 1932 

Abstract. Four different types of cork-warts, the ordinary cork-warts, the d-scars, 
wound cork and trichome scars have been described from E. citriodora and 
E. torcllina. The differences between them are discussed. 

Keywords. Eucalyptus ; cork-warts ; d-scars ; wound cork ; trichome scars ; d- 
stomata. 



1. Introduction 

The occurrence of cork-warts or holes in the epidermis surrounded by radially 
arranged concentric rows of suberised cells has been mentioned among others by 
Solereder (1908), Metcalfe and Chalk (1950) and Stace (1965). Their presence has 
been explained either as a diagnostic character or as a result of mechanical injuries. 
During a general survey of epidermal characters of a number of Eucalyptus species, 
cork-warts were noticed in some of them. As no detailed accounts of their struc- 
ture and development is available it was thought worthwhile to investigate them 
in detail. 



2. Material and methods 

Young and mature leaves of E. citriodora Hook, and E. torcllina F.VM. were obtained 
from Tamil Nadu. Peels of the epidermis were obtained by scrapping and those 
of the cuticle by maceration in a 1 : 1 mixture of nitric acid and chromic acid 
(both 10%). The peels were mounted in Safranin glycerine jelly. Transparencies 
of the leaves were also prepared by the method of Arnott (1959). 



3. Observations 

In both species, numerous black or brown spots are scattered over the upper and 
lower epidermis (figures 1,12,13). A closer examination of these spots and study 
of their early developmental stages show four distinct types of structures. 

289 
P.(B)~2 



290 Parveen Farooqui (nee Kidwai) 

3.1. d-stomata and d-scars 

In the young epidermis, alongwith developmental stages of stomata, hairs etc., a 
few large fully formed stomata are also present. They persist in the mature leaf 
and occur isolated between groups of normal sized stomata (figure 2). Some 
of these large stomata become darker in colour and a dark zone is demarcated 
around them in the ordinary epidermal cells (figures 3,9). Others 
persist as large stomata, without further development. The stomata with a dark 
zone develop further. The surrounding cells divide concentrically around the 
stoma (figure 4). The pore of the stoma enlarges and becomes thin walled 
(figure 4). The guard cells appear to lose their contents. Finally the guard cells 
are ruptured and lost and the surrounding cells become thick-walled and darker. 
Similar stomata have been described in Ilex species by Korn and Fredrick (1973) 
and have been named d-stomata (or developmentally important stomata) and the 
replacement periderm tissue is termed the d-scar. 

3 . 2. Ordinary cork-warts 

Intermixed between the d-stomata and d-scars are other structures with similar 
concentric rings of periderm like tissue which enclose either a single ordinary sized 
stoma, or several ordinary stomata (figures 5,10) or even ordinary epidermal cells 
without stomata. After the thickening of the walls of the periderm like tissue the 
enclosed cells break down and disappear (figure 8). 

3.3. Wound cork 

Occassionally the epidermis also shows rounded or slit like gaps in it which appear 
like punctures by insects or accidental wounds respectively. Later a periderm 
like activity is seen around them (figure 6), the cells finally becoming thick walled 
and cork-like with the hole in the centre. 

3 . 4. Trichome scars 

Young leaves of both these species show multicellular, multiseriate hairs or emer- 
gences with a broad multiceliular base. In E. torellina, some of these trichomes 
persist even on mature leaves especially over the margins and veins. When the 
hair falls its base is surrounded by the thick-walled, somewhat papillate cells 
(figure 7). A periderm like tissue develops around the hair base. These form the 
tricliome scars (figure 11). 



4. Discussion 

Cork-warts have been mentioned by Solereder (1908), Haberlandt (1914), 
Metcalfe and Chalk (1950) and others. Stace (1965) was perhaps the first to sus- 
pect their composite nature and described them under " cork-warts and similar 
structures ". The function of the cork-warts has invariably been attributed as 



Cork-warts in Eucalyptus species 



291 





Figure 1-7. 1. ". torellina : Law power view of the leaf surface showing 
distribution of cork warts (x 20). 2. E. torellina : Large d-stoma surrounded by 
normal sized stomata f x 340). 3. E. citriodora: d-stoma showing a zone of darker 
area around it (x 340). 4. E. citriodora : d-stoma and d-scar (x 340). 
5. '. torellina: Cork-wart showing three stomata within it (xl75). 6. E. torellina: 
hole, presumably an insect puncture showing beginning of formation of wound- 
cork around it (x 340). 7. E. torellina : Hair base with papillate cells and 
beginning of periderm in lower right hand corner (x 340). 



292 



Parveen Farooqui (nee Kidwal) 



mechanical and to heal and repair holes caused by insect punctures or mechanical 
injuries. Stace (1965) has observed that cork-warts are not abundunt in nature and 
that they are present in certain species irrespective of wounds or insect punctures 
and, therefore, are of diagnostic value. He considers them as distinct from those 
caused by mechanical or insect injury. 

d-stomata and d-scars have been reported only from the leaves of some species 
of Ilex (Korn and Fredrick 1973). They are formed around large stomata which 
can be distinguished from the early developmental stages of the leaf. In some 
mangrove genera, Stace (1965) had earlier mentioned cork formation around 
some large stomata, which he suspected to be water stomata. As far as I am aware, 
they have not been reported from any other plant. Hence, their presence in species 
of Eucalyptus is interesting. 

The large d-stomata in the young epidermis, and the few that persist in the 
mature condition without the formation of a periderm like tissue, appear very 
similar to the giant stomata reported in a number of plants (Stace 1965 ; Sitholey 
and Pandey 1971 ; Farooqui 1979 and others). It is possible, that such giant 
stomata at a later stage form a d-scar around them. It may be worthwhile to rein- 
vestigate plants with giant stomata from such a stand point. 

According to Korn and Fredrick (1973), the d-stomata produce a zone of inhib- 
ition that prevents additional d-stomata from forming during subsequent growth. 
Their scattered distribution and absence of any contiguous d-stomata either in 
Ilex or Eucalyptus support,- such an interpretation. 

Wound cork formed as a result of injury may also be distinguished into two 
types. As pointed out by Stace (1965), purely mechanical accidental wounds may 
be variously shaped but are usually long and scar like. On the other hand 
insect punctures are usually more or less rounded. The size of the insect punctures 
may sometime indicate the identity of the insect visitor as it will correspond to the 
size of the stylet of the insect. 

The formation of a trichome scar after the falling out of the trichome is also 
interesting. Usually hair fall off without leaving any mark on the epidermis or 
their bases are left as such or they may become slightly thick walled. Here, 
periderm like tissue is formed around the trichome base. 

In the mature condition, when the inner plug of tissue is completely formed 
or has fallen off, the four types of structures described here, appear very similar 
to each other (figures 12, 13). However, developmentally they are different and 
may be distinguished on the following basis : 



d-stoma and 
d-scar 

(a) d-stoma is 

formed early in 
development and 
later forms the 
d-scar. The posi- 
tion of the d-scar 
is therefore, pre- 
determined. 



Cork-wart 

Formed towards 
the beginning of 
leaf maturation. 
Position random. 



Wound cork 

Formed when 
injury occurs. 
Position deter- 
mined by site of 
injury. 



trichome scar 

Formed towards 
.leaf maturation. 
Position deter- 
mined by site of 
the falling tri- 
chome. 



Cork-warts in Eucalyptus species 



293 




Figures 8-13. E. citriodora : Cork-wart with the central plug fallen (X 400). 9. 
E. citriodom: d-stoma showing formation of a dark zone around it (x 400). 10. 
E. citriodom : cork-wart showing four stonnta within it ( x 500). 11. E. 
torellina : hair scar(X 10DO). 12, 13. E. torellina: cork-warts in different stages 
of development (x600), 



.Cork-warts in Eucalyptus species 



295 



) Only one large 
stoma is involved. 



) Cork forms 
around stoma 
first, stoma 
breaks up later. 



One, several or 
no stomata are 
involved. It may 
include only 
epidermal cells. 

Cork formed 
first and tissue 
internal to it 
breaks up later. 



Tissue involved 
is determined by 
the size of the 
wound. 



Break up or 
injury occurs 
first and cork 
formed later. 



Only one tri- 
chome base is 
involved. 



Trichome falls 
off first. Cork 
is formed later. 
However, some 
basal cells of the 
trichome are 
already thick- 
walled. 



.) May be of 
diagnostic value 
as they have 
been reported in 
rare cases only. 



According to 
Stace (1965) of 
diagnostic value. 



Of no diagnostic 
value as they 
are purely acci- 
dental. 



Falling hairs 
leaving scars in 
the epidermis 
may be of diag- 
nostic value. 



The present study has clearly shown four distinct types of cork-warts. It is 
:erefore, important that in all future studies where cork-warts are found, their 
jvelopmental stages should also be studied in order to assign them to the correct 
pe. 



cknowledgements 

hanks are due to Professor D D Pant, Head of the Botany Depart- 
ent, Allahabad University for facilities and Shri K K Rao for help in 
lotography. 



eferences 



rnott H J 1959 Leaf clearings ; Turtox News 39 192-194 

irooqui P 1979 On the occurrence of abnormal stomata in plants; Curr. Sci.AS 841-849 

aberlandt G 1974 Physiological Plant Anatomy Translated from the fourth German edition by 

M Drummond, (London : Macmillan and Co.) 
orn R W and Fredrick G W 1973 Development of D-type stomata in the leaves of Hex 

crenata var. Convexa , Ann. Sot. 37 647-656 

:etcalfe C R and Chalk L 1950 Anatomy of the Dicotyledons (Oxford : Clarendon Press,) 
tholey R V and Pandey Y N 1971 Giant stomata , Ann. Sot. 35 641 
rtereder H 1908 Systematic anatomy of the dicotyledons (Translated by L A Boodle and F E 

Fritsch revised D H Scott ; Oxford) 
ace C A 1965 Cuticular studies as an aid to plant taxonomy ; Bull Br. Mus. (N.H.) 4 1-78 



3?roc.tidiati Acad. Sci. (Plant Scl), Vol.91, Nfutaber 4, August 1982, pp. 297-301. 
Printed in India. 



Pericarpial sclereids in some Mimosaceae 



S RANGAIAH, I L KOTHARI and G L SHAH 

Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120, 
India 

MS received 17 June 1981 ; revised 17 May 1982 

Abstract. Structure, ontogeny and distribution of sclcrcids in the pericarp of seven 
species of Mimosaceae are studied. Their occurrence is recorded in the epidermis 
hypodermis, mesocarp and endocarp. They may be macrosclereids or brachy- 
sclereids. Their structure and ontogeny are described and their taxonomic utility 
is pointed out. 

Keywords. Pericarp ; sclcreids ; Mimosaceae. 



1. Introduction 

Extensive Itierature is available on foliar sclereids (Metcalfe and Chalk 1979 ; 
Rao 1951, 1980 ; Rao and Bhupal 1973 ; Rao and Bhattacharya 1978 ; Rao and 
Das 1979), but it is very meagre on the occurrence and ontogeny of pericarpial scle- 
reids (Gupta and Lamba 1981), especially in Leguminoseae (Halliburton et al 1975). 
In this paper, therefore, an account of the distribution, structure and ontogeny of 
sclereids in the pericarp of seven species belonging to five genera of Mimosaceae 
is given and their use to delimit the investigated taxa is also indicated. 

2. Materials and methods 

Fresh fruits of Acacia auriculiformis Car., A. pennata Willd., A. torta Craib, Albizia 
lebbeck Bth., Calliandra sp., Pithecellobium dulce Bth. and Samanea saman Merr. 
were collected at different stages of their development from the University Bota- 
nical Garden. The materials were processed through the usual procedures after 
fixing in FAA. Voucher specimens are deposited in the Herbarium of Sardar Patel 
University. Materials were macerated in Jane's fluid (Jane 1956). Histochemical 
localization of lignin was done by Phloroglucinol-HCL method (Johansen 1940). 

3. Observations 

Sclereids occur at various places in the mature pericarp and are found scattered 
among parenchyma. They are recognized by lignified cell walls, orderly pitting 
and size. Their distribution is characteristic in the different species understudy. 

297 



298 S Aangata/i, / L Kothari 'and G L Shah 

There are two categories of sclereids in the investigated taxa : (a) non-idioblastic 
tissue forming sclereid layers and (b) idioblastic sclereids. In all the investigated 
taxa non-idioblastic tissue is present. In Acacia pennata and A. torta it consti- 
tutes 2-3 hypodermal layers (figures 3,4, and 6) whereas in Calliandra almost an 
equal number of layers occur below a hypodermal layer (figure 7). One to three 
layers of sclereids are present in the endocarp of Albizia, Pithecellobium and 
Samanea (figures 9-11). Idioblastic sclereids are present among the parenchyma 
of mesocarp in small or large groups in A. aurlculiformis (figures 1,2) or isolated 
and scattered in A. torta (figure 6 at arrow). Besides isolated sclereids they occur 
in groups of 2-3 in the outer epidermis in the .latter species (figure 5 at arrow). 
In Calliandra they are also present in small patches at the junction of sutural region 
and the valve. 

Sclereids vary in shape. Brachysclereids, which are more or less isodiametric 
(figures 12, 16 at arrow), are more frequent than the other types of sclereids. 
Other types of sclereids observed are the macrosclereids, which are rod-shaped 
(figures 12 at dart, 13 at arrow, 14), fusiform, which are broader at the centre and 
gradually taper at both ends (figure 16 at dart), spindle-shaped, which are also 
broader at the centre but abruptly taper at the ends (figure 15 at arrow) and 
kidney-shaped (figure 15 at dart), 

In transection they may be spherical (figures 1,4,5,8), oval (figure 1), angular 
(figure 7) or columnar and palisade like (figures 9-1 1). Sclereids have thick 
lignified and stratified walls with either broad or narrow lumen. In the endocarpic 
sclereids, however, the lumen is very much reduced and slit-like (figures 9-11). 
Sometimes it is obliterated (figure 2). Usually mature sclereids are occluded with 
tanniferous contents, as evidenced by ferric chloride staining. However, tanni- 
ferous contents are absent or very rare in the endocarpic sclereids (figures 9-1 1). 
Pit canals are visible in the epidermal, hypodermal and mesocarpial sclereids 
(figures 1,3-8) but not in the endocarpial ones (figures 9-11). They may be simple 
(figures 1,3,4) or branched (figure 8 at arrow). 

Differentiation of sclereids is evident at the mature stage of fruit development. 
In early stages a sclereid initial is distinguished by its size larger than the adjoining 
cells, dense cytoplasm and prominent nucleus with a single nucleolus (figure 1 at 
arrow). In the course pf differentiation the cell wall stratification increases with 
the deposition of lignin. Consequently, the lumen size is reduced. Finally the 
nucleus and the cytoplasm are autolyzed. In all the species, except endocarpic 
sclereids in Albizia, Pithecellobium and Samanea, pit canals become prominent 
and the lumen is filled up with tanniferous contents. Further mature sclereids 
are devoid of cytoplasm and nucleus. 

4. Discussion ..-.-.-.,... 

As far as the authors are aware this is the first account of the presence of sclereids 
in the pericarp of Mimosaceae (Metcalfe and Chalk 1979) though endocarpic 

Figures 1-8. All transactions of fruits showing sclereids. 1, 2. Acacia auriculi- 
formis. 3, 4. A. pennata. 5, 6. A. torta. 7, 8. Calliandra sp. (1,3,4,8 X 
1080 ; 2, 5, 7 x 270 ; 6 x-430). 



Pericarpial sclereids in some mimosaceae 



299 




309 



S Rangaiah, f L Kothari and G L Shah 




Figures 9-11. Transactions of fruits. 12-16. Macerated sclereids of fruits. 
9. Albizia lebbeck. 10. Pithecellobium duke. 11. Sanwnea saman 12,16. 
Acacia auriculiformis. 13-15. Calliandra sp. (9-11x270; .12, 13, 15x230; 
14 x 340 ; 16 x 75). 



Pericarplal sclefeicls! in some niimosacede 30 1 

sclereids of three types -vermisosclereid (worm-like), calceusosclereids (boot-like) 
and cruxosclereids (cross or T-like), are reported in Arachis of the Papilionaceae 
(Halliburton et al 1975). The only other detailed report of pericarpial sclereids 
is that of Gupta and Lamba (1981). They have accounted for the structure of 
sclereids in the endocarp of Rauvolfia serpentina. In the species investigated by 
us we have observed them in the epidermis of Acacia torta, hypodermal layers of 
A. pennata and A. torta, and in the mesocarp of A. auriculiformis. In Calliandra 
sp. sclereids occur at the junction of the sutural region and the valve in addition 
to subhypodermal layers. Endocarpic sclereids occur in Albizia, Pithecellobiwn 
and Samanea. 

The position and type of sclereids can also be useful to delineate the taxa studied 
as follows : 

Sclereids only non-idioblastic : 

Only brachysclereids present : 

Sclereids endocarpial * Albizia, Samanea 

Sclereids hypodermal Acacia pennata 

Sclereids fusiform Pithecellobium 

Sclereids idioblastic in addition to non-idioblastic ones : 

Sciereids mesocarpial Acacia auriculiformis 

Sclereids solitary or in 

groups of 2-3 in the epidermis Acacia torta 

Sclereids below a hypodermal layer * . * Calliandra 



Acknowledgement 

One of the authors (SR) thanks the Hoc for the award of a Teacher Fellowship. 

References 

Gupta V and Lamba L C 1981 Sclereids in the endocarp of Aauvolfia serpeiitina (L.) Benth. e# 

Kurz. ; Proc. Indian Acad. Sci. 90 79-84 
Halliburton B W, Glasser W G and Byrne J M 1975 An anatomical study of the pericarp 

of Arachis hypogaea with special emphasis on sclereid component ; Sot. Gaz. 136 219-223 
Jane F W 1956 The structure of wood (New York : Macmillan Company) 
Johansen D A 1940 Plant Microtechnique (London : McGraw-Hill) 
Metcalfe C R and Chalk L 1979 Anatomy of the Dicotyledons Vol. I, 2nd (ed.) (Oxford : 

Clarendon Press) 

Rao T A 1951 Studies on foliar sclereids. A preliminary survey ; J. Indian Bot. Soc. 30 2&-S9 
Rao T A 1980 Aspects and prospects of foliar sclereids in angiosperms. Sym. Vol. : Current 

trends in Botanical research, (eds.) M Nagaraj and C P Mallik, Kalyani Publications, 

pp. 67-72 
Rao T A and Bhattachary J 1978 A review on foliar sclereids in angiosperms ; Bull. Bot. Surv. 

India 20 91-99 

Rao T A and Bhupal O P 1973 Typology of sclereids ; Proc. Indian Acad. Sci. B77 41~55 
Rao T A and Silpi Das 1979 Leaf sclereids occurrence and distribution in the angio?perms. 

Bot. Notiser 132 319-324 

P.(B)~3 



Proc. Indian Acad. Sci. (Plant Sci,), Vol. 91, Number 4, August 1982, pp. 303-308 
Printed in India. 



Viability and infectivity of zoospores of Sclerospora graminicola 
(Sacc.) Schroet in the soil 



C R RAMESH* and K M SAFEEULLA 

Downy Mildew Research Laboratory, University of Mysore, Mysore 570 006, Indja 
* Scientist, Central Plantation Crops Research Institute, Kasargod 670 124, Icdia 

MS received 24 July 19S1 ; revised 7 July 1982 

Abstract. In the present study an attempt has been made to establish the fate of 
sporangia of Sclerospora graminicola (Sacc.) Schroet deposited in the soil. A 
technique has been standardised to demonstrate the germination of sporangia and 
the viability and infectivity of zoospores in the soil under laboratory conditions. 
For how long the zoospores remain motile in the soil is one of the many unanswered 
questions in the zoospore biology. From the present study it is seen that, the 
sporangia can germinate in the soil and liberate zoospores. The zoospores can 
move against gravity, remain viable and infective for 5 hrs in the soil. Survival of 
zoospores in the soil indicated that, they may serve as a potential secondary 
source of inoculum through soil under field conditions. 

Keywords. Sclerospora graminicola', pearl millet ; zcospoe ; soil ; green egr 
disease. 



1. Introduction 

Data have accumulated in evidence of zoospore serving as inoculum through 
water currents in soil-borne plant pathogenic phycomycetes like Phytophthora 
(Bewley and Buddin 1921 ; Klotz et al 1949 ; Mehrotra 1961 ; Nolla 1928 ; 
Mclntosh 1964 ; Zentmyer and Richards 1952). No studies have been 
made on these lines in the host parasite relationship of the "green ear disease" 
of pearl millet caused by Sclerospora graminicola. Turner (1960) found that 
zoospores of Phytophthora palmivora remained viable for six months in the soil 
at 50% water holding capacity. Royle (1963) established that, zoospores of 
Pythiwn aphanidermatum, P. cryptogea 9 P. fragariae and Aphaftomyces euteiches 
retained motility 1-3 hrs in non-sterile soil. Following water column technique, 
Mehrotra (1970) demonstrated that zoospores could move for a limited distance 
through soil towards plant roots, such movement being largely dependent on 
movement of water through the soil. Although infectivity of sporangia and 
zoospores (Thakur and Kanwar 1977)have been established no attempt has been 
made to study fate of sporangia and zoospores in the soil. In the present study 
an attempt has been made to study the germination, viability and infectivity of 
the sporangia of S. graminicola ip the soil . ; . 

303 



304 C R Rarnesh and K M Safeeulla 

2. Materials and methods 

2.1. Zoospore release and viability 

Three corning glass funnels of 10 cm diameter were used in the experiment. 
A cheese cloth with a 1 mm pore was inserted in the funnel and 3/4 of it was 
filled with sterilised red loamy soil. A rubber tubing carrying a pinch-cock 
was introduced at the narrower end of the two funnels, which were used for 
porangial release. The third funnel was used for sowing surface sterilised seeds 
of susceptible bajra cultivar HB-3. Distilled water was added to the funnels 
and allowed to become saturated from the bottom until the soil surface was 
covered with 2-4 mm of water. Inoculum was prepared by following the proce- 
.dures given by Safeeulla (1976). Sporangial suspension was obtained by incubating 
leaf bits collected from systemically infected bajra plants in petri dishes lined with 
moist filter paper and scraping sporangia, thus obtained in distilled water. In 
order to observe the release of zoosporcs from the sporangia in the soil,, freshly 
collected sporangial suspension was added to the soil in the two funnels and 
the water film on the surface of the soil was periodically observed for zoosporc 
release. After the liberation of zoospores, water was periodically drained from 
one of the funnels at regular interval of 1 hr for ten hrs to test the viability of 
zoospores. 

2.2. Infectivity and upward movement of zoospores 

For testing the infectivity and upward movement of zoospores in the soil, the 
following method was used. Three day old susceptible bajra seedlings were kept 
in contact with the periodically drained water to test the infectivity of zoospores. 
The seedlings thus inoculated were incubated for 12 hrs and transferred to pots 
containing sterilised soil. To test the upward movement of zoospores, the second 
funnel containing the zoospores was connected with the help of a rubber tubing 
to the narrower end of the funnel containing 4-day old bajra seedlings and the 
soil was saturated with water so that a continuous column of water was maintained 
in the rubber tubing connecting the two funnels (figure 1). 

3. Observations 

3.1. Release and viability of zoospores 

Zpospores were released from the sporangia 30 min after sowing in the funnels. 
Observation of zoospores at hourly intervals revealed that they were motile for a 
period of 5 hrs from the time of release. 

3.2. Infectivity and upward movement of zoospores 

Pearl millet seedlings kept in contact with drained off water, developed downy 
mildew symptoms indicating the infectivity of zoospores up to 5 hts after their 
release in the soil. However the percentage of infection decreased with the 
increase in the duration of retention following their release. Retention for 
1-2 hrs resulted in 60% infection. At 3rd, 4th .and 5th hrs it decreased to 36%, 
and 9% respectively. Beyond 5 hrs no infection >as noticed. Seedling 



Viability and infect ivity of zoo spores 



305 




Figure 1. Viability and upward movement of zoospores. 



Viability and infectivity of zoospores 307 

raised in the funnel connected to the zoospores source showed downy mildew 
symptoms 6 days after inoculation indicating the upward movement of zoospores. 



Discussion 

Sclerospora graminicola produces a large number of asexual sporangia during 
night under field conditions. Out of the large number of sporangia produced 
and liberated, some get into the air, a few get deposited on the same or on the 
neighbouring plants and a few fall to the ground. The significance of the sporangia 
falling on the soil was not realised so far in Sclerospora graminicola. Sporangia 
are produced during the humid night hours, which is congenial for the release 
of zoospores from the sporangia. The present study has indicated that sporangia 
can germinate in the soil liberating zoospores, which can thrive for 5 hrs without 
loosing their viability. The earlier reports (Haensler 1925; Lockwood and 
Ballard 1959 ; Esmarch 1927 ; Kuhlman 1964 ; Chupp 1917) have failed to 
establish the distance travelled by zoospores in the soil unaided by water move- 
ment. From the present study it is clear that the zoospores have moved up- 
wards along the rubber tube, stem of the funnel and the soil profile to infect 
the seedlings in the funnel. Since zoospores can thus remain viable and infec- 
tive in the soil, they may serve as a potential source of secondary inoculum for 
causing infection in the field. The texture and moisture content of the soil might 
also influence the viability and infectivity of zoospores. The decline in the percen- 
tage of infection of seedlings with the increase in the retention period might be 
due to the reduction in the number of motile zoospores coming in contact with 
the roots. This also indicates that, zoospore loose their motility in the soil when 
they are retained for longer periods. 



Acknowledgements 

One of the author (CRR) acknowledges the Indian Council of Agricultural Research 
New Delhi, for financial assistance. 



References 

Bewley W F and Buddin W 1921 On the fungus flora of gteen house water supplies, relation 

to plant disease ; Ann. AppL Biol. 8 10-19 
Chupp C 1917 Studies on club root of cruciferous plants ; New York State Agric. Exp. Stn. 

Bull. 387 421-452 

Esmarch F 1927 Untersuchungen zur biologic des kartofesl knebser ; Angew. Bot. 9 88-124 
Haensler C M 1925 Studies on the root rot of peas (Pisum satium) caused by Aphanomyces 

euteiches Drechsler. ; New Jersey Agric. Exp. Stn. Rep. 46 467-484 
Klotz C J, Wong P P and De Wolfe T A 1949 Survey of irrigation water for the presence 

of Phytophthora species pathogenic to citrus ; Plant Dis. Rep. Suppl 43 830-832 
Kuhlman E G 1964 Survival and pathogenecity of Phytophthora cinnamomi in several Western 

Oregon soils ; Forest ScL 10 151-158 
Lockwood J C and Ballard J C 1959 Factors affecting a seedling test for evaluating resistance 

of pea to Aphanomyces root rot ; Phytopathology 49 406-410 
Mclntosh D L 1964 Phytophthora species in soils of the Okang^s and imilankameen valleys 

of British Cotyrnbi^ ; Can. J. Bot. 42 1411- 141? 



308 C R Ramesh and K M Safeeulla 

Mehrotra R S 1961 Phytophthora parasitica var piperina on pan piper betel Ph.D. thesis Univ. 

Saugar India p. 181 
Mehrotra R S 1970 Techniques for demonstrating accumulation of zoospores of Phytophthora 

species on roots in soil ; Can. J. Bot. 48 890-892 
Nolla JAB 1928 The black shank of tobacco in Puerto Rico ; /. Dept. Agric. Puerto Rico 

12 185-215 
Royle D J 1963 The behaviour of zoospores of Pythium aphanidermatwn in response to roots, 

root substances and chemical compounds ; Ph.D. thesis, University of Western Ontario, 

London, Ontario, Canada p. 194 
Safeeulla K M 1976 Biology and control of downy mildews of pearl millet, sorghum and finger 

millet ; Downy Mildew Research Laboratory, University of Mysore, Mysore, India. (Final 

tech. report project CR 352, PL 4SO Grant No. FG-IN-414 1969-1975) XV + 304 
Thakur D P and Kanwar Z S 1977 Infectivity of sporangia of Sclerospora graminicola on 

pearl millet downy mildew ; Indian J. My col. Plant Path. 7 104-105 
Turner P D 1960 Saprophytic activity of Phytophthora palmivora Annual Report West African 

Cacao Res. last. 1958-59 25-26 
Zentmyer G A and Richards S J 1952 Pathogenecity of Phytophthora cinnamomi to avocad, 

trees and the effect of irrigation on disease development ; Phytopathology 42 35-37 



roc. Indian Acad. Sci. (Plant ScL), Vol. 91, Number 4, August 1982, pp. 309-318. 
g) Printed in India. 



nitiation, development and structure of root nodules in 
iome members of the tribe Trifolieae (Papilionaceae) 



G L SHAH and M GOPALA RAO 

Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120, 
India 

MS received 16 September 1981 ; revised '14 June 1982 

Abstract. Initiation and development of root nodules are studied in 7 species and 
the structure in 4 species, belonging to 3 genera of the tribe Trifolieae. The shape 
of the mature nodules may be spherical, cylindrical, fan-like or coral-like. The 
bacterial threads enter the root through the intact epidermis and cause proliferation 
in cortex by liberating the bacteria. The origin of nodules in the investigated taxa 
is exogenous and they belong to the * apical * type in Kodama's classification. A 
mature nodule comprises of nuristematic zone, cortex with vascular bundles and 
the bacteroid zone. The bacteroid zone is heterogeneous and is composed of infected 
and uninfected cells. 

Keywords. Trifolieae; toot nodule; exogenous origin ; bacterial thread; proli- 
feration of cortex ; bacteroid zone. 



L Introduction 

fhe root nodules have been a subject of investigation because of nitrogen fixing 
unction. Though extensive studies have been made on physiology, cytology 
rid histology of root nodules in the leguminosae in general, only a few investi- 
;ators have paid attention to the nodule anatomy in the tribe Trifolieae (Peirce 
902; Thornton 1930; Nutman 1948; Dart and Mercer 1963, 1964; Jordan et al 
.963; Mosse 1964; Munns 1968; Tu 1977). The present irivesligation is a 
,upplement to the existing data based on the study of initiation and development 
>f nodules in Medicago orbicularis All., M. scutellata Mill., M. truncatula Gaertn., 
\felilotus officinalis Pallas, M. wolgica Poir., Trigonella corniculata L. and 
r. foenumgraecum L. and structure of nodules in Medicago sativa L., Melilotus 
ilba Med. M. indica All. and Trigonella foenum-graecum L. 



L Materials and methods 

ieeds of Trigonella corniculata were obtained locally from a seedsman whereas 
ully developed nodules of Melilotus indica were collected from the plants growing 
rild in the University Campus. The rest of the species were raised from the 
eeds obtained from Berlin in the University Botanical Garden.. Low viability 

309 



310 G L Shah and M Gopala Rao 

of the seeds precluded the investigation of nodular structure of some species. The 
roots and rootlets of seedlings and root nodules of different stages of develop- 
ment were fixed in FAA (Johansen 1940) and stored in 70% alcohol after 48 hrs. 
Longitudinal and transverse sections of roots and nodules (5-8 /on) were stained 
with safranin O and fast-green FCF (Berlyn and Miksche 1976) and made perma- 
nent in a customary way. 



3. Observations 

3L Morphological description 

The developing nodules are spherical, but the fully developed ones are commonly 
cylindrical and variously lobed often becoming fan-like or coral-like and rarely 
spherical. They occur on primary, secondary and tertiary roots. There is no 
variation in size except that the smallest nodules are in Melilotus alba and the 
largest in Trigonella foenum-graecum (figures 1-4). 

3 .2. Infection., initiation and development 

The entry of bacterial threads (BT) into the root cortex through the intact epidermis 
(E) is observed only in Melilotus officinalis, Medicago truncatula and Trigonella 
foenum-graecum (figure 5). The cortical cells through which the threads pass 
are relatively larger than the remaining cells (figure 6, at arrows). The threads 
are often found in close proximity or in contact with the host cell nucleus (N) 
while passing through the cells (figures 5, 7). Further, they develop bulbous or 
funnel-shaped swellings (s) adjacent to the cell wall in Medicago truncatula, 
Melilotus officinalis and M. wolgica (figures 7, 8). The threads rupture and 
liberate the bacteria into the middle of the cortex. The bacterial infected cells 
contain dense cytoplasm and distinct nucleus, referred here as "proliferation 
cortical initials" (PCI) (figure 9). By repeated divisions these initials produce a 
mass of cells, eaph with dense cytoplasm and a nucleus (figure 10). Gradually, 
the divisions become restricted to the distal end as a result of which the mass 
of cells attains spherical shape (figure 11). The developing spherical nodule is 
now distinguishable into 2-3 layers of peripheral tangentially elongated cells (PL) 
around the inner mass of cells (MC) (figure 12). It is at this stage the inner mass 



Figures 1-10. 1-4. Mature root nodules. 1. Melilotus alba. 2. Medicago 
sativa. 3 and 4. Trigonella foenum-graecum. 5-10. T.S. of roots. 5. Meli- 
lotfts officinalis showing the entry of bacterial thread, into the root. 6. Medicago 
truncatula showing large cortical cells through which bacterial threads pass at arrows. 

7. Melilotus wolgica showing the contact of bacterial thread with host cell nucleus. 

8. Melilotus officinalis showing the swelling of bacterial thread near cell wall, 
note the breaking of thread at arrow. 9. Melilotus wolgica showing proliferation 
cortical initials. 10. Melilotus wolgica showing mass of proliferated cells. 
1-4 line indicates 1 mm ; 5 x 260 ; 6 X 160 ; 7 x 650 ; 8 x 380 ; 9 X 430 ; 
10 X 160. 

Abbreviations : BT, bacterial thread ; N, host cell nucleus ; E, epidermis 5 
s, -swelling; PCI, proliferation cortical initials. 



Intiaition, development and structure of root nodules 



311 




312 



G L Shah and M Gopala Rao 



msm^mtK 




initiation^ development and Structure of root nodules 313 

of cells transforms into the bacteroid zone (BZ) and the peripheral layers into 
nodule cortex (NC) with distinct apical meristematic zone (MZ) (figure 13). The 
ruptured threads even after liberating the bacteria remain in the developing nodule 
(figure 12 at arrow). The developing nodule now protrudes from the root with 
a protective covering of a few layers of the root cortical cells (figure 13). 

3.3. Structure 

A mature nodule consists of meristematic zone (MZ), nodule cortex (NC) with 
vascular bundles and the central bacteroid zone (BZ). 

The meiistematic zone is situated at the apex of the nodule, composed of multi- 
layered, thin walled, tangentially elongated cells with dense cytoplasm and promi- 
nent nuclei, arranged compactly in regular rows (figure 14). 

The nodule cortex is homogeneous comprising of 3 to 6 layers of compact 
parenchymatous cells with vascular bundles (figures 15, 20). In Medicago sativa, 
Melilotus alba and M. indica 2 vascular bundles enter the base of the nodule 
(figures 16, 17 at arrows), but in Tngonella foenum-graecum there are 4 vascular 
traces two of which supply to each side of the nodule (figures 18, 19 at arrows). 
The vascular strands arise opposite to the protoxylem of the root stele (figure 17). 
The vascular strands during their upward course, branch repeatedly within the 
nodule cortex, but do not come in contact with the bacteroid zone (figure .20). 
The vascular bundles are "inversely collateral" and conjoint, surrounded by an 
endodermis (EN) (figure 21). 

The bacteroid zone is heterogeneous composed of approximately 75% infected 
cells (ic) and 25% uninfected cells (uc) (figure 22). The uninfected cells are 
packed with spherical starch grains and interspersed within the tissue of infected 
cells (figure 23 at arrow). The young infected cells are with distinct nucleus and 
contain bacteria (figure 24). In the maturing infected cells small vacuoles (v) 
appear (figure 25) and subsequently their fusion tend to form a large vacuole, 
pushing the contents and the nucleus towards periphery of the cell (figures 26, 27) 
and at this stage the mature infected cells are about four times larger than the 
uninfected ones (figure 22). At a later stage the disappearance of the nucleus 



Figures 11-19. 11-13. T.S. of roots. 11. Medicago orbicularis showing deve- 
loping spherical nodule. 12. Tngonella corniculata showing differentiation of 
peripheral layers and inner mass of cells in the spherical nodule. 13. Tngonella 
foenum-graecum showing protrusion of nodule from the root. 14. L.S. of nodule 
of Tngonella foenum-graecum showing meristematic zone. 15. T.S. of nodule of 
Medicago sativa. 16. T.S. of the basalmost region of nodule of Medicago sativa 
showing vascular bundles, at arrow. 17. T.S. of root with .nodule of Melilotus 
indica showing vascular connections with root stele, at arrows. 18. -T.S. of the 
basalmost region of nodule of Tngonella foenum-graecum showing vascular traces, 
at arrows. 19. L.S. of the root with nodule of Tngonella foenum-graecum showing 
vascular connections with root stele, at arrows. 

11 x 532 ; 12 x 380 ; 13 x 43 ; 14 x 450; 15 x 32; 16 x 100; 17 x 48; 18 x 80; 
19 x 38. 

Abbreviations : PL, peripheral layers ; MC, mass of cells ; NC, nodule cortex; 
BZ, bacteroid zone ; MZ, meristematic zone ; VB, vascular bundle ; RC, root cortex; 
RS, root stele. . 



314 G L Shah and M Gopala Rao 

and clumping of the bacteroids of the infected cells lead to the senescence of 
the nodule (figures 28, 29 at arrow). 



4. Discussion 

The mode of invasion of bacterial thread into the roots is considerably interesting. 
Inmost of the legumes the infection takes place through root hairs (Thornton 
1930 ; Bond 1948 ; Harris etal 1949 ; Arora 1956b, c ; Nutman 1959 ; Dart and 
Mercer 1963, 1964 ; Narayana 1963 ; Narayana and Gothwal 1964 ; Kapil and 
Kapil 1971), but in a few cases it is reported to enter through the intact root 
epidermal cslh (McCoy 1929 ; Sshaede 1940), broken epidermal ceils (Bieberdorf 
1938) and wounded and ruptured cortical cells during the emergence of lateral 
roots (Allen and Allen 1940 ; Arora 1954). Narayana and Gothwal (1964) reported 
the infection thread to enter through root hair in Ttigonella foenum-graecum, 
but we have noted its entry through the intact root epidermis in Ttigonella foenum- 
graecum and also in Medicago sativa and Melilotus officinalis. 

From the data presented regarding the depth of penetration of infection thread 
into the root cortex (Biebsrdorf 1938 ; Bond 1948 ; Arora 1956c ; Narayana 
1963 ; Kapil and Kapil 1971) it appears that there is no correlation between the 
depth of penetration of the thread and the structure (thickness) of the root cortex. 
In the plants of present investigation also the penetration of the infection thread 
is up to middle region of the cortex in primary, secondary and tertiary roots. 

The dissemination of the bacteria can be either by the invasion of the infec- 
tion threads of the newly produced cells (Bond 1948 ; Harris et al 1949 ; Nara- 
yana 1963 ; Narayana and Gothwal 1964 ; Kapil and KapiJ 1971) or by the 
division of the infected cells (McCoy 1929 ; Allen and Allen 1940 ; Arora 1954). 
In our plants it is by the second method. 

Several view points have been put forth to explain the formation of the funnel 
shaped swellings in the infection threads (McCoy 1929 ; Thornton 1930 ; Harris 
etal 1949 ; Arora 1956c ; Narayana 1963 ; Narayana and Gothwal 1964 ; Dixon 
1964). Such swellings are observed in the present investigation and may be due 
to emaciation of the bacterial mass caused by the stretching of the thread during 
the enlargement of the host cell harbouring it (Arora 1956c). The breaking of 



Figures 2(1-29. L.S./T.S. of nodules. 20. L.S. of nodule of Tngonella foertum- 
graecum showing branching of vascular strands. 21. Melilotus indica showing 
vascular bundle. 22. Bacteroid zone of Medicago sativa showing infected and 
uninfected cslls. 23. Melilotus indica showing starch grains in the uninfected 
cells, at arrow. 24-26. Melilotus alba. 24. Young infected cells. 25. Maturing 
infected cell showing vacuoles. 26. Fusion of vacuoles in the maturing infected 
cell. 27. Miture infected cell of Melilotus indica showing large central vacuole 
aid paripheral contents. 28-29. Trigondla foenum-graecum showing infected cells 
at the senescence. 28. Early stage of bacterial clumping, at arrows. 29. Late 
stage of bacterial clumping, at arrow. 

20 x 38 ; 21 x 768 ; 22 X200 ; 23 x 380 ; 24 x 1040 ; 25 x 960 ; 26 X 640 
27 x 350 ; 28 x 1600 ; 29 x 560. 

Abbreviations : NC, nodule cortex ; EN, eadadermis ; XY, xylera ; PH, phloem ; 
uc, uninfected cell ; ic, infected cell ; N, nucleus ; V, vacuole. 



Tnitiation, development and structure of root nodules 



315 




Initiation, development and structure of roo-t nodules 317 

the thread in the middle (Narayana 1963) further supports our conclusion (figure 8 
at arrow). 

The meristematic zone may be situated at the apex or at several places 
surrounding the nodule and accordingly they may be " apical " or " spherical " 
respectively (Kodama 1967). In the species of the present investigation the meri- 
stematic zone occurs at the apex and thereby the nodules confirm to "apical" 
type of Kodama (1967). 

The structure of the nodule cortex is reported to be heterogeneous in some 
legumes (McCoy 1929 ; Harris etal 1949 ; Arora 1954 ; Allen etal 1955 ; Nara- 
yana and Gothwal 1964 ; Kapil and Kapil 1971), but it is homogeneous, compris* 
ing of 3-6 layers of compact parenchyma cells surrounded by 3-6 layers of root 
cortex in the species studied by us. 

There is a great variation in the number and orientation of vascular strands 
connecting the root stele in different leguminous nodules (Bieberdorf 1938 ; Bond 
1948 ; Harris etal 1949 ; Arora 1954, 1956a-c ; Narayana 1963 ; Kapil and Kapil 
1971). We have observed four vascular connections in Trigonella foenum-graecum 
(see also Narayana and Gothwal 1964) and two in Medicago sativa, Melilotus 
alba and M. indica. 

The arrangement of phloem and xylem in the vascular bundles in different 
leguminous nodules is variable (Allen and Allen 1940 ; Allen et al 1955 ; Kapil 
and Kapil 1971). It is inversely collateral in the nodules of the investigated 
species (see Kapil and Kapil 1971). 

All the cells in the bacteroid zone contain bacteria (Allen and Allen 1940 ; 
Arora 1954, 1956a) or some of them may be infected by bacteria and others may 
not (Harris etal 1949 ; Arora 1956b, c ; Narayana 1963 ; Kapil and Kapil 
1971). The latter situation is observed in our plants and the percentage of infected 
and uninfected cells is 75% and 25% respectively. 

We are in agreement with the observations of Allen and Allen (1958) who 
report that the earliest indication of senescence is the change in colour of the 
bacteroid zone from red to green and a change in the nodule surface from smooth 
to wrinkled. Clumping of bacteria is quite common during the nodule senescence. 



Acknowledgements 

We thank the director of Botanischer Garten iind Botanisches Museum, Berlin 
for the gift of seeds and the Government of Gujarat for awarding a research 
fellowship to MGR (11-7-1977 to 10-7-1980). 



References 

Allen E K and Allen O N 1958 Ecological aspects of symbiotic uitiogen fixation ; Handb. 

Pfi-Physiol. 8 48-118 
Allen E K, Gregory K F and Allen O N 1955 Morphological development of nodules on 

Caranaga arborescens Lam. ; Can. J. Bot, 33 139-14S 
Allen O N and Allen E K 1940 Response of the peanut plant to inoculation with rhizobia, 

with special reference to morpholcgical development of the nodules ; Bot. Gaz. 102 121-142 
Aiora N 1954 Morphological development of the root and stem nodules of Aeschynomene indica 

L. ; Phytomorphology 4 211" 21 6 



318 G L Shah and M Gopala Rao 

Arora N 1956a Morphological development of root nodules in Crotalaria juncea ; Proc. 43rd 

Indian Sci. Congr. (Agra) 244 
Arora N 1956b Morphological study of root nodules on Cajanus indicus ; Proc. 43rd Indian 

Sci. Congr. (Agra) 244-245 
Arora N 1956c Histology of the root nodules on Cicer arietinum L. ; Phy to morphology 6 367- 

37$ 
Berlyn G P and Miksche JP 1976 Botanical microtechnique and cytochemistry; The Iowa State 

University Press, Ames, Iowa. 
Bieberdorf F W 1938 The cytology and histology of the root nodules of some Leguminosae ; 

J. Am. Soc. Agron. 30 375-389 
Bond L 1948 Origin and developmental morphology of root Modules of Piswn sativum ; Bot. 

Gaz. 109 411-434 
Dart P J and Mercer F V 1963 Development of the bacteroid in the root nodule of Barrel Medic 

(Medicago tribuloides Desr.) and subterranean clover (Trifolium subterraneum L.) ; Arch. 

Microbiol. 46 382-401 
Dart P J and Mercer F V 1964 Fine structural changes in the development of the nodules of 

Trifolium subterraneum L. and Medicago tribuloides Desr. ; Arch. Microbiol. 49 209-235 
Dixon ROD 1964 The structure of infection threads, bacteria and bacteroids in pea and 

clover root nodules ; Arch. Microbiol. 48 166-178 
Harris J O, Allen E K and Allen ON 1949 Morphological development of nodules on 

Sesbania grandiflora Poir. with reference to the origin of nodule rootlets ; Am. J. Bot. 

36 651-661 

Johansen D A 1940 Plant microtechnique New York, USA 
Jordan D C, Grinyear I and Coulter W H 1963 Electron microscopy of infection threads and 

bacteria in young root nodules of Medicago saliva ; /. Bact. 86 125-137 

Kapil R N and Kapil N 1971 Root nodules of Cajanus cajan : Origin, structure and onto- 
geny ; Phytomorphology 21 192-202 
Kodama A 1967 Cytological studies on root nodules of some species in Leguminosae II ; 

Bot. Mag. Tokyo 80 92-99 
McCoy E F 1929 A cytological and histological study of the root nodules of the bean, Phaseolus 

mlgaris L. ; Centr. Bakt. II 79 394-412 
Mosse B 1964 Electron microscope studies on nodule development in some clover species ; 

/. Gen. Microbiol. 36 49-66 
Munns D N 1968 Nodulation of Medicago sativa in solution culture. I. Acid-sensitive steps ; 

Plant Soil. 28 129 
Narayana H S 1963 A contribution to the structure of root nodule in Cyamopsis tetragonoloba 

Taub. /. Indian hot. Soc. 42 273-280 
Narayaua H S and Gotbwal B D 1964 A contribution to the study of root nodules in some 

legumes ; Proc. Indian Acad. Sci. B59 350-359 
Nutmau P S 194& Physiological studies on nodule formation. I. The relation between nodu- 

lation and lateral root formation in red clover; Ann. Bot. 12 SI -96 
Nutrnan P S 1959 Some observations on root hair infection by nodule bacteria ; /. Exp. Bot. 

10 250-263 
Peirce G J 1902 The root tubercles of bur clover (Medicago denticulata Willd.) and of some 

other leguminous plants ; Calif. Acad. Sci. Proc. 3rd Ser. Botany 2 295-328 
Schaede R 1940 Die kn ollechen der advent; ven wasser wurzelen con Neptunia oleracea und 

ihre bakterian symbiose ; Planta 31 1-21 
Thornton H G 1930 The early development of the root nodule of lucerne (Medicago sativa) ; 

Ann. Bot. 44 385-392 
Tu J C 1977 Structural organization of the rhizobial root nodule of alfalfa ; Can J Bot 55 

35-43 



Proc. Indian Acad. Sci. (Plant Sci.) Vol. 91, Number 4, August 19S2, pp. 319-328 
Printed in India, 



Turnera ulmifolia var. elegans x T. ulmifolia var. angustifolia 
crosses and its bearing on the taxonomy of the species 



K RAJEEV, P I KURIACHAN and C A NINAN 

Department of Botany, University of Kerala, Trivandrum 695581, India 

MS received 17 November 1981 

Abstract. The heterostylous taxon T. ulmifolia var. elegans Urb. (2/z = 20) and 
the homostylous taxon T. ulmifolia var. angustifolia Willd. (2n 30) were selfed and 
intercrossed. Both pin and thrum forms of var. elegans are self incompatible while 
the variety angustifolia is self compatible. Among the intervarietal combinations 
only angustifolia x elegans (thrum) crosses were successful though the hybrid seeds 
were inviable. 

From an analysis of the results of in vivo pollen germination studies in the 
incompatible crosses, it is concluded that angustifolia pollen are of the thrum type 
and its pistil is of the pin type. It is suggested that var. angustifolia has evolved 
by a rare crossing over within the super gene complex for heterostyly in the parent 
taxon, which might be the hexaploid (2/z = 30) heterostylous T. ulmifolia Linn. The 
failure of the apparently ' legitimate ' cross elegans (pin) x angustifolia is suggested 
to be due to the ploidy difference between the two varieties. On morphological 
cytological and biochemical grounds the separation of the elegans element from, 
T. ulmifolia complex and assigning of species status to it is suggested. 

Keywords. Turnera ; heterostyly ; intervarietal crosses ; incompatibility. 



1. Introduction 

Turnera ulmifolia L. is a polymorphic weedy species of the New World Tropics 
with a fairly high caffeine content in the seeds (Raffauf 1970; Tarar and Patil 1974). 
T. ulmifolia var. elegans Urb. and T. ulmifolia var. angustifolia Willd. occur as intro- 
duced weeds in South India (Gamble 1915). The former variety is heterostylous 
and is self incompatible while the latter is homostylous and self and cross compa- 
tible (Barrett 1978). Earlier cytological studies have shown that T. ulmifolia 
var. elegans has 2n= 20 chromosomes (Raman and Kesavan 1963; Barrett 1978) 
and T. ulmifolia var. angustifolia has 2n= 30 chromosomes (Barrett 1978; Tarar 
and Dnyanasagar 1976, 1979). Results of intervarietal crosses between these two 
taxa are reported here. 

2. Materials and methods 

Plants of natural populations of the two varieties occurring in the Kariavattom 
Campus of the University of Kerala were used in this study. The flowers of 

319 



320 K Rajeev, P I Kuriachan and C A Ninan 

T. ulmifolia var. angustifolia open by about 6 am while those of T. ulmifolia 
var. elegans open by 7-30 am. Flowers of the former taxon were emasculated on 
the evening previous to blooming. Emasculation on the day previous to blooming 
caused serious damage to the buds of T. ulmifolia var. elegans and hence these were 
emasculated only about one hour before the flowers open. Flowers of both the 
varieties were pollinated at 7-30 am. 

White's (1954) culture medium solidified with 4% agar and supplemented with 
100 ppm indole acetic acid was used to culture the hybrid seeds. In vivo germination 
of pollen grains was studied by staining the styles and stigmas after treatment in 
lactophenol for 17hrs at 70C. Pollen tubes taken from 9 styles were studied. 
Measurements of leaves, bracteoles, stamens, styles and pollen are given from 25 
observations in each case. 



3. Results 

3-1. Intra- and inter- varietal crosses 

Turnera ulmifolia var. elegans is a profusely branched, bushy, distylous herbaceous 
perennial growing up to a height of 40 cm (figure 1). The leaves are 2-5 cm to 
4 -25 cm long, roundish with crenate margin. The bracteoles are linear, up to 
2 cm long. The flowers are cream coloured with dark violet spots at the base of 
the petals (figures 2,3). The pin styles are on an average 10 -56 mm long while 
the thrum styles measure only 5 -66 mm. The average length of pin and thrum 
stamens were 6 -00 mm and 10 -74 mm respectively. The pollen also showed 
dimorphism with an average measurement of P x E= 64-15/* x 58-93#na in pin 
types and P x E = 70- 12/*jn x 63-35/*m in the thrums. Both the types showed 
an average of 20 seeds per capsule. 

Plants of T. ulmifolia var. angustifolia are sparcely branching erect shrubs, rea- 
ching more than 1 in in height (figure 4). Leaves are elliptic-lanceolate, 10-12- 5 cm 
long with irregularly serrate margin. Bracteoles are foliaceous up to 3- 1 cm long 
and 0- 75 cm broad. Flowers are long-homostylous (figure 5) with bright yellow 
petals. The styles are 19 -2 mm long and the stamens 20 -44 mm. The fruits are 
larger than that of the former variety and contain about 70 seeds in each. 

Self and cross pollinations in T. ulmifolia var. angustifolia and both pin and 
thrum forms of T. ulmifolia var. elegans and the intervarietal crosses were made 
(table 1). T. ulmifolia var. angustifolia was found to be self and cross com- 
patible. In T. ulmifolia var. elegans only crosses between the thrum and pin forms 
were compatible. Among the intervarietal crosses only the cross T. ulmifolia 
var. angustifolia x T. ulmifolia var. elegans (thrum) was found to be successful. 

The hybrid seeds obtained failed to germinate when sown on wet cotton in petri 
dishes and also in soil. The hybrid seeds cultured in White's medium with 100 
ppm indole acetic acid also failed to germinate. 

3-2. In-vivo pollen germination 

In vivo germination of pollen grains in the three unsuccessful intervarietal crosses 
were studied with a view to elucidate the causes of their failure. In the successful 



Intervdrietal crosses in Turnera ulmifolia 



321 




Figures 1-5. 1. vat. elegans, plant habit. 2. Dissected thrum flower of var. 
elegans, arrow points to stigma x 1. 3. Dissected pin flower of var. elegans, 
arrow points to stigma x 1. 4. Var. angustifolia, plant habit. 5. Dissected 
flower of var. angustifolia, arrow points to stigma in a black background x 1, 



322 



K Rajeev, P I Kwlachan and C A Ninan 




Figures 6-11. 6-$ and 11. x $0. lO. x 2. Arrow point to ends of pollen tubes. 
6, Elegans (pin) stigma showing inhibition of elegans (pir>) pollen tubes. 7. elegans 
(thrum) stigma showing inhibited angustifolia pollen tubes. 8, 9. elegans (pin) 
style bases showing angustifolia pollen tubes. 10. elegans (pin) gynoecium after 
(a) pollination with angustifolia pollen and (b) selfing. 11. Angustifolia style 
showing inhibited elegans (pin) pollen tubes, 



intervarietal crosses in "furnerct ulmifotid 



Table 1. Details of inter and intravarietal pollinations in T. ultnifolia var. elegans 
and T iilmlfolia var. angustifolia 



No. of 
Type of pollination flowers 
pollinated 


No. of Total Average 
capsules no. of no. of 
formed seeds seeds per 
capsule 


1. 


Intravarietal 


17 




elegans (pin) x elegans (pin) (cross) 


2. 


efe#w,s(pin) x elegans (pin) (self) 


15 


. . 


3. 


elegans (thrum) x elegans (thrum) (cross) 


17 


. . 


4. 


elegans (thrum) x elegans (thrum) (self) 


15 


.. 


5. 


elegans (pin) x elegans (thrum) 


11 


3. 67 22 


6. 


elegans (thrum) X elegans (pin) 


11 


1 13 13 


7. 


angustifolia x angustifolia (self) 


10 


7 518 73 


8. 


angustifolia x angusti folia (cross) 


10 


9 603 67 


9. 


Intervarietal 


21 




elegaus(v'm) x angustifolia 


10. 


elegans (thrum) x angustifolia 


14 


.. 


11. 


angustifolia x elegans (pin) 


39 


.. 


12. 


angustifolia x elegans (thrum) 


22 


9 117 13 



cross T. ulmifolia var. angustifolia x T. ulmifoUa var. elegans (thrum type), the 
elegans pollen tubes were found to reach the stylar base in 4 to 5 hrs after pollina- 
tion. Therefore, the styles in the unsuccessful intra- and intervarietal crosses were 
examined 7 hrs after pollination. In both pin x pin and thrum x thrum crosses 
in elegans the pollen grains germinated, but failed to enter the style (figure 6). 
The ends of pollen tubes got enlarged and burst at about the basal region of the 
stigma. In the three unsuccessful intervarietal crosses also the pollen germinated 
in vivo. In elegans (thrum) x angustifolia, the angustifolia pollen tubes were in- 
hibited at the stigma style joint. The ends of most of the tubes got enlarged and 
burst at this region (figure 7). In elegans (pin) x angustifolia crosses the angus- 
tifolia pollen tubes were found to reach the stylar base in about 7 hrs after polli- 
nation (figures 8,9). Pollinated flowers were invariably found to fall off, but only 
after 5-7 days during which period the ovary showed some enlargement (figure lOa). 
Long styled flowers of elegans emasculated and bagged for a day to prevent pollina- 
tion took 5-6 days to fall off, but the ovaries in these were not enlarged (figure lOb). 
In angustifolia x elegans (pin) crosses the elegans pollen tubes entered the style 
and grew down ; but most of the pollen tubes stopped growth just below the 
stigma, where their ends ballooned and burst opened (figure 11). The maximum 
length attained by pollen tubes in 9 cross pollinated styles are given in table 2. 
It is seen that the maximum length of pollen tubes observed was 95 % of the length 
of the style. 



K Rajeev, P / guriachan and C A tfinan 

Table 2. In viva pollen tube growth in T. ulmifolia var. angustifolia x 7. ulmifolia 
var. elegans cross 



No. of 
styles 


Length of Maximum growth 
style (mm) of pollen tubes 
(mm) 


Length of pollen 
tube as percent- 
taga of stylar 
length 


1 


15-0 


10-1 


67-33 


2 


15-5 


11-7 


75-48 


3 


15-0 


11-5 


76-67 


4 


16-0 


12'7 


79-38 


5 


15-5 


12-7 


81-94 


6 


15*5 


13-0 


83-87 


7 


16*0 


13-9" 


86-88 


8 


15-5 


13-9 


89-68 


9 


16-0 


15-2 


95-00 



4. Discussion 

4-1. Causes of intervarietal incompatibility 

Intervarietal crosses between angustifolia (female) and elegans thrum (male) were 
successful with. 40-9% fruit set. There was no fruit set in the reciprocal cross : 
though the angustifolia pollen grains germinated in vivo, the tube growth was 
inhibited in the stigma itself. The nature of inhibition of pollen tubes in this cross 
is similar to that in the incompatible intravarietal crosses of the heterostylous 
elegans. Moreover, in elegans pin (female) x angustifolia (male) crosses, angusti- 
folia pollen grew to the base of the styles in pin plants of elegans. These facts may 
suggest that with regard to its incompatibility reaction to the pin and thrum forms 
of elegans, angustifolia pollen are of the thrum type. 

In the intervarietal cross using pin forms of elegans as males, the pin pollen readily 
grew into the styles of angustifolia. But none of the pollen tubes was found to 
grow beyond 95% length of the style and the vast majority of the pollen tubes 
stopped growth much earlier. Their ends became bulged and were burst. It 
is clear from this that this cross is incompatible. This may further indicate that 
thr'long styled Angustifolia is, with regard to the incompatibility reaction of the 
pistil, a pin type. This inference is supported by the observed compatibility of the 
intervarietal cross when thrum plants of elegans were used as pollen parents. 

Since angustifolia is a homostyle with ' thrum type ' behaviour of pollen grains, 
the intervarietal cross elegans pin (female) x angustifolia (male) is an apparently 
c legitimate * cross. In this cross the angustifolia pollen tubes could be traced 
to the very base of pin styles of elegans. Though this together with the enlarge- 
ment and the slightly delayed abscission of the cross pollinated ovaries might 



Intervdrietal crosses in Turnera ulmlfolia 



325 




Figures 12-13. Photomicrographs of somatic chromosomes x 1500. 
elegans ; 13. var. angusti 'folia. 



12. var. 



Intenarietal crosses in Turnera ulmifolia $27 

suggest the possibility of fertilization in the cross, there was no fruit set. Normal 
growth of pollen tube without fruit set have been reported in such * legitimate ' 
crosses between heterostylous (pin) Linum perenne and homostylous L. lewisii also 
(Baker 1961). It is not clearly understood whether the failure of fruit set observed 
in the present cross is related to the interaction between the heterostylous and 
homostylous systems. However, L. perenne and L. lewisii are reported to have 
the same chromosome number (Fedorov 1969) whereas elegans used in this study 
is a tetraploid with 2n = 20 (figure 12) and angustifolia, a hexaploid with 2n = 30 
(figure 13). It is known that plants differing in chromosome numbers may show 
differences between reciprocal crosses and that in such cases greater success may 
be met with when the taxon with larger chromosome number is used as the seed 
parent (Thompson 1930). It is likely that the above cross is unsuccessful due to 
the lower level of ploidy in elegans (pin), used as the seed parent, than in angusti- 
folia. 

4-2. The probable origin of var. angustifolia 

The pistil of angustifolia exhibited incompatibility reactions characteristic of long 
styled plants and its anthers exhibited incompatibility reactions characteristic of 
short styled plants. This shows that this variety is a long homostyle. Lewis 
(1954, 1979) and Baker (1961) have suggested that such long homostyles may be 
formed as segregants in the progeny of polyploids of heterostylous species, as a 
result of rare cross-overs between 1^ and / a genes in the super gene complex 
GSIilaAP, controlling the heterostyly system. Instances of homostylous segre- 
gants have been reported among the offsprings of polyploids of the heterostylous 
species Fagopyrum esculentum (Esser 1953) and Primula obconica (Dowrick 1957), 
It is highly probable that the long homostylous angustifolia is derived from a hetero- 
stylous progenitor. The variety angustifolia is a hexaploid (2n = 30). The only 
other hexaploid taxon so far known in this species complex is T. ulmifolia 
(Hamel 1965). Apart from the fact that T. ulmifolia is heterostylous and T. ulmi- 
folia var. angustifolia is a long homostyle, these taxa resemble each other very 
closely in other morphological characters (Mudaliyar and Rao 1951). Therefore 
it is likely that var. angustifolia is a segregant of T. ulmifolia. 

4 '3. Taxonomic considerations 

Crosses between T. ulmifolia var. angustifolia and short styled plants of T. ulmifolia 
var. elegans yielded some seeds. But seed set per capsule in the cross was found 
to be very low compared to seed set by selfing in the seed parent (table 1). More- 
over, the hybrid seeds were inviable. These facts reveal the existence of a total 
reproductive barrier between the two taxa. Turnera ulmifolia var. angustifolia 
with 2n= 30 chromosomes (Tarar and Dnyanasagar 1976; Barrett 1978) is cytolo- 
gically distinct from T. ulmifolia var. elegans with 2n = 20 (Raman and Kesavan 
1963; Barrett 1978). T. ulmifolia var. elegans is endowed with a stable distyly 
system having self incompatibility. The plants of this variety are of a spreading 
type, with roundish leaves, 2- 5 to 4-25 cm long having crenate margin with promi- 
nent scarlet spots in the basal parts of petals. Plants of the variety angustifolia 



328 K Rajeev, P t Kuriackan and C A tfinart 

on the other hand are erect and shruby, plants lack the corolla spots, have ctfaftge 
yellow petals and elliptic-lanceolate leaves, 10 to 12- 5 cm long and with irregu- 
larly serrate margin. Besides, with respect to their foliar phenolic constituents 
these two varieties showed isolation values as high as 66-66% for elegans and 
68 -18% for angustifolia, which denote significant difference between them (Rajeev, 
Kuriachan and Ninan, unpublished). The foliar phenolic components of T. ulmi- 
folia (s.s) are not known. Though it is heterostylous (Martin 1965) as T. ulmi- 
folia var. elegans, in other morphological characters it resembles I 7 , ulmifolia var. 
angustifolia more closely than T. ulmifolia var. elegans (Mudaliar and Rao 1951). 
Cytologically also T. ulmifolia with 2n = 30 (Hamel 1965) is nearer to T. ulmifolia 
var. angustifolia than T. ulmifolia var. elegans. Thus evidence from crossability, 
cytology, morphology and foliar phenolic constituents support the separation 
of the ' elegans ' element from Turner a ulmifolia complex and assignment of species 
status to it. 



References 

Biker H G 1961 Rapid spsciation in relation to changes in the breeding system of plants, in 

Recent Advances V Botany Vol. 1 8S1~885 (University of Toronto Press : Toronto) 
Barrett S C H 1978 Heterostyly in a tropical weed : the reproductive biology of Turnera ulmi- 

folia complex (Turneraceae) ; Can. J. Bot. 56 1713~1725 
Dowrick V P J 1957 Cited from Baker H G (1961) 
Esser K 1953 Cited from Baker H G (1961) 

Fedorov A 1969 Chromosome Numbers of Flowering Plants. (Komarov Botanical last. : Lenin- 
grad) 
Gamble J S 1915 Flora of the Presidency of Madras Vol. 1 2nd edn. (Botanical Survey of India. 

Calcutta 1967) 
Hamel J L 1965 Le noyan et les somatiques di Turnera ulmifolia L. ; Mem. Mus. Nat . Hist. 

Natur. Now. Ser. B. Bot. 16 3-7 
Lewis D 1954 Comparative incompatibility in Angiosperrns and Fungi in Advances in Genetics 

6 235-285 (ed) M Demerce (Academic Press Inc. : New York) 
Lewis D 1979 Sexual incompatibility in Plants (Edward Arnold Ltd. : London) 
Martin F W 1965 Distyly and incompatibility in Turnera ulmifolia; Bull. Torrey Bat. Club 92 

185-192 
Mudaliar C R and Rao J S 1951 A contribution to the taxonomy of the genus Turnera ; Madras 

Agric. J. 38 319-371 
Raman V S and Kesavan P C 1963 Meiosis and nature of polyploidy in Turnera ulmifolia ; /. 

Indian Bot. Soc. 43 495-497 
Raffauf R F 1970 A Handbook of Alkaloids and Alkaloid Containing Plants. (Wiley Interscience : 

New York) 
Tarar J L 1974 Cited from Tarar J L and Patil K J Qualitative and quantitative estimation 

of caffeine in Turnera ulmifolia L. ; Indian J. Bot. 2 118-119 (1979) 
Tarar J L and Dnyanasagar V R 1976 Karyotype studies in Turnera ulmifolia Linn, var angusti* 

folia wllld. Botan ; que 7 217-222 cited from Tarar and Dnyanasagar, 1979 
Tarar J L and Dnyanasagar V R 1979 Meiotic studies in Turnera ulmifolia Linn. var. angusti* 

folia Willd. ; /. Indian Bot. Soc. 58 167-175 
Thompson W P 1930 Causes of differences in success of reciprocal interspecific crosses ; Am. 

Nat, 64 407-421 
White P R 1954 Cultivation of Animal and Plant Cells (2nd edn) (The Ronald Press Co. : New 

York) 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 4, August 1982, pp. 329-350. 
Printed in India. 



Airborne pollen grains of Visakbapatnam : A combined field and 
air sampling study 



A JANAKI BAI* and C SUBBA REDDI 

Department of Environmental Sciences, Andhia University, Waltair 530003, India 
* Botany Department, Andhr? University, Waltair, India 

MS received 9 December 1981 ; revised 21 June 1982 

Abstract. Field assessments at regular intervals from April 1975 to March 1979 
recorded 61 plant species comprising 13 grasses, 20 weeds and 28 trees and shrubs 
as emitting appreciable amounts of pollen into the atmosphere of Visakhapatnam. 
The data also showed relative prevalence of these taxa in different zones of the 
city and their flowering periods. Pollen output in terms of mimbei per anther and 
per flower was determined for 29 taxa. Air sampling with rod traps of 0-53 cm 
diameter enabled the identification of 23 different pollen types in the atmosphere 
with Poaceae accounting for Ca. 37% of the total pollen load. Casuarina contri- 
buted to 13% followed by Cyperaceae 6%, Eucalyptus 5-8%, Dodonaea 3-8%, 
Amaranth-Chenopod and Phoenix each 3-4%, Borassus 2-4% and Peltophorum 
2%. Of the total identified pollen, ca 85% belonged to anemophilous taxa. There 
was no pollen-free day. The total pollen and individual types displayed seasonally 
quite closely corresponding with the blooming seasons of the source plants. Three 
pollen peaks, two in the wet period (June -November) and one in the dry period 
(December May) were evident. Year to year variations in pollen abundance 
occurred and urban growth affected pollen frequency pointing to the need for routine 
[V; monitoring of the atmosphere. 

Keywords. Pollination calendar ; .pollen pioductivity ; airborne pollen ; atmos- 
pheric biopollutants ; Poaceae ; Casuarina. 



1. Introduction 

Allergic responses to airborne biogenic particles impose major adverse effects on 
the physical and economic health of mankind (Davis 1972). Airborne pollen 
grains belong to this group of particles and have long been recognised as the inci- 
tants of rhinitis and asthma. In order to identify the offending pollen agent(s) 
it is necessary to monitor the pollen particles in free air, their prevalence and 
emission patterns through systematic air sampling. Field assessments recording 
the relative abundance and blooming phenology of source plants complement 
and greatly increase the value of such information. In areas of industrial atmos- 
pheric pollution there is every likelihood that the airborne pollen and chemical 
pollutants interact with each other and result in the aggravation of human dis- 
comfort (Newmark 1970 ; Nilssow and Nybom 1978). 

329 



330 A Janaki Bai and C Subba Reddi 

Visakhapatnam, situated in Andhra Pradesh on the east-coast of India is an 
industrial area but which also has a rich vegetation (Venkateswarlu et al 1972). 
It is thus likely that the air over the city is being charged with pollen from vegeta- 
tion and gaseous emanations from industries that may cause suffering to the in- 
habitants. The experience of clinicians in the King George Hospital and of private 
practitioners support this supposition. The situation thus calls for serious efforts 
at routine atmospheric monitoring so that necessary measures can be taken to 
alleviate the human affliction. Efforts at trapping the airborne pollen were 
begun a decade ago (Subba Reddi 1970), but the information at hand is still far 
from complete. In this paper we present data describing the composition of 
the airborne pollen, relative prevalence of the constituents and their seasonal emis- 
sion patterns over a period of four years from April 1975 to March 1979 together 
with the distribution, blooming phenology of source plants and their pollen pro- 
ductivity per anther and per flower. We suggest these data serve as a basis for 
local clinical strategy and as a comparison with pollen spectra established 
elsewhere in India and abroad. 



2. Materials and methods 

2*1. Physiography and climate of the study area 

The physiography of Visakhapatnam (latitude 17 42' N and longitude 82 18 ' E) 
and its surroundings is shown in figure 1. On two sides the city is bounded by 
hill ranges ; on the north-eastern side the famous pilgrimage centre Simhachalam 
or Kailasa hill range with an average height of 300 m and about 16 km long and 
on the southern side Yarada hill range running for about 8 km w ith an average 
height of 300m and projecting prominently into the Bay, and popularly known 
as Dolphin's nose. The shore of Bay of Bengal forms the eastern boundary and 
the western side is bounded by the tidal basin called locally Vuppateru. 

The most important feature of the climate is the alternation of monsoon seasons 
classified by Indian Meteorologists as : (i) The northeast monsoon season 
(December-February), (ii) The hot weather season (March-May), (iii) The 
southwest monsoon season (June-September) and (iv) The retreating southwest 
monsoon (October-November). 

Figure 2 shows the mean distribution of temperature and rainfall for the period 
when spore trapping was done. The mean monthly temperature was 27-4 C and 
variation of the mean from month to month was approximately 2C. The diffe-. 
rence between mean monthly minima was no more than 9C. The hottest weather 
occurred in May, while the coolest in January. Most precipitation fell during. 
June-November. The total precipitation during the .4-year period declined in 
each successive year. 
^l;-g:, 

22. Field assessments 

Visakhapatnam was divided into seven zones (figure 1). Zone-I constitutes the 
thickly built up area ; zone-II a sparsely built up area with vacant land patches ; 



Airborne pollen 



331 




332 



A Janaki Bai and C Subba Reddi 



220- 



180- 



140- 



60- 



20- 




-15 



N 



M 



252 
o 



Figure 2. Mean monthly, mean monthly maximum and mean monthly minimum 
temperature and rainfall at Visakhapatnam for the period of the study, April 1975 
to March 1979. 



zone-Ill the areas adjacent to the Railway line ; zone-IV the industrial belt ; 
zone-V a swampy area ; zone-VI the Yarada hill range and zone-VII the Kattasa 
hill range. The last three zones were not included in the survey as the vegetation 
of the two hill ranges is sparse scrub with Dodonaea occurring in considerable 
frequency and the swampy area has mainly Avicennia plants of stunted growth. 
Trips were made to the other four zones at approximately weekly intervals during 
1975-1979 to record the prevalence of anemophilous and such entomophilous 



Airborne 

plants capable of liberating sizeable amounts of pollen into the ambient air. 
Arbitrary units like sparse, common, more common and abundant were used to 
indicate the prevalence. Concurrently observations were made on the onset, 
intensity, duration and termination of flowering of these plants. 

Taxonomic identification of the plants was done using the Flora of Visakhapatnam 
by Venkaterwarlu et al (1972) as well as comparison with the authenticated 
herbarium specimens available in the Botany Department", Andhra University. 

2-3. Pollen productivity 

The output of pollen per anther was assessed by the method described by Subba 
Reddi (1976). Mature and undehisced anthers were taken into clean, dry, stop- 
pered tubes with the aid of forceps and allowed to dry. Care was taken not to 
injure the anthers while transferring them into tubes. Afcer the completion of 
dehiscence, a measured volume of 50% alcohol containing methyl green stain was 
added to each tube with a small quantity of the wetting agent ' Tween 20 ' and 
the contents shaken thoroughly to get a uniform suspension of pollen grains. From 
the suspension thus obtained, one ml was pipetted out into a counting chamber and 
the pollen counted. Three counts were made for each sample and from them was 
calculated the number liberated by the anthers in that sample. Afterwards the 
emptied anthers were mounted on a microscope slide and examined for remaining 
pollen to make any necessary correction. 

Knowing the number of pollen grains produced by the number of anthers in- 
cluded in a particular sample, the pollen productivity was then estimated per anther 
as well as flower. 

2-4. Air sampling 

This was done using glass rods of 0- 53 cm diameter and 23 cm long wrapped with 
18mm square sticky cellophane strips. Each rod was supported vertically and 
exposed for 24-hour periods (1700-1 600 hr) in shelters 'of the type used by Hyde 
and Williams (1945) so as to protect the cylinder from rain other than that experi- 
enced during violent storm. Airborne pollen grains are impacted by the wind 
onto the traps. 

The details of preparing, exposing cylinders, mounting of the trace and scanning 
were the same as described by Ramalingam (1968) and Subba Reddi (1970). The 
pollen counts are expressed as no/cm 8 of the trap surface. 



3* Results and discussion 

3 - 1 . Plant species, their prevalence and flowering periods 

A variety of plant taxa was reported to occur in the study area (Venkateswarlu 
et al 1972). However, field observations on the mode of pollination indicated that 
only a relatively small number would emit pollen into the ambient air in sizeable 
amounts. Table 1 lists such plants with data on their relative prevalence in diffe- 



334 



A Janaki Bai and C Subba Reddi 







| 4. _j- ^ -!- + -f 4- 

+ 4- + 



44 44444 + 4- 

44 4 



4 | -|- + _ h 1 4. 4- 4 I 1 



4- 4, 4 4 . f 4. 4 4 4 4 -1- 4- 

4 4- 4. 4 H . 4 4- -I- 4- 4- 4- 4- 

4 4.. 4. 4. 4. 4 4- 

4 4 4 






2 

5 

5 



+ 



4- 



a 

1 




7T <D O 5 

llfl 



Q, ft 



45 o 
H to 



4- + 



4- 



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4 4. 

4- 



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4. 

4- 





cfl 

u 



> 

CO < 






|;a. 



^ ( * 



. 
.a S . 



M 

I 




si I I 



Airborne pollen 



335 



If 

1! 



o > 
>* > 



60 



ft 
II 

H H 



' ctf 
K O 

43 *> 



O O 

II 



.1 



I 





S o 

J-s 



o 

*- 

S 



> 



C3 



cC 

es 

5 fi 






O 



I S 



r- 'g 



?. 

s 

3 > 
in i 



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&o ^S 



4 



4 



+ + H- 4- 



4- 
4- 
4- 



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'I $t 
+ + 



-h + 4 4 + 4 
4 4 H- 4 + 
4 4 + 

'4 



I I 



+ 



4- + 



444.+ I + 
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+ +.+ -h 



4 4 
'4 



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4 



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4- 



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444 4 + 
' 4 + + 
' 4 



4 






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4- 



+ 4- 



4 4- 




: 

8 25.2^ 




1155 



336 



Jflnarfcj Sai and C Subba Reddi 







i 




t_j 


H > 






Flowering period 


. > S s 

1.1 f? tit 

o^5 & a, &^ 

V< I <& 60 cG 

11 |.I ill 
s< ^s ^^s 


November-Apr! 
January-June 


Throughout yeai 


f i! ! 
Ifl * 
III I 


ophilous . 














H 














8 








1 4- 




, , + . 


11 




> 


++ i + ++ + 


1 -y" 


+ 


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H 














IK 

a 




H 

M 


+ + i + + + + 


I + 


+ 


+ + + , 


1! 
1 




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4- + 






'+ + 


M 




a 














JH 










03 




? 










a 




*** 3 


++ l+ i + $ 


+ -f 


* 


+ + i 


II 




M 




+ + ++ ++.4- 


+ H- 


t 


ttf + 


5 












+ 


i 





























U 

II 














rt 








"o 






I 








J 






o 

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a 


* PH uSf 






a 

1. 


' .i 

tn 


yrtaceae 
Eucalyptus globulus, Labill. 
Syzygium }ambolanum> DC, 
potacae 
* Madhuca indica, Gmelin 
. * Mimusops elengi, Linn, 
iphorbiaceae 
* Cicea disticha, Linn, - 
*Emblica officinalis Gaertn. 
*- Ricinus communis, Linn. 


maceae 
* Baloptelea imegrifolia, (Ro 
oraceae 
* Morus alba, Linn. 


^uarinaceae 
* Casuarina equisetifolia, Lini 


eoaceae 
rt Boras wsflabellifer, Linn. 
* Coco* nucifera, Linn. 
* Phoenix sylvestris, (Linn.) 

ndanac^ae . 
* Pandanus fasdcu laris, Lam 


11 
-h 
-f 

i 

1 

If 






I S. A . w 




TO 

O 


<! ' ' 





Airborne pollen 



337 



snt zones of Visakhapatnam and their periods of flowering. The pollination 
alendar of some of the more important taxa is graphically presented in figure 3 
iiich shows clearly that, unlike temperate regions of the world, one or the other 
f the diverse plant taxa was in flower at all times of the year with consequent 
beration of pollen into the ambient atmosphere. During two periods a maximum 
umber of wind-pollinated plants were in bloom. Most Poaceae and other weeds 
roe in bloom during late June-early December, while most of the trees bloom 
uring January-April. Some of the weeds and grasses flowered in both periods 



&6donaea 

Eucalyptus 

Artemisia 

Xan.thlum 

Amaronthac* 



Cyperaceae 

Pandanus 

Gramineae 

Syzygium 

Peltophorum 

Emblica 

Madhuca 

Borassus 

Holoptelea 

Casuarina 

Phoenix 

Mimusops 

Crotoh 

Cicca 

Riclnus 





P [ M { A | M ( J 



f \ maxim at flowering - 'duration of flowering 

Figure 3, Pollination calendar of some plant species at Visakhapatnam. 



A Janaki Sai and C $ubba Reddi 

but they were more, numerous during late Jute-early December, Thpse obser- 
vations are in general agreement with the. earlier records of the blooming periods 
of the plant specie of this area (Subba Reddi 1974), . . 

3-2- Pollen productivity ^ '' ... 

Table 2 giv&s the estimated pollen productivity .per antfcer .as well as .flower 
determined for 29 of the -61 plant- taxa listed iti'table L The sample, size (the 
number of anthers included in a sample, percent of pollen that failed to get 
liberated into the medium together with the size of individual pollen types are 
also included in the table. 

Among these 29 plants, the highest pollen output per anther was for Phoenix, 
whereas jnaximum number per flower was for Ricinus. The lowest production 
both per anther and per flower was for Cyperus compressus. It might be expected 
that pollen productivity in different plants would be influenced by the size of 
grains as well as anthers, but this is not borne out by the results (table 2). 

The pollen productivity data given in table 2 indicate the relative capability of 
the different taxa to pollute the ambient air with their pollen. However, in some 
cases all the pollen produced might not be emitted into the air because of the 
hindrance offered by the plant habit. This is so in the case of Phoenix which was 
first for pollen productivity but the thick crown of leaves obstructs the free dissemi- 
nation of pollen. 

Distinguishing plant species by light microscope examination of pollen is not 
always possible, e.g., the pollen of stenopalynous groups like Poaceae and 
Amaranthaceae-Chenopodiaceae are not readily separated. In such cases the 
relative importance of the taxa contributing to the airspora might be determined 
by a knowledge of their pollen productivity coupled with tfyeir distributional data. 
For instance, the two species of Amaranthus greatly vary in' the quantity of pollen 
produced (table 2) and by knowing their relative abundance in a region, one can 
easily assess which species is important in the area. Assessed in this way, at 
Visakhapatnam A. spinosus is more important than A. gracilis. 

3 3 . Components of airborne pollen flora and their relative contributions 

Over the 4-year period a total of 34976 pollen/cm 2 of trap area were counted, Of 
these, 30851 pollen were identified and assigned to 23 different pollen types which 
are listed in table 3 alongwith the percent contribution of each to the total pollen 
flora. Numerically Poaceae ranked first with a mean contribution of 37-32% 
followed by Casuarina (13-11%), Cyperaceae (6-15%), Eucalyptus (5-83%), 
Dodonaea (3-8%), Amaranth-chenopod and Phoenix each 3-44%, Borassus 
(2- 45%) zn&Peltophorum (2-21 %). It may be noted here that the Cardiff aero- 
biologists are of the opinion that the numbers alone are misleading as glides to 
the relative importance of aeroallergens (Hyde and Adams 1960 ; Hyde and 
Williams 1961). They stressed how the relationship between number and pollen 
volume might be important in allergy, because some pollen although numerically 
minor, might assume dominance in terms .of * bulk concentration'. However, 
they did, not. stress enough that allergens that are superficial or migrate through 



Airborne pollen 



'339 



BU 



,2 



^ a 

! 

O O 



o 

A 

1 1 

Sd 



s 



.-< 



OVOO O 00 



OOOOC4OOCOO VOOOOOOOOOOO*?- 
* Y O<N OO 



ch'ol 

T *T 



00 

I 



I 
d. 



-H-H-W ^41-ttHH-H-H-H^ -H 



-H - 



41 -H 



o 

*s 

O 00 



I 



a 



o 

g> 



o 
3> 

o 

cC 



I 



60 

cC 



- 





' 



Ill 




i ^ ?5 ?T r5 r5 



c< r* r* 



00 OS 
tN C* 



340 A Janaki Bai and C Subba Reddi 

Table 3, Components of the airborne pollen flora and their relative contributiont 



4 year average 

Pollen type seasonal Contribution 

total 



Gramineae 


3263 


37.15 


Casuarina 


1146 


13.05 


Cyperaceac 


536 


6.10 


Eucalyptus 


510 


5.81 


Dodonaea 


33S 


3.85 


Phoenix 


301 


3.43 


Araaranth-chenapod 


300 


3.42 


Borassus 


214 


2.44 


Peltophorum 


193 


2.20 


Syzygium 


178 


2.03 


*Muntingia 


142 


K62 


Ricinus 


130 


1.48 


Embliea 


99 


1.13 


Asteraceae 


98 


1-12 


Ooco& 


73 


0.83 


Mimosoideae 


56 


0.64 


Holoptelea 


53 


0.60 


Tamarindus 


29 


0.33 


Croton 


25 


0.28 


Artemisia 


25 


0.28 


MoruS 


19 


0.22 


Cicca 


15 


0.17 


Xanthiutn 


9 


0.10 



Damaged and unidentified 

pollen 1031 11.74 



pollen surface might be more potent in unit weight of small rather than large 
pollen (Hirst 1973). In this connection it is worth mentioning that the variety 
of size was one of the features of the complete airspora that impressed Gregory 
in 1951 (Gregory 1973) and led him to stress how size would influence dispersal. 

3-4. Pollen groups counted 

In allergy literature plant species are generally grouped into grasses, weeds and 
trees (Duchaine 1959). Following this . tradition, the pollen recognised, were 
differentiated into these three groups (table 4). Further, a division of these 
pollen according to the mode of pollination ;pf the source plants was made. The 
average contribution of grass, weecl and tree (including shrubs) pollein to th$ totaj 



Airborne pollen 341 

Table 4. Pollen group counts 

% contribution to the total identified pollen 

Name of group 

I II III IV 



Gramineae 


50-69 


39-08 


38-82 


41-35 




Weeds 


9-11 


17-26 


10-89 


13-33 




Anemophilous 


8-04 


25-24 


9-96 




10-98 


Entomophilous 


1*06 


2-02 


0-93 




2-35 


Trees and shrubs 


40-22 


43-66 


50-28 


45-33 




Anemophilous 


30-57 


27- lg 


32-52 




34-60 


Entomophilous 


9-63 


16' 4S 


17-77 




10-73 


Total anemophilous 


81-31 


81-50 


81-30 


86-93 




Total entomophiloust 


10-70 


18-50 


18-70 


13-07 





I 1975-76 II =* 1976-77 HI = 1977-78 IV 1978-79. 

identified pollen were 42, 13, 45% respectively. The tree pollen were distributed 
over 16 types and the weed pollen over 6 types. Of the four seasons, the 1st 
year witnessed the highest incidence of grass pollen (50- 69%) ; it even excelled the 
tree pollen count (40-22%) in this year. The 2nd year registered maximal inci- 
dence of weed pollen (17-2%), and in the 3rd year, tree pollen (50-28%). As 
expected, pollen from anemophilous taxa were predominant in the catches. On 
average, 84-7% of the total identified pollen were of wind-pollinated taxa. 

3 5 . Seasonal periodicity in airborne pollen 

Table 5 and figure 4 give the trends in the seasonal emission of the different pollen 
types recognised and of total pollen. While arriving at the data of table 5 each 
month was considered to consist of a uniform 30 days and the monthly totals were 
got by applying a correction factor. In figure 4 the counts of the same month over 
the four-year period were averaged and plotted as a function of time. Additional 
curves were also drawn through the higher and lower points. Such a graphical 
representation of the data gives an indication of the most likely density of pollen 
to be expected and the probable range about the mean (Ogden etal 1974 .Solo- 
mon 1976). 

As was thought of from a consideration of the pollination calendar, no pieriod 
altogether free from airborne pollen occurred. The total pollen frequency was 
significant all through the season with three periods of pronounced occurrence* 
These were July-September, November-December and February-March. Corres- 
ponding to these periods of higher pollen incidence three peaks : two in the wet 
period and one in the dry period, were evident. Of the two peaks in the wet period, 
one was confined to the southwest monsoon. Qf the three peaks, the one in 
August is the highest and, the February and November peaks follow in that 



342 A Janaki Sal and G Subba Reddi 



*-> 




T^- sj rH ^t" "*"* ^5 vi ON ^* t"^" cn ^3" CM c? CM ^d" 

\O \O VO \O O CM O VH t^- t Tf O rH C- rH 


t^-r^ot VDt^rn^ 
CM SJD en ^ en 


1 


"5s. 
"^ 


en en <NT CM 


rH rH 


*3 


H 


'."*', , . ' - * ' 




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o 


t-rHrJ--^ CMfM-^Ti" t-r-HOO O O O O 

en rH Tf Tf CM >0 r-t 


OOOS> .^en^tcM 


1 


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rH rH rH 




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rHViON-'t -<t^OO OOO^-tCM OCMOO 
O l> SO 00 CM - ' rH 

<s| i-H rH 


enoOO <^cMoON 

en ' . ' 


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o 


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l> t- CM ^ ( . 


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TJ- do m ^ vo-^tenso eMsoost^- OOOO 


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VO <"n ^ ^ CMCMONOs dxfen 1 ^" 


oooo c^vo-^tvo 


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' . .- : ' ^ VH <j- *-t I- en fM r-i O Tt- O TM O O O O 
en v> VO , en tM "rH co . . <-HrH 


OCMOO OCMOO 
l/^ 


53 tj 

Si 


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' CSf -^ Hf t*- ' ' <M _M 


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vot-ooos vo^ooos jg-p:g;gi" -t2-f:jsg 


VO t^ 00 ON SO J^ Op ON 


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rHrHrHrH rH'^rHrH 


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Airborne pollen 



343. 



^ CSi <M <N i-H 



o o o o o vo o <* 

O oo oo ON 



oooo 



\o 



tncxioo 



. coovocf 06 vo 



OOOO 



rt- co 
co en 



O O O o 



O<>Oo 



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VO O 't 00 ^ VO 00 



O , O O 



f O \o 

^ 



O O 



2 "* r3 ^ O O O VO . o . O O co 



O O O o 



OOOO O O O 



O O 

" 



^-tf<|>>0 



oOO' 'O O O fc 



' O O 



OOC^OO OOO'OOOO 



O-O O rt O CS O 



O <N,.- ; .OS O-'* C<|. 



"O -O. O * O CO (* O 



OOC4O VO.cOOO'-H 



'OOoO 



!.?* O O OOOO 



!> t-~ Tr** t 1 *- ^ ^. i>* i>. 


t^- V- "^ ^ 

v O\ ON ON G 


r\ ONONONON ON^ON ON' Ox ON ON PN 


fh, ^** f. 

ON ^ c 


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r\ ON 






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344 A Janaki Bai and C Subba Reddi 



a 

I 



I 



I 



I 



00 00 t-( ON - 

vo vo Tt- 



o o 



o o o 



vo oo "H 



oooo oooo 



oo j o vo 5} o 



o vo o o o o <M oooo 



O O OOOO 'OOOO OOOO 



OOOO OOOO OOOO 



OOOO 



CSNOO oooo oooo oooo asr^oo 

* i *~< T < 



ooooo oooo o^t<NO oooo oooortso 



OOOO OOOO OOOO OOOO 



O'OOO OOOO OOOO t>-<NOOOOOO 



OOOO OOOO 



O'co*'O O OOOO SOOOOVO 



ooon 



t^ooo oots-^- 



I VO _< 00 O O CJ O 
i "d" 00 



OOOO (N O 00 <N O O4 O 

Tf 



^ o o o o^oc^ o^o. 



111 h \o -00 ' r- oo ; ' ' 

o\ ON" ON ON ov ON OA^ ON ONONONON ONONOA ON J ON ON ON ON 



s 



I 



Airborne 



345 



h- O rH 
rH O VO 



>rHO > 00 ON rH C4 C* VO VO 

** 25 ^ ^ 52 S" ^ ON <o r- <o 

! rH <N rH OOONO rt-,-nr- 

rH rH rH OO O OO IT~ 



O O O (N OOOO 



o o o r- o o 



2* oo . r- 
ON in ;jfr rn 



o oo co o 
oo o t- o 



OOrHON OOOO 



ooo ooo oo o4 TH o en ON t- oo 

^ -<t OO CO ,-H rH rH CS 

r " 1 rH Cl <M O 



o o o o 



OOO OOO C4CSOVO OOcOONrH' 

> *fr O rH 4fO Tj" VO 



oS{cn OOOO 
^ C4 C* 



OOO OOOO rH^rHCO VOrMOOVi 

(Nvorn^f rnrqvooo 

rH rH ^/j ^j g^ ^j. 



O O C4 O 



<sJC<i O O Tj- ON 00 VO 00 

oo oi t^> C"4 ON i 



o o o o 



SO CS */*) 
ON fo C4 
CS C*-" VO V) 



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ON \0 ^ 



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^ CS O ^) O rt 00 



o vo o o o <N o 



S 

o 



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1 ^ 10 t- vo 



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ON ^ |> 



O 

SO 



00 



o c^ 



oe o 

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o o o o o i 



oso 



rH t rH C- ^ OO OO 

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rH rH d ^ Tf rH 



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. t> f*> t^- 

, ON ON ON 



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ag 

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0f a 

p 



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n. 

I 



346. A Janaki Sai and C Subba Reddi 




Airborne poll&i - - - , , - - - r. 

f he. chief denors to the pollen geak in (August. were Poaceae, .^asuarina^ Cyperaceae 
in that order. In February the chief donors were Casuarina, -.Rh'oenix and 
Poaceae and to that in November, Poaceae, Dodonaea, Eucalyptus and Cyperaceae 
contributed more. 

The pollen of Poaceae, CyperacQae/.Amaranth-chenopod and Casuarina were 
encountered all through the year. Poaceae pollen exhibited two distinct seasons : 
one major and the other minor, the former practically started in early July with a 
great uprush in the counts from late July to niid September folio wed' by 1 ' a- gradual 
decrease till the 2nd week of October. On the average, 50% of the-aiiimal catch 
was recovered in this season. Peak incidence was recorded in the 2nd ; week of 
August. The 'minor season can be said to have started by the 2nd \tfefck of 
October and continued up to the 3rd week of December; maximal ntinrbe^'s- usually 
occurred in November. The main Cyperaceae season occurred in' July-October ; 
on average, 60 % of the annual datch was recovered here. Relatively 'high/numbers 
of Amarantli-ChenopDd type were noticed during June-November peribd with 
maximal incidence in September. With Casuarina, two periods of higher inci- 
dence were apparent ; the main period extending from January to* April arid the 
subsidiary from July to' October. Peak incidence occurred in February' during the 
main and iti August during the subsidiary period ; on average, 80% and 19% of 
the annual catch were recovered in the respective seasons. : 

Pollen types like Asferaceae, Cocos and Ricinus were caught rather irregularly 
all round the 'year. Types other than 'these were more or less confined to- a parti- 
cular period of the year, thus the Syzigium pollen season was from late April 
to early July 'with maximal incidence in June. Emblica pollen were caught from 
early March to early June with higher numbers during April-May.' *Peliophomm 
pollen were registered "from" March tb November (though not regularly) with 
higher frequency from' April" to September. Tamarindus pollen were- -mainly 
caught from May to August' 1 ; greater frequency was from June r to 'July. -Muntingia 
pollen season was from April* to- October ;. the numbers abounded in August-* 
September. Mtmosoidcae pollen though encountered occasionally in. se^tfral-rftonths, 
were rather more common from August to December. Eucalyptus pollen were 
noticed from July to M^rch with maxima,! incidence during November-December, 
The Dodonaea pollen season .extended from September until March, but was at 
its maximum during November-January, Phoenix pollen began to appear in 
air from late December, reached, a peak in February, decreased, through Mardj 
$nd v , disappeared by April. Borassus pollen were trapped ia all months (though 
not evejy year) except in 'December- January. They abounded during Marchr* 
May with a ppak in Ajfril. ' ' / *.'.<' 

The pollen of Holvptelea, Artemisia, Xanthium, Croton, Moms and Cicca were 
of spasmodic occurrence over a major part of the year. ' 

3.6- Between-year' variation in 'pollen abundance ' ' ' ' ; 

The different pollen types, recognised as well as total pollen counted, varied consi- 
derably in their abundance froin year to ye.ar (tables 5 and 6)- The. catches 'of 

' : :; Figure 4. Seasonal curves of maximum, minimuin abd avi&rage incidence -x>Jf major 

.,-;. :..' ., pdlen.typeSasi wdi; as total pollen 'at Visakhapatnam, ' -^ ;',' , / ' 



348 A Janaki Bai and C Subba Reddi 

Table 6, The four-year average annual figure* and toe range of variation for the 
airborne pollen 



Pollen type 



4-year average Range of 
(Ko./sqom) variation (%) 



Difference 



SOYOSSUS 


214 


90-65-115*42 


24-77 


Ofcramineae 


3263 


84-71-115-35 


30 k 64 


Casuarina 


1146 


76-79-116-06 


39-27 


Phoenix 


301 


80-40-142-19 


61-79 


Amarantfrchenopod 


301 


58-80-125-25 


66*45 


feltophorum 


193 


72-54-145*60 


73-06 


MimoSoideae 


56 


67-86-142-86 


75-00 


Asteraceae 


98 


54-08-139-80 


85-72 


Cocos 


73 


67- 12~157- 53 


90-41 


Cyperaceae 


536 


74-81-171-64 


96-83 


Tamarindus 


29 


68-97-186-21 


117-24 


Dodonaea 


338 


39-64-162-13 


122-49 


Croton 


25 


52-00-188-00 


136-00 


Syzygiim 


178 


27-53-206-74 


179-21 


Eucalyptus 


510 


22-94-203-92 


180-98 


* Muntmgia 


142 


33-10-232-39 


199-29 


Emblica 


99 


40-40-275-76 


235' 36 


Artemisia 


25 


16-00-280-00 


264-00 


ffohptelea 


53 


15-09-320-75 


30$- 66 


Riciwts 


130 


12-31-327-70 


315-38 


Total pollen grains 


8744 


88-70-116-10 


27-40 



average of 3 years 



Poaceae were higher in 1975-76 than in the other years monitored, and decreased 
progressively in the subsequent years. These varied between 85% of 4-year 
average in 1978-79 and 115% in 1975-76. The total weed pollen registered in 
1976-77 were greater than in other years. They ranged from 68% to 158% of 
average. The pollen of anemophilous weeds preponderated almost equally in 
1976-77, and 1977-78. They ranged from 69% to 160% of average. The pollen 
of entomophilous weeds of 1976-77 outnumbered others. They varied from 
58% to 150% of average. The pollen of trees preponderated in almost equal 
magnitude in 1976-77 and 1977-78. They ranged from 86-115% of average. 
The total anemophilous as well as total entomophilous tree pollen were more in 
1976-77 ; the former ranged from 95% to 104%, and the latter from 67% to 
140% of respective averages. The total pollen preponderated in the 2nd year 
of this study and ranged from 89% to 116% of average. From the range of 
variations it becomes evident that group-wise, the pollen of trees and of grasses 
remained relatively steady. Much more steady were the pollen of anemophilous 
trees. This observation regarding tree pollen incidence is at variance with Dua 



Airborne pollen 



349 



and Shivpuri (1962), Subba Reddi (1970), Mittre and Khandelwal (1973) from 
India and Hyde (1972) from UK, Solomon and Buell (1969) from USA who 
reported a high degree of annual variation. 

Meteorological differences from year to year may account at least in part for 
the recorded variation in annual pollen emission rates. This is especially so with 
Poaceae and other weeds which grow and subsequently flower most abundantly 
in response to the amount and distribution of precipitation. Vertical cylinder 
traps are somewhat sensitive to wind speed and this may also account for some 
of the observed variations, but the constancy of tree pollen counts suggest this 
was not important. 

3-7. Effect of urbanisation on airborne pollen 

A comparison of the results of an earlier survey conducted during April 1966- 
March 1968 (Subba Reddi 1970) and those of the present study revealed a consi- 
derable decline in pollen abundance between the two study times (figure 5) indi- 
cating that increasing urbanisation has had an effect on the airspora. Due to 
increased industrialisation of the city the demand for residential houses increased 
with the result the neighbouring suburban and rural areas have been intensively 
used for extensive building construction. This has drastically curtailed the sites 
available for plants especially of herbaceous flora. 



12- 


- 






































- 














- 


_ 






O 


















- 




- 




o 








9- 








3 
























o 
















H 


















6- 






CM 








- 








D 






- 


















It 














- 




















- 






D 








* 








*" 






- 












- 














6- 










" 


r~i 


6- 


~i 




o 






- 








"" 






- 





















o 






4- 















- 














- 






D 






- 














3- 














3- 












1- 














i- 














I- 






n 




nn 



8- 



4- 



Totol pollen Gramineae. 



Am-ch 



Figure 5. The frequency of some pollen types at Visakhapatnam in six years from 
1966 to 1979 (A, 1966-67 ; B, 1967-68 ; C, 1975-76 ; D, 1976-77 ; E, 1977-78; fr, 1978-79). 



4. Conclusions 

As is true of other tropical regions, no season was altogether free of pollen at 
Visakhapatnam. Nor were any clear-cut tree, grass and weed pollen seasons 
characteristic of spring, summer and autumn of temperate zones distinguished. 
The different pollen types encountered, are to be considered as pernicious and 
important pollutants of biological origin in the air of Visakhapatnam adding to 



350 A Janaki Bal and C Subba Reddi 

the overall daggers of atmospheric pollution of this growing industrial city. This 
wan-ants an urgent inquiry into the clinical significance e of these biopollutants as 
well as the interaction between these and the chemical pollutants, and the possible 
resulting synergistic adverse effects on human health. 



RefereaceS 

Davis D J 1972 r NIAID initiatives in allergy research ; /. Allergy Clin. ImmunoL 49 323 

E>i*a K L nd Shwpurj D N 1962 Atmospheric pollen studies in Delhi area in 1958-59 ; 

/. Allergy 33 507-512 
Duchaine J 1959 Allergy of the upper respiratory tract. In International Text-book of Allergy 

(ed) J M Jamar Copenhagen pp. 154-195 

Gregory P H 1973 The Microbiology of the Atmosphere 2nd (ed) Leonard Hill pp. * 377 
Hirst J M 1973 A Trapper's line ; Trans. Br. Mycol. Soc. 61 205-213 

Hyde H A 19-72 Atmospheric pollen and spores in relation to allergy I ; Clin. Allergy 2 
' . 153-179 ' 

Hyde H A and Adams K F I960 Airborne allergens at Cardiff, 1942-59 ; Acta Allergol. 15 

159-169 

Hyde H A and. Williams I> A 1945 Studies in atmospheric pollen II. Diurnal variation in the 
: incidence of grass pollen; New Phytol. 44 83-94 

Hy^e H A and Williams D A 1961 Atmospheric pollen and spores as causes of allergic disease : 
* hay fever, asthma and the aerospora ; Adv. Sci. (L r ondon) 11 525-533 
Mittre V and Khandelmal A 1973 Airborne pollen grains and fungal spores at Lucknow during 

1969-70 ; Palaeobotanist 22 111- 135 

Newmark M F 1970 Recent developments in pollen Aerobiology; Ann. Allergy 28 149-152 
Nilsson S and Nybom R 1978 Particles on pollen ; pp. 14-15, In International Aerobiology News 

Letter (Special Issue) No. 8 
Ogden E C, Raynor G S, Hayes J V, Lewis M D and Haines.J H 1974 Manual for sampling 

airborne pollen ; (New York : Hafner Press) pp, 182 

Ramalingam A 1962 The construction and use of a simple air sampler for routine aero- 
biological surveys: Environ. Health 10 61-67 
Solomon W 1976 Volumetric studies of aeroallergen prevalence I ; Pollens of weedy forbs 

at a midwestern station ; /. Allergy Clin. ImmunoL 57 318-327 
Solomon M A and Buell F M 1969 Effects of suburbanization upon airborne pollen ; Bull. 

Torrey Bot. Club 96 435-444 
Subba Rsddi C 1970 A comparative survey of atmospheric pollen and fungus spores at two 

places twenty miles apart ; Acta Allergol. 25 189-215 
Subba Reddi C 1974 A study of potentially allergenic pollen producing plants of Visakhapatnam ; 

/. Palynol 1 155-157 
Subba Reddi C 1976 Floral mechanism, pollen productivity and pollen incidence in Madhuca 

Mica Gmelin. with remarks on the mode of pollination ; New Botanist 3 11-15 
Venkateswarlu J, Bhiravamurty P V and Narasimha Rao P 1972 The Flora of Visakhapatnam ; 

Andhra Pradesh Akademi of Sciences, Hyderabad, pp. 260 



Proc. Indian Acad, Sci. (Plant ScL), Vol. 91, Number 4, August 1982, pp 351-356 
Printed in India. 



The floral anatomy of Kniphofia uvaria Hook. (Liliaceae: Kniphofieae) 



N P VAIKOS and R M PAI 

Plant Morphology Laboratory, Depaitment of Botany, Marathwada University, 
Aurangabad 431 004, India 

MS received 24 July 1981 

Abstract. The floral anatomy of Kniphofia uvaria Hook, is described. The tepals 
are anatomically similar and one-traced. The stamens are one-traced. The 
outer whorl consists of shorter stamens. The placentation is parietal, nectary ovarian 
and septal. The extension of the carpellary ventrals into the style is an important 
anatomical feature. The trend towards development of an inferior ovary is noted. 
Evidence from floral morphology and other disciplines is discussed and it is inferred 
that the alleged affinity of Kniphofia and the Kniphofieae with the Aloineae and the 
Hemerocallideae is rather remote. 

Keywords. Floral anatomy ; Kniphofia uvaria. 



I. Introduction 

In earlier contributions, the floral anatomy of the Aloineae and Hemerocallideae 
was presented (Vaikos, Markandeya and Pai 1978, 1981). The tribe Kniphofieae 
is thought to chiefly comprise of the genus Kniphofia (cf. Stebbins 1971) although 
two more genera Blandfordia and Notosceptmm are included in it by Hutchinson 
(1959). The present paper deals with the vascular anatomy of the flower of 
Kniphofia uvaria Hook. 



2. Materials and methods 

The flowering material was obtained from the Curator, Government Botanical 
Gardens, Ootacamund. The flower buds were fixed in FAA. The usual paraflBn 
method was followed. The microtome sections cut at 9-12/* were stained with 
crystal violet using erythrosin as counter stain. 



3. Observations 

The pedicel contains a ring of three large bundles (figure 1). These are laterally 
connected upwards to develop an anastomosis (figure 2). The six tepal strands 

351 



352 ' N--P Vaiko's and R M P<ti r 

emerge first (figure 2). The six staminal strands emerge quickly upwards 
(figure 3). The remainder of the vascular tissue resolves into three carpellary 
dorsals and six carpellary ventrals (figure 3). 

The hypanthium is adnate to the ovary for a short length (figures 4, 5). The 
tepals and stamens separate out simultaneously (figure 5). The tepals are united 
into a tube for a considerable length (figures 5-11). Each of the tepals receives 
a single vascular bundle (figures 5-11). 

The stamens have cylindric filaments (figures 5-8). Those of the outer whorl are 
antheriferous first and are also shorter (figures 9-10). The inner three stamens are 
antheriferous at a much higher level (figures 10-11). Each of them receives a 
single vascular bundle which continues upwards into the connective without a 
division and ends beneath the tip of the anther (figures 5-10). The connective 
splits and ends together with the anther lobes (figure 10). The anthers are introrse 
and two-celled. 

The ovary is trilocular at the base with the ovules arranged in two rows in each 
loculus (figures 4-6). The carpellary ventrals of adjacent carpels are united to 
form the composite placental bundles which are lodged on septal radii (figures 4-6). 
These split into the constituent ventrals at the beginning of the ovuliferous zone 
and bear traces to the ovules of adjacent carpels (figure 6). Upwards, the placen- 
tae separate in the centre rendering the ovary unilocular (figure 7). The ventrals 
of adjacent carpels extend at the inner end of the septa, continue to bear traces to 
the ovules of adjacent carpels (figure 7) and enter the base of the style (figure 8). 
The carpsllary ventrals end in the basal half of the style. . 

The septal nectaries are developed from the base of the ovary (figure 4). These 
open at the base of the style (figure 8). The ovarian loculus is continued upwards 
into the style as a triradiate canal (figure 8). The stylar canal is closed in the centre 
in some flowers to result in three cavities (figure 9). It is lined with transmitting 
tissue. The carpellary dorsals extend into the style (figures 8-9) and end much 
beneath its tip (figure 10). The style has three grooves along which it splits into 
three non-vascular stigmatic lobes (figure 11). 

3 1 . Abnormal flower 

This flower has eight tepals, eight stamens and four carpels. The vascular supply 
is derived in conformity with the tetramerous structure (figure 12). The septal 
glands are four (figure 13). The style receives four carpellary dorsals and it is 
four-lobed (figure 14). 



4. Discussion 

'. ' ' f . ' ' ' " 

The six tepals are arranged in two whorls and are united to develop a prominent 
tube. Each of them receives a single bundle and, anatomically, both the whorls 
are similar. This is a condition observed in many liliaceous genera, e.g., Ophiopogon, 
Convallaria, Polygonatum, Maianthemum, Eucomis, Asphodelus, Urginea, etc. In 
the allegedly allied Aloineae and Hemerocallideae (Vaikos, Markandeya and Pai 
1978, 1981), the tepals are three-traced ; the three traces may arise separately 



Floral anatomy of Knlphofia uvaria 



353 



from the stele or the laterals of the outer and inner tepals may.be derived through 
a bifurcation of the common or commissural bundles. 

The stamens are also one-traced ; this is. characteristic of most lilies studied. 
The similarity in the vascular supply to the tepals and the stamens does not seem 
to indicate a staminal origin for the perianth as is sometimes inferred (cf. Leinfellner 
1963). 




U 



Kniphqfja uvaria Hook. 



Figures 1-14. Knlphofia uvaria : 1-11. Transections of the flower from 'the base 
upwards. 12-14. Transactions of the abnormal flower, showing teframerous 
condition. 

Abbreviations D, carpellary dorsal ; is, inner staminal strand ; L, lociile ; MIT* 
median bundle of an inner tepal ; MOT, median bundle of an, outer tepal ; N, 
nectary; os, outer staminal strand; PL ? placental bundle; sc ? stylar canal ; 

' '' ' ' 



354 ff P Vaikos and R M Pal 

The outer stamens are shorter than the inner ones. This condition is charac- 
teristic of species of Aloineae, Allieae and some other genera (Markandeya 1978 ; 
Vaikos 1974; Vaikos et al 1978, 1981). It represents a trend towards diffe- 
rentiation of the two androecial whorls and ultimate reduction of one or more of 
the shorter stamens or a whorl of stamens as occurs in Allieae (Markandeya 
1978). 

The outer floral whorls are adnate to the base of the ovary indicating a trend 
towards the development of an inferior ovary. This is a trend which sporadically 
occurs throughout the many genera and tribes of the family (Markandeya 1978 ; 
Vaikos 1974 ; Vaikos et al 1978, 1981). 

The gynoecium is tricarpellary and trilocular at the base and unilocular upwards- 
The carpellary ventrals are lodged on the alternate septal radii and bear traces to 
the ovules of adjacent carpels. The placentation is anatomically and morpho- 
logically parietal (cf. Puri 1952). 

The nectaries are ovarian and are typical septal glands. They develop at the 
base of the ovary and open at the base of the style. The placental bundles which 
lie close to the glands have to be associated with them in their function (Agthe 
1951 ; Frei 1955 ; Pai and Tilak 1965). 

The style receives the carpellary dorsals and the carpellary ventrals, although the 
latter end early. The extension of the carpellary ventrals into the style is a less 
advanced feature. 

Tetramerous structure is noted in a few flowers. The vascular supply is derived 
in conformity with tetramery. Tetramerous flowers occur normally in Aspidistra. 

The genus Kniphofia forms a component of the Aloineae in the Englerian scheme 
(cf Melchior 1964), whereas Hutchinson (1959) erects a tribe in its name. In the 
old Bentham and Hooker's (1883) system it is placed under the tribe Hemerocalleae. 

This paper demonstrates that the tepals in Kniphofia are one-traced while in 
Aloineae and Hemerocallideae they are three-traced (Vaikos et al 1978, 1981). In 
the studied plants of the tribes Aloineae and Hemerocallideae, the placentation 
is axile, while it is parietal in Kniphofia. 

In the taxa of the Aloineae studied, all or few stamens are adnate to the base 
of the ovary and, as a variation, the stamens and the style may also be fused up to 
the top to develop a prominent column- the gynostemium (Vaikos and Markandeya 
1976 ; Vaikos et al 1978). In Hemerocallis, the stamens are adnate to the 
perianth (Vaikos et al 1981). However, in Kniphofia the stamens are neither 
adnate to the ovary nor to the tepals. 

Embryological evidence shows that Kniphofia is best treated distinct and not as 
a component of the Hemerocallideae (cf. Di Fulvio and Cave 1964). The study 
of vessel structure shows the vessels in Kniphofia are less specialised than those 
in the Hemerocallideae, as also the Aloineae (Cheadle and Kosakai 1971). 

Anthraquinones are detected in Aloe, whereas they are absent in Kniphofia (Van 
Oudtshoorn and Van Rheede 1964). 

The karyotype of Kniphofia is symmetric with n = 6 (Moffett 1932 ; Stebbins 
1971). The Aloineae have the characteristic bimodal 4L + 3S karyotype (Brand- 
ham 1971 ; Darlington 1963; Stebbins 1971), whereas Hemerocallis of the 
Hemerocallideae has n = 11 and the karyotype is not very asymmetric and shows 
rather a close similarity with Amaryllis (Sato 1942). 



Floral anatomy of Kniphofia uvaria 355 

Hutchinson (1959) considers the further development of the Kniphofieae to the 
Aloineae. Cheadle and Kosakai (1971) consider this as a plausible suggestion 
for they find the vessels in the Kniphofieae less specialised than those in the Aloi- 
neae. Furthermore, " no member of the Aloineae has vessels less specialised than 
those most specialised in the Kniphofieae ". 

Stebbins (1971) infers a karyological affinity between the Kniphofieae and 
Aloineae and suggests that, " increasing asymmetry and heterogeneity of the rela- 
tively symmetrical karyotype of the Kniphofieae, consisting chiefly of the genus 
Kniphqfia, together with the addition of a chromosome to the complement through 
fixation of a centric fragment, would lead to the asymmetrical karyotype of the 
Aloineae with n=7". This appears to be too speculative a statement at the 
present stage of our knowledge and further karyological studies of more species 
of the genus should be in order. It may be noted that the bimodal karyotype of 
Aloineae is clearly marked with chromosomal markers for it. The present study 
would demonstrate that Kniphofia is best placed distinct from the Aloineae as also 
the Hemerocallideae. Further studies on more species of the genus are obviously 
in order for a categorical conclusion. 

Hutchinson's treatment of the tribe may also need a review. Di Fulvio and 
Cave (1964) doubt the inclusion of Blandfordia in the tribe. Both Blandfordia 
and Notosceptrwn merit a floral anatomical study. 



Acknowledgements 

The authors are grateful to Prof, K B Deshpande, ex-Head of the Botany 
Department, for his encouragement. They thank Thiru G Kuppuswamy, Ootaca- 
mund, for the supply of the material* 



References 

*Agthe C 1951 Uber die physiologische Horkunft des Pflanzennektars ; Ber. Schweiz. Bot. Ges. 

61 240-277 

Bentham G and Hooker J D 1883 Genera Plantamm Vol. HI Part 2, London 
Brandham P E 1971 The chromosomes of the Liliaceae. II. Polyploidy and karyotype variation 

in the Aloineae ; Kew Bull 25 381-399 

Cheadle V I and Kosakai H 1971 Vessels in Liliaceae ; Phytomorphology 21 320-333 
Darlington C D 1963 Chromosome botany and the origins of cultivated plants (London : Allen 

and Unwin) 
Di Fulvio T E and Cave M S 1964 Embryology of Blandfordia nobitis Smith. (Liliaceae), with 

special reference to its taxonomic position ; Phytomorphology 14 487-499 
*Fra E 1955 Die innerviernng der floralen Nektarien Dikotyler Pflanzenfamilien ; Ber. Schwiez. 

Bot. Ges. 65 60-114 

Hutchinson J 1959 The families of flowering plants H. Monocotyledons, Oxford 
Leinfellner W 1963 Das Perigon der D'liacean ist Staminaler Herkunft ; Ost. Bot. Z. 110 

448-467 
Markandeya S K 1978 Morphological studies in the monocotyledons IV ; Ph.D. Thesis, Marath- 

wada University 



* Original not seen. 



356 jf p Vaikos and R M Pal 

Melchior H 1964 Liliaceae ; In Engler's Syllabus der Pflanzenfamilien II Bd. Gebruder Born- 

traeger, Berlin 
Moffett A A 1932 Studies on the formation of multiimclear giant pollen grains in Kniphofia ; 

J. Genet. 25 315-336 

Pai R M and Tilak V D 1965 Septal nectaries in the Scitamineae ; /. Biol ScL 81-3 
Puri V 1952 PJacentation in angiosperms ; Bot. Rev. 18 603-651 
Sato D 1942 Karyotype alteration and phytogeny in Liliaceae and allied families ; Jpn. J. Bot. 

12 57-161 

Stebbins G L 1971 Chromosomal evolution in higher plants (London : Edward Arnold) 
Vaikos N P 1974 Morphological studies in the monocotyledons- HI. The Liliaceae ;Ph.D.Thesis 

Marathwada University 
Vaikos N P and Markandeya S K 1976 An incipient gynostemium in the Aloineae (Liliaceae); 

Oar. Sci. 45 112-113 
Vaikos N P, Markandeya S K and Pai R M 1978 The floral anatomy of the Liliaceae. The 

tribe Aloineae ; Indian J. Bot. 1 61-68 
Vaikos N P, Markandeya S K and Pai R M 1981 The floral anatomy of the Liliaceae. The 

tribe Hemerocallideae /. Indian Bot. Soc. <50 222-231 
Van Oudtshoorn M C B and Van Rheede 1964 Chemotaxonomic investigations in Asphodeleae 

and Aloineae (Liliaceae); Phytochemistry 3 383-390 



&:oc. loitau Acad. Sci. (Plant goi.), Vol. 91, Nfunibsr 4, AiigUst 19&, 0p. 3$7-37d 
Printed in India. 



Transmission of seed-borne inoculum of Macrophomina phaseotina 
from seed to plant 



TRIBHUWAN SINGH and DALBIR SINGH 

Department of Botany, University of Rajasth?n, Jaipur 302004, India 

MS received 20 February 1981 

Abstract. Macrophomina phaseolina is a serious pathogen which is externally as 
well as internally seed-borne. It causes failure of seed germination and browning 
and rotting of seedlings. The presence of pathogen in infected and healthy looking 
seedlings was tested by clearing, sectioning and incubation techniques. After 8 
weeks almost every surviving plant developed pale yellow to brown circular or oval 
concentric spots on leaves, stem and capsules. Mycelium and micro sclerotia were 
observed in the peripheral region of the lesions. Splitted root, stem and capsule 
also showed the presence of microsclerotia. Cleared wholemounts of leaf and stem 
and T.S. and L.S. of stem showed inter- and intracellular mycelium in cortex, xylem 
and pith cells. Microsclerotia were also observed. In capsule, infection was 
recorded on its inner wall, septum, placenta and seeds spreading from base to apex. 

Keywords. Macrophomina phaseolina ; seed-borne transmission ; Sesamum indicum. 



X. Introduction 

Root, stem or charcoal rot caused by Macrophomina phaseolina (Tassi) Goid 
is a serious seed-borne disease of sesame in India (Pearl 1923 ; Me Rae 1930 ; 
Sundararamau 1931, 1932 ; Mehta 1951 ; Jain and Kulkarni 1965 ; Parasar 
and Suryanarayana 1971 ; Mishra et al 1973 ; Gcmawatand Verma 1974 ; Verma 
and Daftari 1974). It has been reported from many parts of the world and Meiri 
and Solel (1963) regarded it as the most destructive disease of sesame in Israel. 
It is soil as well as seed-borne (Noble et al 1958 ; Meiri and Solel 1963 ; 
Richardson 1979). Singh and Singh (1979) have shown that in sesame M. phaseo- 
lina is surface as well as internally seed-borne. The effects of seed-borne inoculum 
of M. phaseolina on germination and the mode of disease transmission from 
seed to plant have not been precisely investigated so far and therefore this study 
was taken up. 



2. Material and methods 

Six samples ac. nos. 7, 9 (Ajmer), 15, 17, 18 (Chittorgarh) and 11 (Udaipur 
investigated for deep location of M. phaseolina were selected for the study. Of 

357 



358 fribhuwan Singh and tidlbir $ingti 

these ac . no. 11 was free from the infec ti on (figure 1) and used as c ontrol, whereas, 
the remaining samples carrying various degrees of infection were categorized into 
three groups (i) seeds without microsclerotia, (ii) seeds with moderate number 
of microsclerotia, and (iii) seeds with abundant microsclerotia (figure 2). The 
following three methods were used to investigate the transference of disease from 
seed to plant. Seeds harvested in experimental plants were tested by standard 
blotter method. 

2-1. Petriplate method 

One hundred seeds of each sample were treated with 2% available chlorine aqueous 
solution of sodium hypochlorite and tested by standard blotter method. Obser- 
vations were made on percentage of M. phaseolina infection, radicle emergence 
germination, rotting and deformed seedlings, seedlings, with pycnidia andungerl 
minated seeds at 24 hr intervals. 

2-2. Seedling symptom test 

Ten ml of 2% agar water was poured into each test tube and sterili23ed. One 
hundred seeds of each sample after pretreatment with 1% available chlorine were 
transferred aseptically to test tubes (one seed per test tube) and incubated for 
8 days at 22 C under 12 hr of alternating cycles of day light and darkness. 

2-3. Growth test 

One hundred seeds of each sample untreated and pretreated with 2% and 5% 
available chlorine for 5 min were sown in pots containing sterilized soil. Morta- 
lity, seedling survival, symptoms and transmission of disease were recorded at 
weekly intervals. Isolations were made from wilting and healthy looking seed- 
lings and plants at regular intervals. For isolation, seedlings were carefully 
uprooted, washed in running water, segmented and after surface washing with 
1% available chlorine were spaced on blotters and incubated for 7 days. For 
this study different parts, of seedlings (radicle, plumule, hypocotyl, cotyledon) 
and plants (roots, stems, leaves, flower buds, flowers, green and dry capsules 
and seeds) were incubated. 

After 14 weeks dried plants were uprooted and the capsules were harvested 
for further observations. The root system was separated, washed thoroughly and 
splitted longitudinally. External and internal surfaces were observed by naked 
eye and under stereobinocular microscope. 

Stem portions were also split longitudinally and divided into three parts basal 
middle and apical. 

To detect the presence of M. phaseolina on the surface and inside the dried 
capsules, they were kept in 70% ethanol overnight Capsule surface and its parts 
showing microsclerotia were photographed while under 70% ethanol. 

Clearing of plant parts carrying infection was made by using lactic acid, 
potassium hydroxide and ethanol. Hand sections were also cut and stained with 
safranin and fast-green combination. . 



Seed-borne transmission in M. phaseolina 



359 




Figures 1-3. 1. Uninfected seed of sesame, 2. Seeds infected with Macrophomina 
phaseolina ; note pin head like microsclero<ia on seed surface, 3. Leaves infected 
with M. phaseolina ; note dark brown leaf spats with pale centre. Shot holes also 
seen. 



Seed-borne transmission in Af. pkaseotina 361 

3. Results 
3-1. Effect on germination and survival 

In blotter test germination was only slightly affected (table 1). The radicle emerged 
out in 19-29% seeds in five samples within 24 hr and in 55-75% seeds by 48 hr. 
The germination ranged from 73-92%. Seedling abnormalities such as seedlings 
without radicle and seedling with short radicle were observed and the abnorma- 
lities ranged from 1-8%. Germination was found to be better in seeds with 
superficial infection (ac. nos. 15,18) in comparison to other samples having a higher 
percentage of embryonal infection (ac. nes. 7, 9 and 17). Pycnidia formation 
was frequent on rotted seedlings and in ungerminated seeds. 

The growth test using categorised seeds, seeds without microsclerotia and with 
microsclerotia in five samples, showed that seedling emergence was considerably 
affected (table 2) and ranged from 75-^82% in seeds without microsclerotia (untrea- 
ted) and 56-78% in seeds with m'crosderotia. The germination was only 56-66% 
in samples with deep infection and was 77 and 78% for remaining samples with 
superficial infection. Survival of seedlings in samples with superficial infection 
was also high. Seedlings obtained from seeds without nr.crosclerotia were more 
vigorous to start with than those from mlcrocclcrotial seeds. 

Chlorine pretreatment in growth test promoted emergence. Pretreatment with 
2% Cl accelerated germination in both the categories and was 70-97% in seeds 
without m'croselerotia and 60-93% in seed with microsclerotia. 5% Cl-pretrcat- 
ment was phytotoxic and germination showed significant decline (61-88%) in seeds 
without microsclerotia and was only 27-75% in seeds with microsclerotia. Seed, 
ling survival was also higher in 2% Cl pretreatment (table 2). The Cl-pretreat- 
nient (2%) brought about a slight improvement in germination even in the control 
sample but the phytotoxic effects of higher concentration of Cl pretreatn^nt caused 
36 and 37% decrease in germination and survival respectively as compared to 
those of 2% Cl treatment. 5% Cl treatment probably inhibited root growth as 
in petriplate experiment in 12-15% seeds. The embryo was observed to emerge 
after rupturing the seed coat at the chalazal end. The cotyledons in such cases 
turned green later on. The radicle remained lodged in the seed coat and its growth 
was negligible. 

3-2. Seedling symptom test 

Three categories of seeds v/z., (i) seeds without m'croselerotia (ii) seeds with mode- 
rate number of m'.cro:clerotia and (iii) seeds with abundant m'.croscltrotia were 
used in the water agar seedling test (table 3). Seedling from first category showed 
an initial vigorous growth but subsequently 20-25% of them became diseased. 
Seeds of third category failed to germinate in sample ac. nos. 7,9,17 and gave 
only 9 and 7 percent germination in seeds of ac. nos. 15 and 18 respectively, 
Ungerminated seeds were covered with heavy mycelial growth of M. phaseolina^ 
In moderately infected seeds of second category the percentage of ungtrmiLattd 
Seeds was 5-12. The seedlings were usually stunted as compared with those from 
seeds without microsclerotia. The incidence of seedling infection of M. phaseo* 
kna was high in ac. nos. 7,9,17 ranging from 51-70% and 37 and 19% in ac. 
nos. 15 and 18 respectively. The percentage of deformed seedlings was also 



P.(B)~S 



362 



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Seed-borne transmission In M. phaseollna 



363 



Table 2. Data an seedling emergence and seedling survival beyond 10 weeks in six 
samples (I control and 5 infected) in pot experiment. 100 seeds used per treatment 



Seeds without microsclerotia 

Sample 

number Emergence 



Seeds with microsclerotia 



Survival 



Emergence 



. , . Sur^iv&l 

' 



Un- Pretrcptcd 

treated 2% 5% 


Un- Pretreated 
treated 2% 5% 


Un- Pretreated 
treated 2% ' 5% ' 


Un- iPrctrcated. 
treated 2% 5% : 


11 93 


98 


62 


9 


95 


58 














(Control) 
























7 75 


79 


65 


46 


48 


45 


65 


70 


' 55 


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25 


9 79 


84 


67 


66 ; 


69 


55 


56 


60 


27 


36 


27- 


19 


15 SI 


97 


88 


67 


75 


59 


77 


93 


75 


59 


60 


50 


17 76 


70 


61 


40 


66 


34 


66 


74 


53 


36 


44 


26 


18 82 


90 


74 


76 


78 


70 


78 


86 


70 


68 


76 


54 



Table 3. Percentage of ungerminated seeds (US) and healthy (HS) deformed 
(DS) aid infected seedlings (IS), infected with Mcicrophomina phaseollna^ from 
nvw-mlaro *clerotiat and microsclerotial seeds having mild and heavy infection 
(100 seeds/sample). 



Sample 
No. 




Seeds 






Non-microsclerotial 


Microsclerotial 


US HS DS IS 


Mild 


Heavy 




US HS DS IS 


US HS DS 


IS 


11 
(Control) 


... 100 














7 


H 60 9 20 


12 26 11 51 


100 00 00 


00 


9 


9 56 13 22 


10 10 10 70 


100 00 00 


00 


15 


7 62 9 22 


8 53 6 37 


91 3 00 


6 


: .17 . 


10 . 50 15 25 


,.10 . 10 . 10 .70, 


1.00 oo ; oo 


00 


18 


00 69 S 23 


5 '.-67.;;, 9 ".19 


^93 . 00 7 


00 



Tribhumn Singh and Dalbir Singh 



in the former as 



f the latter It may be mentioned 
m and therefore, seeds.with micro- 
of a high incidence of 

in tho 



test. 

3-3. Symptoms and effect on host 

and rotting of seedlings in blotter 

M . jtatffea caused seed rot, browiun^ ^^ ^^ at ^ root 

(figuts 4) and water agar tests. The inxu u ^ rQot and fihoot tlps 

St transition zone (fibres 5,6) ^ progress^ ito^ ^ ^ rf 

(figure 7). Browning and damping of ^root toot pi ^^ ^ ^ 

kfshoot. ^^"Tf^Lwlpy in some cases when the seed coat 
on the whole root turned bro ^* C0tyle dons during germination and 
having **<<f f t^^ downwards from 

remain attached with for a longel peno ofm i c rosclcrotia wasqmte 

l-ral roots) system (^s 8, 9). 




Figures 4-9. Diagrams to show symtoms of M. phaseolina on sesame ^ 

in blotter test. 4. Rotting of seed with pycmdia and imcrosderotia* 5, o, 
Soedlings 'showing streaks and microsclerotia. 8,9. Seedlings showing disti 
batioa of microsclcrotia ; note infection of primary w .yrolt as secondary toots m C* 



Seed-borne transmission in M. phaseolina 365 

In growth tests first disease symptoms were observed on the cotyledons after 
a fortnight. In a few days (by 20th day after germination) these became well 
marked as pale yellow to brown or blackish circular or oval and concentric spots. 
Each spot had a whitish area in the centre. The spots enlarged, became irregular 
and necrosis caused shot-holes. Similar spots were recorded on leaves after six 
weeks of germination (figure 3). Infected plants were weak and had leaves smaller 
than those on healthy plants. After 8 weeks almost every surviving plant from 
untreated as well as Cl-pretreated seeds developed symptoms of M. phaseolina. 
infection on stem appeared as yellowish brown discoloured patches. Their peri^ 
phery turned dark brown to black subsequently (figure 12). Pedicel and green 
.capsules also revealed the occurrence of yellowish patches. 

Under stereobin ocular microscope mycelial growth was observed on leaf, stem f 
pedicel and capsule. Although no microsclerotia were seen on leaf surface, they 
occurred in abundance in black peripheral region of the patches on stem. Scattered 
microsclerotia were also seen on other parts of stem. 

3-4. Isolation and observation of fungus 

Isolations were made from seedlings and plants raised from untreated and 
pretreated seeds at regular intervals. They wera classified into two categories 
viz., rotted and healthy looking seedlings. The two types of seedlings, were treated 
separately for incubation. For the latter, 25 seedlings were used but for the rotted 
all the seedlings available on the day of setting the experiment were used which 
were usually more than 10. 

Ten-day old rotting seedlings yielded M. phaseolina from all the parts including 
cotyledons. The incidence was more than 80% and usually higher in transition 
zone than the other components. Rotted seedlings from pretreated seeds yielded 
the pathogen in slightly low incidence in comparison to those of untreated seeds. 
In healthy looking seedlings, M. phaseolina was recorded in not more than 41% 
cases. Interestingly in such cases the incidence of pathogen recovery was higher 

"in sample nos. 7 and 17 as compared to that of sample nos. 9, 15, 18. 

Similar data were also collected on 50-day old plants which yielded M. phaseolina 
in very high percentage. No significant difference in percentages was recorded 
in plants from untreated and pretreated seeds in all the five samples. The in- 
fection percentage on different plant parts were also comparable, 

Mature but not dead dry plants (75-day old) yielded the fungus in 100% cases 
from roots (figure 10) and transition zone in sample DOS. 7,9,17 and 18. It 
was found to be 80 and 83 (roots) and 87 and 86 (transition zone) in plants 
from untreated and pretreated seeds of sample no. 15. The recovery of fungus 
was usually high from stem and leaves than the other plant parts i.e., flower buds* 
flower, (figure 11) and capsules. 
Af. phaseolina was also recovered in very high percentages from root,stem, whole 

-capsule and its parts (Placenta, seed and septum) from dry plants in blotter test. 
Visibility of microsclerotia was slightly difficult on the hairy surface of capsule, 

.whereas, the placenta and septum turned black. 

* Dry plant parts were also examined for the presence of pathogen. Dry plants 
of all the .five samples were harvested, and examined with unaided eye or 10 x 

*ha&dtens.. Microsclerotia were observed on and inside the tap root and stem from 



366 Tfibhuwan Singh and Dalbir Singh 

base to tip (table 4). Lateral roots could not be split. The incidence of micro* 
sclerotia on roots and basal part of stem surface ranged from 78-100% and 
7(MOO% respectively. In the in ten or part of root and basal stem it ranged from 
9-^14% and 24-^53% respectively. Maximum infection (86%) was recorded on 
the entire surface of the root and stem (figure 12) (base to apex). However, a 
gradual decrease in its incidence was recorded inside the stem from its basal to 
the apical part (figure 13), incidence being 53% (base), 33% (middle) and 22% 
(apical part). In the remaining sample apical region yielded low percentage of 
infection on the outside as well as inside. 

The surface and the interior of the capsules showed abundant microsclerotia 
on different parts including seeds (figures 14,15), A higher incidence of micro- 
sclerotia was observed in the basal and distal region of capsule surface (outer and 
inner), septa, placenta and seeds (table 5). Most of the seeds showed abundant 
microsclerotia and a thick net of black mycelium on the micropylar region 
(figure 15). The chalazal end was either free or showed mild infection. In septum 
the incidence of fungus was rather low (19-67%). 

3-5. Clearing and section cutting 

Cleared wholemount preparations of infected leaves showed abundant dark brown, 
branched and septate mycelium running across the leaf spot (figures 16,17). It 
traversed mostly parallel to veins* The host cells in infected regions were small, 
poor in pigments, cytoplasm and food materials. Microsclerotia were not 
observed* 

The wholemount preparations (figure 18), transactions and longisections 

(figures 19,20,21 and 22) showed the presence of mycelium in different tissues 

including xylem (figures 21,22) and pith ; inter and intracellular mycelium was 

, observed to travel along the vessel length (figure 22). Microsclerotia were also 

observed in the pith cells (figure 19) and vessels (figure 20). 

Seeds harvested from experimental plants also showed the occurrence of micro-* 
sclerotia and mycelium in seed coat, endosperm and embryo as revealed by 
maceration and sections. . 



4. Discussion 

Water agar seedling test and the growth test have revealed the transmission of 
M. phaseolina from seed to plant in all the five sesame seed samples tested pres- 
ently. The fungus was isolated from rotting, wilting and healthy looking seed* 
lings. The vegetative parts, floral btids, flowers and green fruits of experimental 
plants also yielded the fungus on incubation. Careful examination of root, stem 
and fruits from mature and dried plants showed the presence of mycelium and 
microsclerotia. The fungus was recovered from pedicel, calyx, septum, placenta, 
inner surface of capsule and seeds attached to the placenta. The infection usually 
spread from nr.cropylar end onwards in the seeds of infected capsules. The 
systemic transmission of Af. phaseolina from infected seeds is strongly supported. 
The mycelium, running longitudinally as well as horizontally and the microsclero- 
tia, were recorded in pith, cortex and vascular cells in stem. The pith became 
hollow in infected plants probably due to the production of cellulolytic and pecto-* 



Seed-borne transmission in M. phaseoih 



ma 



367 




Figures 10-15. 10, 11. Root and flower after incubation on blotter. 10. Root 
surface showing pycnidia and microsclerotia. 11. Bilabiate flower surface of sesame 
showing microsclerotia. 12-15. Stem and capsule of sesame showing infection of 
M. phaseolina in dried plants harvested at the end of growing season. 12. Surface 
view of healthy and infected stem. 13. Longitudinally split stem showing micro- 
sclerotia. 14. Partially dehisced capsule ; note the microsclerotia on surface. 
15, Interior of an infected capsule. Note the infection of placenta and seeds. 



368 



Trlbhuwan Singh and D alb! r Singh 




Figures 16-22. 16-18. Wholeniounts of cleared infected leaf and stem peeling. id- 
Part of leaf surface showing mycelium in stamatal region. 17. Part of leaf surface 
showing septate mycelium and its association with vein. 18. Cleared peeling, 
from stem surface showing micro sclerotia and inter- as well as intracellular nrycelium 
19-22. T.S. and L.S. of infected stem. 19. T-S. stem showing mycelium and 
microsclerotia. 20-21. T.S. stem showing mycelium and micros clerotia in vessel, 
22. L,S, stem ; note mycelium traversing in vessel, 



Seed-borne transmission in M. phaseolina 



369 



Table 4. Percentage occurrence of raicrosclerotia of Macrophomina phaseolina 
vcgetafwc parts of dried and harvested plants. 



Vegetative 
parts 


Root 


Stem 


sample No. 


tap 


Basal 


Middle 


Apical 




Surface 


Inside 


Surface 


Inside 


Surface 


Inside 


Surface 


Inside 


7 


86 


13 


86 


53 


&6 


33 


86 


2C 


9 


100 


10 


100 


35 


100 


30 


75 


15 


15 


&2 


9 


76 


26 


41 


5 


30 


5 


17 


7g 


11 


70 


24 


60 


15 


40 


11 


IS 


100 


14 


92 


40 


82 , 


34 


60 


10 



Table 5. Percentage occurrence of Macrophomina phaseolina on dried capsule parts 
(after soaking in 70% ethanol for 24 hr) 



Capsule 



Placenta 



Capsule 

parts - 

sample Outer surface Inner surface Basel Distal Basal . Distal 



Seeds Septum 



No. Stalk 



Basal Distal Basal Distal 



J 


84 


82 


73 


80 


40 


80 


33 


93 


33 


26 


9 


80 


70 


70 


65 


50 


70 


42 


70 


40 


30 


15 


69 


67 


67 


,56 


33 


60 


30 


73 


45 


20 


17 


63 


52 


35 


47 


40 


51 


40 


50 


30 


19 


18 


92 


91 


89 


85 


77 


92 


79 


81 


71 


67 



lytic enzymes in large quantities. This also accounts for easy breaking of stem 
in these plants. 

M. phaseolina is a well-known soil inhibitant and Cougnee (1963) reported 
the disease to be soil borne, Mehta (1951) suspected the possibility of transmis- 
sion of Sclerotiniabataticola by sesame seed in India. "Noble et al (1958) recorded 
the seed-borne nature of M. phaseolina on the basis of investigation of Mehta in 
their list of seed borne diseases, but were doubtful. 1 Meiri and Solel (1963) 
observed that infected seeds of < Ranner 15 > yielded diseased seedlings and 
also observed the first site .of infection in the collar region. 

Fakir et al (1976) working with sunflower kernels have shown that M. pha- 
seolina is highly pathogenic and pathogen could be isolated from seeds of some 
of the inoculated plants, but seeds of symptomless sunflower plants from naturally 
infected seeds did not yield M. phaseolina. "This is in contrast to the present "study 
and does not support the transmission of disease from seed to seedling. However* 
in their description it is not clearly stated whether these symptomless plants grew 
out of microsclerotial seeds. The present study not only provides the adequate 



370 Tribhuwan Singh and Dalbir Singh 

evidence of seed transmission of M. phaseolina in sesame but also clearly reveals 
that the seed-borne inoculum plays an important role in the spread as well as in 
the manifestation of disease. 

Pycnidia formation was rare in blotter as well as agar test. Only on rotted seeds 
and seedlings, and in lesions on stem, pycnidia were frequently formed. Chidam- 
baram and Mathur (1975) reported the production of pycnidia in 20 out of 58 
non-sporulating isolates in water agar nr.dia. They tested 58 isolates of 
M. phaseolina from 20 plants. Present isolate from Sesamum Indicum could be 
described as a moderately sporulating one. 



Acknowledgements 

The authors thank the Council of Scientific and Industrial Research, Now Delhi 
for providing a research fellowship to one of them (T.S.). 



References 

Chidambaram P and Mathur S B 1975 Production of pycnidia by Macrophomina phaseolina ; 

Trans. Br. Mycol. Soc. 64 165-167 
Cougnee M 1963 fitudes sur L' arrthraenase des Cotoniers ; G. barbadensc ; Cotton ct Fibres 

Trop. 18 149,450 
Fakir Golara A, Rao M H and Tliiruraalachar M J 1976 Seed transmission of Macrophomina 

phaseolina in sunflower; Plant Dis. Rep. 44 221 
Gemawat P D and Verma O P 1974 Root and stem rot cf Sesamum in Rajasthan. Evaluation 

of varieties (Macrophomina phaseolina) Indian J. Mycol. Plant Pathol. 4 76-77 
Jain A C and Kulkarni S N 1965 Root and stem rot of Sesamum ; Indian Oil Seeds J. 9 

201-203 
McRae W 1930 Report of the Imperial Mycologist Scient ; Rep. Agric. Res. Inst. Pusa 

1928-29 51-56 
Melita P R 1951 Observations on new and known diseases of crop plants of Uttar Pradesh; 

Plant Prat. Bull New Delhi 3 7-12 
Meiri Aliza and Solel Z 1963 Transmission of charcoal rot by sesame seeds ; Phytopathol. 

Mediterr. 2 90-92 
Mishra R P, Singh B P and Joshi L K 1973 Pod susceptibility of different varieties of till 

(Sesamum orientale) to Macrophomina phaseoli (Maubl.) Ashby ; J.N.K.V.V. Res. J. 7 

2S8-289 

Noble Mary, De Tempo J and Neergaard P 1958 An annotated list of seed-borne diseases ; 
- C.M.L Kewl59 
Richardson M J 1979 Annotated list of seed-borne diseases ; ?rd edition, Proc. Int. Sqed Test. 

Ass. 23 1-320 
Parasar R D and Suryanarayaaa D 1971 Polygalecturooase activity of Macrophomina phaseolina 

the incitant root rot of Sesanum "indicum ; Indian Phytopath. 24 559-562 
Pearl R J 1923 Report of the mycologist to the Govt. of the* Central Provinces and Berar. 

Rept. D:ptt. A^ric. Cettral Provinces and Berar for the year ending 30th June 1922 19-20 
Singh T and Singh D 1979 Anatomy of penetration of Macrophomina phaseolina in seeds of 

sesame ; Recent researches in plant sciences (ed.) S S Bir (Ludhiana : Kalyani Publisher) 

pp. 603-606 
Sundarajraman S 1931 Administration report of the mycologist for the year 1929-30 ; De.pt. Agric. 

Madras 

Suodararaman S 1932 Admbtstration report of the mycologist for the year- 1930-31 ; Dept. 

Agric , Madras . 
Verma O P and Daftari L N 1974 Amount of seed-borne inoculum of Macrophomina phaseolina 

and its effect on mortality and growth of Sesamum seedlings ; Indian Phytopath. 27 130-131 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 5, October 198?, pp. 371-378. 
Printed in India. 



Effect of water stress and sucrose on opening and longevity of 
flowers in gladiolus 

I V RAMANUJA RAO and H Y MOHAN RAM 

Department of Botany, University of Delhi, Delhi 110007, India 

MS received 20 January 1982 ; revised 9 August 1982 

Abstract. The percentage of buds opening and flower longevity as affected by the 
availability of water and sucrose to cut spikes of gladiolus were studied. Uptake 
of sucrose solution and fresh weight changes in spikes were dependent on sucrose 
concentration. Marked reduction in uptake and fresh weight occurred when poly- 
ethylene glycol (PEG) was used as the stressing agent. In comparison, PEG failed 
to induce any significant change in the percentage of flower buds op.en.ing. 
Sucrose was essential for opening since the buds that failed to open in the control 
were caused ta open in sucrose. Induced water stress did not curtail flower 
longevity at any given concentration of sucrose. Thus flower opening and longe- 
vity in gladiolus appear to be limited more by the availability of sucrose than 
water. 

Keywords. Flower longevity ; flower opening ; gladiolus ; polyethylene glycol ; 
sucrose ; water stress. 



1. Introduction 

Studies have been carried out on the factors affecting water uptake and vascular 
blockage in cut flowers on account of their crucial role in maintaining freshness 
(Durkin and Kuc 1966 ; Marousky 1969 ; Oilman and Steponkus 1972 ; van Meeteren 
1978 ; Rao and Mohan Ram 1982a). Water deficit causes early wilting of flowers 
(Marousky 1969; Paulin 1972; Mayak et al 1974). Mayak et at (1974) noted a 
sharp decline in the water potential of petal tissues in wilting cut roses but not in 
intact flowers. Water stress has been identified as the cause of failure of flower 
opening in the spikes of iris stored at low temperature for four days and 
then for one additional day at 22 C (Mayak and Halevy 1971). Appreciable bud 
opening has, however, been recorded in gladioli and chrysanthemums stored in cold 
using sucrose alone or in combination with silver nitrate or gibberellic acid 
(Kofranek et al 1975; Kofranek and Halevy 1976; Rao and Mohan Ram 1979, 
1981, 1982b). 

In spite of the development of successful techniques to handle cut flowers, 
our understanding of water requirement of opening flowers and their ability to 
withstand storage has remained incomplete. The property of sucrose to act as an 
antidesiccant when supplied before storage, in addition to its metabolic role is 

i 

371 



372 / V Ramanuja Rao and^H Y Mohan Ram - ' 

still unclear. For example, gladiolus spikes, given a pulse treatment with sucrose 
before storage, open satisfactorily on subsequent transfer to water (Mayak et al 
1973; Bravdo et al 1974). However if the spikes are first stored dry, it becomes 
necessary to provide gibberellin plus sucrose subsequently to ensure full opening 
(Rao and Mohan Ram 1979, 1982b). This paper discusses the importance of 
water and sucrose in flower opening and longevity. 



2. Material and methods 

Spikes of Gladiolus natalensis Hort. were obtained from a commercial grower in 
New Delhi at the green-bud stage (harvested one day before the corolla of the 
lowermost bud emerged from the enveloping bracts and the tip just became visible). 
The spikes were stored dry for 24 hr after harvest at 20 C to facilitate a larger 
uptake of the pulsing solution (Rao and Mohan Ram 1981a). Twenty spikes 
each were pulsed for 48 hr with sucrose solution (0'25 M and 0-5 M) to eliminate 
any effects caused by a low amount of carbohydrates in the spike. These were 
then transferred to (i) water (control), (ii) sucrose solution of the same concen- 
tration as was used for pulsing and (iii) polyethylene glycol solution (PEG, MW 
6000, Sigma Chemical Co., USA) having a water potential similar to the pulsing 
solution (0 25 M sucrose = 7 bars ; 5 M sucrose = 15 bars). In all six sets, 
each with 20 spikes were set up in glass tubes (2*5 x 15*0 cm) containing 40 ml 
of the test solution. The solutions were prepared using glass-distilled water. 
One additional set of spikes held continuously in water served as the control. 
The spikes were kept in a chamber at 20 2 C with 14 hr photoperiod (under 
cool-white daylight fluorescent tubes giving 500 lux). 

The number of flowers opening and withering per spike were recorded daily. The 
longevity of individual flowers on the spike (the period between flower opening 
and withering) was recorded. The term 'uptake * used here refers to the volume 
of the solution taken up and 4 fresh weight change ' to the differences in fresh 
weight of a spike over a given period. Tubes containing different solutions but 
without spikes served as controls to measure loss caused by evaporation, 
Confidence intervals of the means were determined at P < 0*05. 



3. Results 

3.1. Rate of uptake 

During the period of pulsing a high initial uptake was recorded for the control 
(table 1). Spikes kept in 0-25 M sucrose showed a greater uptake than those in 
0-5 M during this period. By day 2 the spikes pulse-treated with sucrose and 
transferred to water took up a larger volume of solution than those continuously 
held in sucrose and the control.' A marked decrease was observed in the amount 
taken up by the pulsed spikes on day 4 as compared to that on day 2. It was, 
however, still higher than that for spikes kept continuously in sucrose. A decrease 
in uptake was noted in the latter during the same period, although it exceeded 



Effect of water stress and sucrose on flowers of Gladiolus 373 



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374 / V Ramanuja Rao and H Y Mohan Ram 

the amount in the pEG-treated spikes. Spikes transferred from sucrose (both 
concentrations) to PEG showed practically no uptake during the first two days. 
Subsequently, however, a very low uptake was recorded in these spikes ; the 
uptake by spikes held in PEG at 7 bars was slightly higher than that 
at 15 bars. 

During the entire period of experimentation pulse-treatment with sucrose 
(0*25 M) followed by transference to water resulted in a significantly greater 
uptake over the control (table 1). However, in the spikes held continuously in 
sucrose the magnitude of uptake was lower. Treatments with PEG markedly 
curtailed uptake as compared to the control and other treatments, 

3.2. Changes in fresh weight 

As with uptake, the maximal fresh weight increment during the pulsing period 
was noted in the control, followed by that in spikes treated continuously or pulsed 
with sucrose at 0*25 M and 0'5 M, respectively (table 2). Whereas after two days 
the control spikes attained a negative fresh weight change, the spikes which were 
pulsed with sucrose and transferred to water or sucrose continued to show 
a positive fresh weight change. Spikes transferred to PEG showed a negative 
fresh weight value, much lower than that of the control. In general the spikes 
which were held continuously in 0'5 M sucrose showed a lower fresh weight change 
as compared to the spikes treated with 0-25 M. In all the treatments, the lowest 
fresh weight change was recorded on days 6 and 8. 

The overall highest fresh weight was observed in spikes pulsed with sucrose 
at either concentration and transferred to water (table 2). Thus the control exhi- 
bited the lowest fresh weight. Low fresh weight was also observed in spikes 
treated with sucrose (0'5M) continuously or stressed with PEG at either con- 
centration. 

3.3. Percentage of flower buds opening 

A significantly higher percentage of flower buds opened in all the treatments 
over the control (table 3). Among the treatments the percentage of flower 
buds opening did not vary except in spikes pulsed with sucrose at both the 
concentrations and transferred to PEG which showed slightly lower opening as 
compared with those pulsed with sucrose (0"25M) and transferred to water. 

3.4. Flower longevity 

A study of the longevity of individual flowers at different positions on the spike 
showed differences between the control and those pulsed with sucrose and trans- 
ferred to either water or sucrose (table 4). In spikes stressed with PEG at both 
the concentrations there was a continuous increase in longevity from flowers 1-5. 
Interestingly when the mean longevity of flowers 1-5 was compared, it turned 
out that the lowest longevity was recorded for the control (2 '8 days). The longe- 
vity of flowers in spikes pulsed with a particular concentration of sucrose and 



Effect of wafer stress and sucrose on flowers of Gladiolus 375 



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376 / V Ramamija Rao and H Y Mohan Ram 

Table 3. Effect of' water stress on the percentage of flower buds opening in 
gladiolus. 

Treatments 

Control SlW S2W SIC S2C SlP S2P ~~ 

xcr xci xcr xci xci xcr xcr 

54-0 3-0 80*0 5*0 75-0 5'0 76*0 4-0 75'0 4-0 69'0 3'0 69-0 4-0 



X Mean values. 

CI Confidence interval calculated at P^O'05. 

SlW, S2W Spikes pulsed with 0-25M and 0-5M sucrose for 48 hr and later transferred to 

water. 

S1C 3 S2C Spikes held continuously in 0-25M and 0-5M sucrose. 
SIP, S2P Spikes pulsed with 0'25M and 0-5M sucrose for 48 hr and later transferred to 

PEG at. 1 bars and 15 bars, respectively. 



Table 4. Effect of water stress on flower longevity* in gladiolus. 





Treatments 


Flower 
number 


Control 


SlW 


S2W 


SIC 


S2C 


SIP 


S2P 


1 


2-6 


3'8 


4-2 


4-0 


4-2 


3-2 


3-8 


2 


2*6 


3"6 


4-2 


4-0 


4-2 


3'4 


4-0 


3 


2-6 


3-4 


4-2 


3'8 


4-6 


3'6 


4-6 


4 


3-0 ' 


3-6 


4-2 


3-8 


4-8 


3-8 


4'8 


5 


3-2 


3'8 


4.4 


4-0 


4-6 


4-0 


5-0 


6 


2-6 


3-8 


4-4 


4-4 


4-4 


4-6 




7 




4-0 




... 






... 


X 


2- & . 


3-6 


4-2 


3-9 


4-6 


3-6 


4-4 



* in days. 

X Mean longevity of flowers (1-5). 

SlW, S2W Spikes pulsed with 0*25 M and 0'5M sucrose far 48 hr and later transferred to 

water. 

SIC, S2C Spikes held continuously in 0'25M and 0* 5 M sucrose. 
SIP, S2P Spikes pulsed with Q'25M and 0'5M sucrose for 48 hr and later transferred to 

PEG at 7 bars and 15 bars, respectively. 



transferred to water, sucrose or PEG was more or less similar. For example/ at 
0*25 M of sucrose, it ranged from 3-6 to 3 -9 days and at 0'5 M of sucrose it 
varied between 4 -2 and 4-6 days in different treatments. 



Effect of water stress and 'sucrose on flowers of Gladiolus 377 

4. Discussion 

A study of the effect of water stress on gladiolus indicated that uptake of the 
test solution and fresh weight change during the initial period of pulsing with 
sucrose were related to the concentration of the solution. Whereas the lower 
concentration of sucrose itself reduced initial uptake compared to the control, 
doubling it did not result in a proportional decrease. A similar result has been 
recorded by Bravdo et al (1974) who observed uptake even from a solution with 
50% sucrose concentration. The subsequent absorption of liquid was dependent 
not only on the water potential of the transfer solution but also on the nature 
of the transfer osmoticum. Thus, when the spikes were held in PEG instead of 
sucrose of similar water potential, there was a steep drop in uptake. 

The fresh weight change of the spikes showed a direct relationship with the water 
potential of the transfer solution. The sucrose-pulsed spikes which were transferred 
to water maintained higher fresh weight. Halevy and Mayak (1974) have shown 
that sucrose decreases the water potential of the petals and enhances their ability 
to absorb water. 

In comparison with water, the uptake of which was markedly curtailed by 
PEG, the availability of sucrose was found to be a major factor in bud opening. 
In all the treatments a higher percentage of opening over the control was ob- 
tained. Stress pronouncedly affected uptake and fresh weight but not the per- 
centage of flower buds opening. This is quite remarkable in the light of the 
finding by Goldschrnidt and Huberman (1974) that citrus petals have a very large 
water requirement during opening (highest fresh weight was recorded) and in 
view of the reported failure of flower bud opening under water stress condi- 
tions (Mayak and Halevy 1971), Thus, flower opening in gladiolus appears to 
be limited more by the availability of sucrose than water, especially because of 
the ability of the newly opening buds to draw out water from the older open 
flowers and cause their premature withering (Rao and Mohan Ram 1982a). Our 
recent study has also shown that green-bud spikes lack adequate reserves of carbo- 
hydrates and that this is one of the principal causes of poor opening (Rao and 
Mohan Ram 1981). 

It is significant that in the present work induced water stress did not curtail 
flower longevity at any given concentration of sucrose. In addition to its role 
as a respiratory substrate (Coorts- 1973), -sucrose has been shown to enhance the 
effect of cytokinins, and counter the deleterious effects of ethylene and abscisic 
acid (Borochov et al 1976a; Mayak and Dilley 1976). Sucrose also reduced 
the endogenous levels of abscisic acid in cut rose flowers (Borochov et al 1976b). 
Spikes treated continuously with sucrose showed higher longevity than PEG-treated 
spikes probably because of greater availability of sugar. 

References 

Borochov A, Mayak S and Halevy A H 1976a Combined effects of abscisic acid and sucrose 
on growth and senescence of rose flowers ; Physiologia Plant. 36. 221-224 

Borochov A, Mayak S and Halevy A H 197 6b Abscisic acid content of se n escing petals on 
cut rose flowers as affected by suerose and ; water stress ; PI. Physiol 58 175-178 



378 / V Aamanuja Rao and H Y Mohan Ram 

Bravdo B, Mayak S and Gravrieli Y 1974 Sucrose and water uptake from concentrated sucrose 

solutions by gladiolus shoots and the effect of these treatments on floret life ; Can. J. 

Bot. 52 1271-1281 

Coorts G D 1973 Internal metabolic changes in cut flower? ; Hort. Sd. 8 195-198 
Durkin D and Kuc R 1966 Vascular blockage and senescence of the cut rose flower ; Proc. 

Am. Soc. Hortic. Sti. & 683-689 
Oilman K F and Steponkus P L 1972 Vascular blockage in cut loses ; /. Am. Soc. Hortic. 

Sci. 97 662-667 
Goldschrnidt E E and Huberman M 1974 The co-ordination of organ growth in developing 

citius flowers : a possibility for sink type regulation ; J. Exp. Bot. 25 534-541 
Halevy A H and Mayak S 1974 Improvement of cut flower quality, opening and longevity 

by pre-shipment treatments ; Acta Hortic. 43 335-347 
Kofranek A M, Halevy A H and Kubota J 1975 Bud opening of chrysanthemums after long 

term storage ; Hort. Sci. 10 378-380 
Kofranek A M and Halevy A H 1976 Sucrose pulsing of gladiolus stems before storage to 

increase spike quality ; Hort. Sci. 11 572-573 
Marousky F J 1969 Vascular blockage, water absorption, stomatal opening and respiration 

of cut "Better Times" roses treated with 8-hydroxyquinoline citrate and sucrose ; /. Am, 

Soc. Hortic. Sd. 94 223-226 
Mayak S and Halevy A H 1971 Water stress as the cause for failure of flower bud. opening, 

in iris ; /. Am. Soc. Hortic. Sd. 96 482-483 
Mayak S, Bravdo B, GuilU A and Halevy A H 1973 Improvement of opening o.f cut gladioli 

flowers by pretreatment with high sugar concentrations ; Scientia Hortic. Amsterdam 1 

35T-365 
Mayak S, Halevy A H, Sagie S, Bar-Yoseph A and Bravdo B 1974 The water balance of 

cut rose flowers ; Physiohgia Plant. 31 15-22 
Mayak S and Dilley D R 1976 Effect of sucrose on responses of cut carnations U> kind in 

ethylene and abscisic acid ; J. Am.Soc. Hortic. Sci. 101 583-585 
van Meeteren U 1978 Water relations and keeping-quality of cut Gcrbera flowers. 1. The 

cause of stem break ; Scientia Hortic. Amsterdam 8 65-74 
Paulin A 1972 Influence d'un deficit temperaire en eau sur le metabolisms azote des flours 

coupees d'/m gennanica ; C. r. Hebd. $eaitc. Acad. Sci. 9 Paris 275 209-212 
Rao I V Ramanuja and Mohan Ram H Y 1979 Interaction of gibberellin and sucrose in 

flower bud opening in gladiolus ; Indian J. Exp. Biol. 17 447-448 
Rao I V Rarcanuja and Mohan Ram H Y 1981 Nature of differences between green-bud and 

tight-bud spikes of gladiolus : basis for a postharvest bud-opening treatment ; Indian J. 

Exp. Blol 19 1116-1120 
Rao I V Ramanuja and Mohan Ram H Y 1982a Prevention of compensatory withering and 

stem-break in cut spikes of gladiolus by overcoming vascular blockage ; Proc. Indian Natl 

Sci. Acad. Part B (in press) 
Rao I V Ramanuja and Mohan Ram H Y J982b Specificity of gibberellin and sucrose promoted 

flower bud growth in gladiolus ; Ann. Bot. 50 473 --479 



fooc. Indian AcacL Sci. (Plant ScL), Vol. 91, Number 5, October I$g2, pp. 3t9-38& 
Printed in India. 



Petal venation in Trigonclla (papilionaceae) 



MOHINI GUPTA 

Department of Botany, Institute of Advanced Studies, Meerut University, 
Meerut 250001, India 

MS received 7 March 1981 ; revised 12 June 1982 

Abstract. Petal venation of nine species of Trigonella has been worked out. A 
positive correlation has been found between length or area and the number of 
dichotomies but no correlation is found with breadth. In all the species corolla is 
of simple type except T. polycerata in which it is of medicagoid type. Among 
the different types of anastomoses C and D types are of most frequent occurrence 
and other types are species specific with a low range of variation. 

Keywords. Petal venation ; papilionaceae ; trigonella. 



1. Introduction 

The analysis of petal venation received the attention of various workers in tracing 
phylogeny after the remark that petal venation shows both simplicity and diver- 
sity. Petal venation of regular corolla received the attention of various workers 
(Arnott and Tucker 1963, 1964 ; Banerji and Mukherji 1970 ; Banerji 1972) 
but that of irregular corolla received only the attention of Datta and Saha (1968) 
and Subramanyam and Nair (1973). Datta and Saha (1968) reported important 
differences at specific level in types of anastomoses and their pattern of distribution 
on standard, wing and keel petals of four species ( Butea frondosa, Cajanus cajan, 
Dolichos lablab and Erythrina indica) belonging to tribe phaseolac. Hence from 
the perusal of the literature it appears that venation of petals may be significant 
at specific level and since ho work is available on the species of Trigonella^ there- 
fore, in this paper petal venation of nine species has been worked out. 



2. Materials and methods 

Flower buds for the present study were either collected locally or procured from 
places .as mentioned below : . 

Species Place of collection 

1. Trigonella arabica Dehile Bet Dagan, Israel. 

2. T. caemlea Ser, Ontario, Canada, 

3. r. calllcerasoites Fish Ontario, Canada. 

379 
P.CW-2 



380 -Afohini Gupta 

Species Place of collection 

4. T. corniculata Linn. Meerut, India. 

5. T. cretica (L.) Boiss. Ontario, Canada. 

6. T. gmciliSy Benth. Nainital, India. 

7. T. polycerata, Linn. Meerut, India. 

8. T. stellata Forsk. Jerusalem, Israel. 

9. r. suavissima Lindl. Canberra, Australia. 

Petals from fully mature flowers were cleaned in lactic acid, stained in, 1% 
aqueous safranin and mounted in glycerine. Twenty-five petals of each, species 
were studied. Drawing of each petal was subdivided equally into basal, central 
and peripheral regions. Mean number of dichotomies and anastomoses was calcu- 
lated in each sector. Area of the petal was measured by planimeter. For descri- 
bing the types of anastomoses classification of Arnottand Tucker (1963) is mainly 
followed. 



3* Observations 

Each flower consists of a standard, two keel arid two wing petals which vary in 
shape, size and structure (figures 1-3). Standard petal is symmetrical while the 
other petals are asymmetrical. Keel and, wing petals are clawed (figures 2-3, 
5-6), but standard petal is generally nonclawed except T. arabica, T. gradlis 
(figure 7) and T. callicerasoites. Each petal receives a single trace which branches 
after the separation of the individual petal (figures 1-2). 

Standard petal is generally obovate in shape (figure 4) except T. gmcilis, 
T. cretica (figure 8) and T. callicerasoites in which it is elliptic. Apex is notched 
in all the species. The bundle that enter the standard petal branches into one 
median and two costal veins in the basal region (figures 4, 8). However, in 
T. gracilis (figure 7) it branches at the base of the limb. Out of three veins, costal 
vein branches further dichotomously but the median vein first branches trichoto- 
mously and then dichotomously (figures 7-8). However, in few petals of 
r. polycerata median vein shows first dichotomous branching then trichotomous 
and further dichotomous branching. 

The bundle of the keel and wing petals branches dichotomously at the junction 
of limb and claw (figures 2-3, 5-9). In T. cderulea and T. arabica wing petals 
possess ridges and grooves at the distal end and the veins end freely in the groove 
region. The wing petals of T. polycerata (figure 5) and T. callicerasoites are serrated 
at the distal end. 

In all the species wing petals at the junction of the limb and claw possess a 
spur like process which is generally tubular except T. stellata and T. cretica (figure 9), 
in which it is flattened. This spur like process receives supply from the main 
bundle of the petal (figure 3). Wing petal of T. polycerata has a tooth like process 
also at the distal end. This tooth like process is nectariferous and vascular 
bundles in this region are found to be inversely oriented with respect to the petal. 
Keel petals of this speciei has a pocket Jike structure which receives the tooth 
like process of the wing petal (figures 5-6). In all the types pf petals venation 
is mainly open dichotomous (figures 1-9). 



Petal venation' in fngomlia 



381 




Figures 1-9. Venation in petals of Trigonella. 1-3. standard, keel and wing petals 
of T. caerulea, 4-6. standard, wing and keel petal of T, polycerata, 7. standard petal 
of T. gracilis, 8. standard of T. suavissima, 9. wing 'petal of T, cfetica. 



Mohini Gupta 







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04 VO ON 


04 O S VO Q S 




A* ,!H 4f PO VD </* 


tio o 00 


m po co vo o O 




^ C^ TH CO CS TH 




O4 T-t TH O4 TH "f^ 1 


W 








w 








41 * 


ro xo o O TH 'no 
xn ^ SO \O CJI 04 


vo S o 


ON 00 O4 VO rH 


,.. c3 

1 S 

cj ti 


41-H^I -H-H4I 


^ 41 4| 


-H 4i 41 -H 41 




VQ ^ VO "4" OO 


04 rt O 


VO O4 C4 O *t 


1 I 


4t n ^ ON v> o 


CO TH t^H 


co o4 <N TH O 


.H 








O 


"n ^f e^ o 04 ON 


04 00 00 


04 t* ON oo ON 


M 1 


TH O ^\j co ON ON 






** "T3 








H ** 


41.4J4J 41441 


-H4J41 


^-^ 333 


C rj 


^ vo ^ oo so oo 


o 04 oo 


04 o4 T> VO O O4 


a 


Vj ^ ^ XO TH 


*n ON 00 


00 TH 'b- ON V> t^ 

rH 


08 

O 








S 










xn vi r^ ON co oo 

VO ON g 00 00 CNi 





fO VO "^t" "nn "^Jf* oI 

oo ON vo O vo 




" '*H 


TH 


" ' " TH O 





4) 41 4f -H 41 41 


41 


41 4j 4^ -H 4i -H 


tt 




p 


00 vo Jn S S 




^^ c^ rH 


t- 


xt* 4f- xr> c4 "vf co 

TH TH 


.2 








ffi 

JN 

a g 


S s s r ? 

^^-H ^ -H -H -H 


??? 

414141 

Tt 1 00 Ovl 


00 

04 t> o O "d- o^ 

ro o O TH O O 

41 41 -H 41 41 -H 

VO ^J" ON O-4 t> TH 
TH oo O r-' ON r4 


E 


5-^ *-^ 


04 6 TH 


Xf" TH O4 O4 O TH 


.s 


ON ^ 00 Jj ^ ^ 


5h co c5 


xn o r- c4 xn TH 
cO co o4 C*4 C4 TH 


fw 


* * JH 




.... -* 


^ -H 




41 4^ 41 41 -H 41 


4] 4| 4| 


41 41 41 -H 44 41 

^t- ON vo vo oo Tr- 


(- H 








K M 


xn v >, v> v> vb 


"* 


vo \n \c> co cn co 


.S 








cS 








S-s 


2SS S^S 


^8S 


00 TH O fn TH C4 
oo oo n oo co r> 


li 


"** *" 


oo 04 c4 


xn vb oo vb r<l <N 

TH 


5- 


S? 






o 


%} 






1 




1 




s 

Q 


amo of tlw if 
type of Petal 


li is 

,g "8 M^ "ifg-tt-^ 
^ S.S 8 ^ S.-S g 
Hgfc'M h'S^'M 


T. caerulea 
Standard 
Wing 
Keel 


S S 1 ts g "g 

8a>M '-aPM 


fc 


TH c4 


CO 


^f xn 



s 

4 



Petal venal ion in Trigonella 



383 



QO r\ ^f 
m xi" o 

-H 4- 41 



-H 4- 



so ro rt- 

CO f- O 



-H 4- 41 
o n o 

P* 
06 en <fxj 



VO ON 
l> n 

^* 6 



CO ON ON 
*H CO 

^ o b 

-H 41 41 



Tf ON in 4 i 

O >/> nt O i 



41 41 4^ -H 41 4- 



ON 66 rf **- "^ ON 

Vi ^ tN V> CO c^ 

-H 4i 41 41 41 4- 

O Q O o O O 

Tfr O 00 Tf 06 



C^ ^H 

-H 41 -H 

OoQ 



41 41 
o o o 
o ^ ^o 



-H4-1 

O 
Tt- VO 



41 4- -h 
o u-> vo 
O m o 



SO ^H 
<D CO 



o n- -i 
<s o <s 

4J4I-H 

OQ CO CO 

CO *H i-H 



4I-H44 

St- go 
00 t 



O O 



cn co 



00 ^ 
ON O\ 



-^ -o 

15 

frl 8 
l- 



1 

^ 



2' 
5" 

11 

o 9 to 

* a.s g 
f^l?^ 



2 

5s 

1-S- 

S5 g a 

rt-S'ifi 



rf ON ^ 

co M ON 

*H r^H 

-H4^4- 

t^ b b 



-H414- 
o o 

<S Ci 

os. -4t- b 



vo ro ro 
O o 



H-H + 









384 Mohini Gupta 

3.1. Dichotomies 

Table 1 shows mean area, mean length and mean breadth of the different types 
of petals and mean number of dichotomies in different regions with standard error. 
A positive correlation has been found in all the species except T; caemka, 
T. comiculata and T. callicerasoites between area and number of dichotomies 
(figure 10). Similarly a positive correlation of dichotomies is also found with 
length except T. comiculata^ T. gracilis and T. suavissima (figure 11). No corre- 
lation is found with breadth (figure 12), 



cr 



CC 

< 




Us 

| 4 
i 3 

K O 

a 2 

jj i 



Figure 10. Histogram comparing the average area with mean number of dichoto- 
mies of standard (s), wing (w), keel (k) and total corolla (T) in different species. 



140 

1202 

,00 




80 
60 
40 
20 

o 



Figure 11. Histogram comparing the average length with the mean number of 
dichotomies of standard (s), wing (w), keel (k) and total corolla (T) in different 



species. 



JOO o 




Figure 12. Histogram comparing" the' average breadth with the mean number of 
dichotomies of standard (s), wing (Mr), keel -Oc) and total corolla (T) * '-different 
species. 



Petal venation in Tngonclla 



385 




Figure 13. Histogram comparing the average area with the mean number of anasto- 
moses of standard (s), wing (w), keel (k) and total corolla (T) in different species. 




Figure 14. Histogram comparing the average length with the mean number of 
anastomoses of standard (s), wing (w), keel (k) and total corolla (T) in different 
species. 

Abbreviations : A = T. arabica , B = T. caerulea ; c . = T. callicerasoites ; D = T. 
corniculata ; E = T. cretica ; * = T. gracilis ; o = T. polycerata ; H = T. stellata ; 
i --= T. suavissima. 



3 . 2. Anastomoses 

The open dichotomous venation becomes complicated at places by vein fusions. 
The percentage of petals showing vein fusions varies from 20-66-6% (table 2). 
A slight positive correlation has been observed between the length or area of 
petals and the number of anastomoses (figures 13-14). In most of the petals 
anastomosing is generally found at one point. Among the different types of 
anastomoses C and D types are of most frequent occurrence in all the species 
(table 3), In all the species vein anastomoses are generally concentrated in the 



386 Mohini Qupta 

Table 2. Percentage of petals shown g 



SI. Total 

No. Name of the species (%) 



Points of anastomosis 



5-10 



i. 


T. arabica 


40-0 


66-6 


33-34 




2- 


T. callicerassoites 


53-4 


37-5 


25-00 


25-0 12-5 


3. 


T. caerules 


40-0 


66-6 


33*34 




4. 


T m corniculata 


20-0 


66-0 


33-0 


. . . . . 


5. 


T. cretica 


20-0 


66-0 


33-0 


. . 


6. 


71 graeilis 


71-4 


80-0 


10-0 


to-o 


7. 


T. polycerata 


66-6 


20-0 


30-0 


10-0 40-0 


S. 


T. stellata 


33-3 


80-0 




20-0 


9. 


T. suavlssima 


6<5*6 


60-0 


20-0 


10-0 10-0 



Table 3. Percentage of different types of anastomoses. 



SI. 



No . ^atne of the species 



D 



h . 


21 arabica 


25-00 


25-00 


25-00 


. * 


25-00 


2. 


r. callicerasoites 






66-60 


.. 


33-34 


3, 


T. caerulea 




11-11 


77-78 




11-11 


4; 


T. corniculatd 


.. 


25-00 


50-00 


.. 


25-00 


5. 


T. cretica 


.. 




25-00 


75-00 




6. 


T. gracilis 


11-11 




44-44 


ll'H 11-11 


22-22 


7. 


T. polycerata 


6-96 


6-96 


41-86 


4*64 2-32 


37' 21 


8. 


T. Stellata 






57-14 


.. 


42-86 


9. 


T.suavissima 


11-76 


35 '89 


35-89 


5-08 


11-76 



central region except the standard petal of T. polycerata in which they are more 
in the peripheral region. 



4. Discussion 

The petal venation of this genus has shown some interesting features. Arnott 
and Tucker (1964) reported significant correlation between size (length, width, 
area) and number of dichotomies. In this paper a positive correlation is found 
with the length and area only. They have also given importance to the position 
of the dichotomies. In the present study maximum number of dichotomies is 
found in the central region except the standard petal of T. cretica and T. corniculata 
in which it is found in the basal region. Generally no correlation is found 
between the frequency of anastomoses and length of the petals. HoweveF, the 



Petal venation in TrigmiolUt 387 

petals of T. arabica, T. polycerata and T. gmcilis have shown a positive correlation. 
Just like the length no correlation is found with the area excepting the petals of 
71 caemlea, T. comiculata and T. polycerata. Thus from above it is clear that 
frequency of anastomoses is not dependent upon the area or the length of the 
petals. In all the species highest frequency of anastomoses is found in the central 
region but the wing petals of 7". ambica and keel petals of T. comiculata and 
T. stellata possess highest frequency in the basal region. Datta and Saha (1968) 
reported highest frequency in the peripheral region in the members of phaseolae. 

Among the different types of anastomoses C and D types are of most frequent 
occurrence in all the species studied except T. crelica in which C and C are 
common. Earlier Dalta and Saha (1968) have also reported the frequent occur- 
rence of these types for the members of phaseolae and Arnott and Tucker (1963) 
for Ranunculus repens. In addition to these types only B type is found in 
T. caendea and T. comiculata, A and B in T. arabica, A, B, C in T. suavissfma, 
A, C, C" in T. gmcilis and all types in T. polycerata. Thus in this genus distri- 
bution of anastomoses appears species specific. Among the different species 
more points of fuisons are found in T. polycerata, T. callicerasolt.es, T. gracilis 
and T. suavissima. 

Foster and Arnott (l960),Bnerji and Mukherji (1970), and Subramanyam 
and Nair (1973) are of opinion that open dichotomous venation is primitive while 
on the other hand Chertek (1962, 1963) views that anastomosed venation is primitive. 
The present study supports the former view and among the species of this genus 
T. callicerasoites, T. gracilis, T. polycerata and T. suavissima appears to be more 
advanced as these species have more points of fusions and more number of 
petals showing vein fusions. 

Earlier reports indicate that species of Trigonetta are characterized by presence 
of both simple as well as medicagoid type of corolla. In the present investigation 
simple corolla is found in all the species except T. polycerata in which it is of 
inedicagoid type. The wing petal of T. polycerata possesses a tooth like structure 
arising from the inner surface of the petal and receiving supply from the main 
bundle of the petal. In this region vascular bundles are inversely oriented with 
respect to the petal bundle. The same inversely oriented supply was reported 
by Arber (1936) for the nectary of Ranunculus and for the corona by Narcissus 
(Arber 1937). Thus this tooth like structure may be regarded as petalline nectary 
or corona. Earlier Larkin and Graumann (1954) named this structure as horn 
in Medicago sativa, Thus it can be concluded that among the different species 
of this genus T. polycerata is the highest evolved species. 



Acknowledgements 

The author expresses her gratitude to Prof. Y S Murty for giving valuable 
suggestions and facilities, to Dr R Loiselle, Prof. A H Gibson and Prof. C Heyn 
for providing materials and to CSTR for giving Post-Doctoral Research 
Fellowship. 



388 Mohini Gupta 

References 

Arber A 1936 Studies in flower structure U, OB the vascular supply to the nectary in Ranun- 
culus ; Ann. Bot. 50 305-319 
Arber A 1937 Studies in flower structore HE. On the carcma and androecium in certain 

Amaryllidaceae ; Ann. Bot. (NS) 1 293-304 
Arnott H J and Tucker S C 1963 Analysis of petal venation in Ranunculus I. Anastomoses in 

R. repens, V. plenlflorus ; Am. /. Bot. 50 821-830 
Arnott H J and Tucker S C 1964 Analysis of petal venation in Ranunculus II. Number and 

position of dichotomies in R. repens var. Pleniflorus ; Bot. Gaz. 125 13-25 
Banerji M L 1972 Morphological studies on petal venation of Ranunculus diffusus DC. and its 

affinities with fossil materials ; Biology Land Plants pp. 1-8 
Banerji M L and Mukherji M 1970 Petal venation in Ranunculus scleratus Linn.; Castama 35 

157-161 
*Chertek J 1962 Die Verlauf der Nervatur in den Kronblattern bzul kronen der Dikotyledonen ; 

Novitates Bot. Hort. Bot. Univ. Carolineae Pragensis pp. 3-10 
*Chertek J 1963 Die Nevatur der Kronblattern bei den Vertretern der Ordnung Rosales s.L; 

Acta Horn. Bot. Pragensis pp. 13-29 
Datta P C and Sana N 1968 Specificity o.f distribution of venation anastomosis patterns in 

petals of phaseoleae (Leguminosae) ; Ann. Bot. 32 791-801 
Foster A S and Arnott H J 1960 Morphology and dichotomous va&culature of the leaf of 

.Kingdonia umflora ; Am. J. Bot. 47 684-698 
Larkin R A and Graumann, H O 1954 Anatomical structure of alfalfa flower an explanation of 

tripping mechanism ; Bot. Gaz. 116 40-52 
Subramanyam K and Nair N C 1973 Dichotomous venation and anastomosis in corolla of 

an orchid ; Proc. Indian Acad. Sci. (Plant Sci.) 78 195-202 

* Not consulted in original. 



Proc. Indian Acad. Sci. (Plant Sci.) Vol. 91, Number 5 y October 1982', pp. 389-395. 
Printed in India. 



Responses of cotton-cultivars to long day conditions 



J G BHATT and M R K RAO 

Central Institute for Cotton Research, Regional Station, Coirabatore 641 003, India 

MS received 29 August 1981 ; revised 1 July 1982 

Abstract. Flowering of cultivated varieties of cotton belonging to G. arboreum, 
G. herbaceum and (?. hirsututn was delayed by over 14 hrs of daylength because of 
increase in number of days for square formation. The long day treatment in 
general increased height, production of fruiting branches, leaf area and dry weight 
per plant. The number of fruiting forms, bolls retained, yield of seed cotton and 
fruiting coefficient decreased under long day conditions. These characters were 
affected more in upland varieties and short day Cambodia derivatives. The most 
of G. arboreum and G. herbacewn varieties became more vegetative in growth but 
their boll number and yield per plant increased. 

Since the varieties 1998 F (G. hirsutum) and Gaorani 1111 (G. arboreum) were 
tolerant to long photopejriod and grew satisfactorily, it is suggested that these 
may be used as donor parents for improving the quality of cottons grown in northern 
India. 

Keywords. Photoperiod ; flowering ; long day ; boll ; square ; yield. 



1. Introduction 

The flowering of cultivated and wild species of cotton is governed by both, day- 
length and temperature as shown by Waddle et al (1961) and Mauney and Phillips 
(1963). They found that most of the varieties flower under short days and cool 
nights. The importance of low night temperature in promoting flowering was 
further stressed by Mauney (1966) using a non-photoperiodic upland cotton 
variety. Bhatt (1977) and Bhatt et al. (1976) found that long days and high tem- 
perature singly or in combination delayed flowering of upland cottons of northern 
India whereas the photosensitive Cambodia varieties did not flower .udder long 
days alone or long ; day plus high temperature. The upland genotypes when gro.wn 
at latitudes 29 N,21 and 11 N flowered progressively earlier and at lower nodes 
at more southern latitudes because of reduction in daylength and temperature. 
Though the cotton crops in the northern cotton zone in India are grown under 
irrigation and thus give higher yields, they are of short and medium staple. An 
attempt was therefore made to screen some of the promising varieties (belonging 
to both G. hirsutum and G. arboreum) for tolerance to long days so. as to identify 
donor parents for improving quality. , . : 

389 



390 / G Bhaii and M R K Rao 

2. Materials and methods 

The cultivated varieties in India representing 
hybrid developed through genetic male sterile 



Species 

G. hirsutum 
G. hirsutum 
G. hirsutum 
G. hirsutum 

G. hirsutum 

G. arboreum 
G. arboreum 
G. arboreum 
G. arboreum 
G. arboreum 
G. herbaceum 
G. herbaceum 



Variety 

LSS 
320 F 
CP 1998 F 
170 Co 2 

MCU 1 

AK 235 
G 27 
K 9 

Gaorani 1111 
Gaorani 1187 
V 797 

Jayadhar 
CPH 2 



three species of cotton and an i\ 
line were taken as shown below : 

Area 

Upland type from North zone 
Upland type from North zone 
Grown in South zone 

An Itfdo-Americart type grown 
in Central zone 

Photosensitive short day deriva- 
tive of Cambodia cotton from 
South (used as check) 

From Central zone 
From North zone 
From South zone 
From South zone 
From South zone 
Grown in Central zone 

Grown in South zone 

An intraspecific (G. hirsutum) 

hybrid 



The cotton varieties were raised in large (45 cm diameter and 105 cm depth) 
pots adequately manured. There were six plants per treatment one in each pot. 
The daylength around latitude 30N in northern cotton zone in India when cotton 
is sown and even thereafter until about 80 days is over 14 hours. The normal 
dayteiigth in summer at Coimbatore (latitude 11 Nf> varies from ll-50hrs to 
12-20hrs. It was extended to 14-5tes through 60 watt incandescent lamps. 
The treatment were (i) Conti^l, i.e., normal dayleagth of 11-50 h*s to 12'201u?s, 
and (H) long photopetiod of 14* 50 his. Under both the treatments the day and 
night temperatures were the same. The ctey temperature was between 34-0 C 
to 35- 5 C from germination to square formation for about 35 days and thereafter 
until flowering did BOt rise above 3'6-8 C. The night temperature during this 
period fluctuated between 17- 5 C to 21-8 C to 24- 6 C and subsequently remained 
around 23>0C. The long day treatment was discontinued after 75 days after 
soig. Taking cotyledonary node a$ zero, the node on the main stem producing 
first sympodiuin was taken as the first fruiting node. The fruiting coefficient is 
defined as the yield of seed cotton produced pee lO&'gm of total dry matter 
(Crowtter 1944). 



Effect of long days on cotton 



391 



3. Results 



The long days delayed formation of flower buds in all the varieties irrespective 
of the species (table 1). The short day Cambodia derivatives MCU 1 and 170 
Co 2 set squares just after the long day treatment was discontinued. The early 
types CPH 2 and 1998 F took only 4 and 6 days more respectively to square 
when compared with the delay of three weeks in LSS and 320 F. Among the 
diploids both the Gaorani types were early whereas in the rest, delay in square 
formation ranged from 15 to 20 days. 

Most of the varieties took significantly more days to flower when photoperiod 
was extended except CPH 2 and 1998 F with a delay of 4 and 9 days respectively 
and of 6 days in Gaorani 1111. With a difference of only 2 days Gaorani 1187 
appeared to be practically photoinsensitive. The photosensitive types MCU 1 
and 170 Co 2 took maximum days to flower followed by the upland types LSS 
and 320 F, and the Asiatic type AK 235. The square period, i.e., the number of 
days from initiation of flower bud to opening of the flower remained more or less 
the same between the treatments except that it increased by 3 days in 1998 F and 
reduced by 3 days in G 27, 5 days in K 9, 6 days in Gaorani 1187 and 3 days 
in Jayadhar. 



Table 1. Flowering behaviour of cottoii-cultivars as affected by extended photo- 
period. 



.Species 


Variety 


Number of days 
to square 


Number of days 
to flower 


First fruiting 
node 


Days for first 
boll bursting 



G. hirsutum 


LSS 


34 


56 


52 


75 


4- 


3 


9- 





us 


150 


G. hirsutum 


320 F 


33 


54 


56 


75 


4- 





6- 





120 


145 


G. hirsutum 


CP1998F 


25 


31 


46 


55 


4- 





6- 





S5 


95 


G. hirsutum 


170 Co 2 


35 


72 


54 


39 


3- 





7- 


6 


120 


147 


G. hirsutum 


CPH 2 


22 


26 


42 


46 


6- 





6- 





86 


84 


G. hirsutum 


MCU1 


39 


73 


59 


95 


2- 


3 


10- 


3 


110 


130 


G. arboreum 


AK235 


33 


52 


53 


72 


3- 





7- 





122 


140 


G. arboreum 


027 


35 


50 


55 


67 


5- 


3 


7- 


7 


118 


130 


G. arboreum 


*C9 


34 


54 


59 


74 


2" 


6 


7- 





125 


142 


G. arboreum 


Gaorani 1H1 


35 


42 


53 


59 


2- 





6- 


6 


130 


139 


G. arkoreum 


Gaorafli 1187 


35 


43 


53 


55 


4- 


6 


7- 


>7 . 


125 


132 


G. herbaceum 


V797 


45 


60 


65 


82 


5- 


o 


6 


-o 


130 


150 


G. herbaceum 


Jayadhar 


35 


50 


65 


77 


2- 


6 


5 


o 


120 


135 


S.E. foe varieties 


1-90 




&-61 






! 


40 




4' 84 


S.E. for treatment 


1-10 




3-51 






o- 


58 




1-97 



Control T = Treatment 



392 / G Sfiatt and M A K Rod 

The node at which the iirst synipodial branch appeared was also higher, the 
notable exceptions being CPH 2 and V 797. Other varieties flowered 2-8 nodes 
higher . under .long day conditions. 

An increase in daylength also increased boll maturation period, i.e., the days 
from opening of the first flower to dehiscence of carpellary wall or boll bursting 
thus adding to farther lateness. The trend was more or less similar to flowering. 
The upland type LSS took the maximum number of days whereas CPH 2 was 
unaffected. It was interesting to note that the Gaorani * cultures 1187, 1111 
and 1998 F were late by 7, 9 and 10 days respectively. 

Except for CPH 2 and Gaorani 1111 plant height increased significantly in all 
the varieties reaching the maximum in 170 Co 2 and LSS (table 2). The number 
of sympodia per plant also increased similarly except in CPH 2, V 797 and 
CP 1998 F. The long day conditions reduced production of fruiting forms with 
notable exceptions of G 27 and K 91 showing an increase whereas CP 1998 F 
was not affected significantly. Maximum reduction was found in MCU 1, AK 235 
and V 797. 

There was a significant increase in leaf area per plant in all the varieties. It 
was over three times in LSS and over twice the normal area in 320 F, MCU 1 and 
G 27 followed by 170 Co 2. Increase in leaf area was comparatively less in 
CPH 2, V 797 and CP 1998 F. 



Table 2. Effect of extended photopcriod on Some growth characters. 



Species Variety Height (cm) No. of sympodia No. of fruiting Leaf area per 

per plant forms per plant plant (cm 8 ) 



G. hirsutum 


LSS 


60- 


2 


117-1 


12 


20 


41- 


6 


30-7 


2983 


11581 


G. hirsutum 


320 F 


65- 


3 


95-2 


9 


15 


33- 


3 


39*0 


2950 


6170 


G. hirsutum 


GP 199$ F 


40- 


3 


60-8 


13 


14 


42- 





39-0 


2507 


3575 


G. hirsutum 


170 Go 2 


55- 


3 


120-7 


12 


22 


34- 


2 


22-6 


4210 


8179 


G. hirsutum 


GPH2 


54- 


1 


54-3 


11 


12 


38- 





30-0 


2668 


3548 


G. hirsutum 


MGU 1 


51- 


3 


90-1 


12 


17 


35- 


6 


17-0 


6170 


13217 


G.arboreum 


AK235 


97- 


3 


144-2 


22 


32 


59* 


6 


38-7 


2850 


4879 


G. arbareum 


G 27 


108- 





130-8 


19 


25 


65 


6 


70' 


2217 


4717 


G.arboreum 


K9 


72- 


3 


143-6 


' 17 


25 


59- 


3 


73-3 


2980 


5316 


G. arboreum 


Gfaoraiti 1111 


72- 


6 


77-0 


16 


18 


51- 


3 


35-0 


2950 


4850 


G.arboreum 


Gaorani 11 87 


52- 





85-1 


11 


15 


49. 





31-0 


2170 


3950 


G. herbaceum 


V797 


56- 





83-3 


12 


15 


62' 


3 


40-1 


2007 


2970 


<j. herbaceum 


Jayadhar 


59 


1 


101-2 


14 


20 


54- 


3 


41-6 


2350 


' 4317 


S.E.'for varieties 


7-39 




4-28 






8-61 




159-80 


S.E. for treatments 


3*02 




1-75 






'3-51 




65-24 



C = Control T = treatment 



Effect of long days on cotlari 

At maturity dry matter per plant increased in ino&t of the varieties under long 
day treatment (table 3). But CP 1998 F, AK 235 and Gaorani 11 87 were not 
affected. It was, however, highly significant in CPH 2, LSS 320 F, MCU 1, K 9, 
Gaorani 1111 ajad Jayadhar. 

In G. hirsutum varieties LSS 170 Co 2 and MCU 1, the number of bolls per 
plant at maturity decreased with consequent decrease in yield of seed cotton. Both 
320 F and CP 1998 F were unaffected whereas CPH 2 recorded higher yield 
under long days. Except Gaorani 1187, all G. arboreum varieties and V 797 had 
more number of bolls and yielded more. Jayadhar remained unaffected. Fruiting 
coefficient of most of the varieties decreased because of higher dry weights of their 
vegetative parts when grown under long days. 



4. Discussion 

The enhanced photoperiod delayed flowering because of increase in the number 
of days required to initiate square formation. The square period was more or 
less unaffected in G. hirsutum varieties confirming the previous findings of Bhatt 
(1977). G. arboreum varieties were similarly affected except Gaorani cultures 
where flowering was delayed by 2 and 6 days only. The G. arboreum variety 
used by Mauney and Phillips (1963) showed essentially no reaction to the environ- 
ments in their study whereas flowering was delayed from 12 to 19 days under 
long day conditions in the present study with the first fruiting node pushed up 
significantly. Differences in flowering responses of different varieties reported 
by Mauney and Phillips (1963) and in our experiments may be attributed to differ- 
ences in day and night temperatures though long day conditions were similar. 
Day temperatures in their experiments varied from 27 C to 32 C whereas the 
night temperature was fixed at 15 C or 30 C, while in our experiments day tempe- 
ratures varied from 34 C to 36 C and night temperatures from 17 C to 
24 C. 

An increase in leaf and stem dry weight and a decrease in yields of seed cotton 
of most of the varieties under long days reduced their fruiting coefficients (Bhatt 
1970). In G. hirsutwn group CP 1998 F responded exceptionally well in terms 
of its vegetative and reproductive growth maintaining high fruiting coefficient. 
This variety was late in flowering by 9 days under extended photoperiod and took 
95 days for the first formed boll to burst as against 150 and 145 days taken by the 
northern upland varieties LSS and 320 F respectively. Because of its superior 
fibre characters, better yielding capacity and high degree of tolerance to long day 
conditions, it may prove as a suitable donor parent for improving the quality of 
northern hirsutums in India. Next in performance was the intm-hirsutum hybrid 
CPH 2. But the short day Cambodia derivatives MCU 1 and 170 Co 2 failed 
miserably in relation to flowering and growth (Hutchinson 1959; Bhatt 1977). 

The G. arboreum varieties except Gaorani 1111 took 12 to 19 days more to flower 
and then 4 bolliAg period also increased by 12 to 18 days. The new culture Gaorani 
1111 in this respect took only 6 to 9 days more for flowering and boll bursting 
respectively. It compares well with tie northern arboreum variety G 27 in terms 
of yield of seed cotton and similar fruiting coefficient. By virtue of its long staple, 
it may prove useful for improving the quality of northern arboreums in India* 



394 



J G Bltatt and M R K &ao 



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Effect of long days on cotton 395 

Among the G. herbaceum varieties, V 797 though late in flowering and boll 
bursting appeared tolerant to longer photoperiod as its growth and yield improved 
But the fruiting efficiency of Jayadhar was reduced markedly. 



Acknowledgement 

We thank Dr M R H Qureshi, IARI Regional Station, Hyderabad, for kindly 
supplying the Gaorani cultures developed by him. 



References 

Bhatt J G 1970 Yield capacity of cotton plant in relation to the production of dry matter ; 
Indian J. Plant Physiol. 13 219-224 

Bhatt J G 1977 Growth and flowering of cotton (Gossypium hirsutum L.) as affected by day- 
length and temperature ; /. Agric. ScL (Camb.} 89 583-587 

Bhatt J G, Shah R C, Patel B D and Seshadrinathan A R 1966 Responses of cotton genotypes 
to latitudi nal differences ; Turrialba 26 247-252 

Crowther F 1944 Studies, on growth analysis of the cotton plant under irrigation in the Sudan. 
III. A comparison of plant development in Sudan Gezira and Egypt ; Ann. Bot. (NS) 
8 213-257 

Hutchinson J B 1959 The application of genetics to cotton improvement (Cambridge University 
Press) 

Mauncy J R 1966 Floral initiation of upland cotton Gossypium hirsutum L. in response to tempe- 
ratures ; /. Exp. Bot. 17 452-459 

Mauney J R and Phillips L L 1963 Influence of daylength and night temperature on flowering 
of Gossypium ; Bot. Gaz. 124 278^283 

Waddle B M, Lewis C R and Richmond T R 1961 The genetics of flowering response in 
cotton. III. Fruiting behaviour of Gossypium hirsutum race lati folium in a cross with 
a variety of cultivated American upland cotton ; Genetics 46 427-438 



P. (B)-3 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 5, October 1982, pp. 397-407. 
Printed in India. 



Seed germination and seedling establishment of two closely related 
Schima species 

RAM B.OOJH and P S RAMAKRISHNAN 

Department of Botany, School of Life Sciences, North-Eastern Hill University, 
Shillong 793 014, India 

MS received 15 December 1981 ; revised 13 July 1982 

Abstract Seed germination of S. khasiana from Upper Shillong and S. wallichii 
from Shillong, Umsaw and Burnihat in Meghalaya, north-eastern India, and 
seedling establishment and growth of these species/populations in reciprocal 
cultivation were studied. Seeds lost viability and germinability gradually within 
a year when stored at 5 cm below the soil surface under natural conditions or at 
20 C in the laboratory. Storage at 0C permitted retention of 15-25% viability. 
Seeds germinated better on the surface layers (0-2 cm) of the soil. S. khasiana 
had a lower temperature optimum (15 C) of germination while the populations 
of S. wallichii had a higher temperature optimum (20/25 C). At a temperature 
of 30 C, the lower altitude population of S. wallichii from Burnihat gave higher 
germination than the high altitude population from Shillong. A given species/ 
population of Schima gave better seedling establishment and growth in its own 
natural habitat as compared to the introduced populations from the other alti- 
tudinal sites. This is indicative of the close adaptation of the natural populations 
to their habitat and ecotypic differentiation in this species. 

Keywords. Tree adaptation ; seed germination ; tree seedling establishment ; alti- 
tudinal ecotype ; Schima. 



1. Introduction 

Germination and establishment represent two critical phases in the life-cycle of 
a plant species and these two aspects have been related to adaptation and distri- 
bution pattern of species in space (Koller et al 1962 ; Harper 1965 ; Cohen 1967 ; 
Ramakrishnan 1972 ; Ross and Harper 1972 ; Thompson 1973 ; Boojh and 
Ramakrishnan 1981a) and in time (Kapoor and Ramakrishnan 1973). However, 
this aspect of the problem in relation to adaptive strategy of tree species has 
received little attention (Kozlowski 1971 ; 1979). Although the size of a single 
species population is to. some extent related to seed supply, it is ultimately deter- 
mined by favourable conditions available for germination and establishment 
(Harper et al 1970). Further, a large gap often exists between the seeding poten- 
tial of a species and the actual number of seedlings established ' in that area, 
depending upon environmental conditions. 

397 



398 R am Boojh and P S Ramakrishnan 

S. khasiana Dyer and S. wallichii (D C) Korth family Ternstroemiaceae are 
two closely related and economically important timber tree species of north- 
eastern hills of India. These species show a distribution pattern on an altitudinal 
basis where S. khasiana is restricted to higher elevations (1800-1900 m), while 
S. wallichii shows a wide distribution ranging from 100 to 1600m. These two 
species are early successions and come up in the secondary fallows after slash 
and burn agriculture (Jhum). These are light-demanding and regenerate pro- 
fusely in the open, through light wind-dispersed seeds. The present study is a 
comparative investigation of seed germination and seedling establishment of these 
two species along an altitudinal gradient in the Khasi Hills of Meghalaya. 



2. Climate 

All the three sites are characterised by marked seasonal changes in climate. The 
year could be divided into 3 distinct seasons : (i) Monsoon season of high tempe- 
rature and humidity extending from May to October when over 80% of the rain- 
fall occurs, (ii) Winter season (November to February) of lower temperature which 
is comparatively dry except for a few winter showers, and (iii) A warm, dry and 
windy summer in March-April (table 1). 



3. Methods of study 

Mature fruits of S. khasiana were collected from Upper Shillong and that of 
5. wallichii from 3 sites at Shillong, Umsaw and Burnihat, in the months of 
February-March, 1978. Seeds were separated out by air drying. The fruit and 
seed weight measurements were based on 100 fruit/seed with 20 replications. 



Table 1. Comparison of climatic data at study sites during 1978-79. 



Upper Shillong Shillong Umsaw Burmhat 



Location 










Latitude (N) 
Longitude (E) 
Altitude (m) 


25-34 
91 '56 
1900 


25-34 
91-56 
1400 


25-45 
91-54 
800 


26-02 
91-52 
300 



Temperature ( C) 

Mean monsoon maximum 22 24 30 32 

Mean monsoon minimum 15 16 22 24 

Mean winter maximum 16 16 20 25 

Mean winter minimum 3 6 10 12 

Precipitation (mm) 2400 2000 1800 1600 



Germination and establishment of Schima species 399 

Seeds were stored in the laboratory at 20 5 C and C in BOD incubators 
in tightly closed plastic bottles. In nature, seeds were similarly stored 5 cm below 
soil surface. The moisture content of seeds at the time of storage was 10%. 
Viability and germinability of stored seeds were tested at intervals of 3 months 
with four replicates of 50 seeds of each species/population. For testing the viabi- 
lity of seeds a freshly prepared 5% aqueous solution of 2, 3, 5-triphenyltetra- 
zolium chloride (TZ) was used. Seeds were first soaked in water for 10-12 hrs, 
then seed coats were punctured to facilitate entry of the xz solution and were left 
in the dark at 30 C for upto 24 hrs. Seeds with completely stained (red colour) 
embryos were scored as viable. Germinability was tested by placing seeds in petri- 
dishes over moist filter-paper at a constant temperature of 15 C for S. khasiana 
and 20 C for S. wdllichiL 

Seeds were tested under two conditions, of continuous light under an incandes- 
cent fluorescent tube (500-600 1 X ) or under continuous darkness by covering 
the petri-dishes inside thick black paper, at a constant temperature of 20 C. Seed 
germination in dark was counted under green light. Germination at different 
constant (15, 20, 25, 30 and 35 C) and alternating (25/15 and 25/20 C) tempe- 
rature regimes were tried in BOD incubators maintained at these temperatures. 
The effect of different soil depths of 0, 2, 4, 6, 8 and 10cm on germination was 
tested in pots filled with soil, by placing seeds at the appropriate depth. 

All germination experiments were replicated 4 times with 50 seeds in each test. 
The emergence of radicle was taken as an indicator of germination. Tests in all 
cases were done for 20 days after the seeds were placed for germination. 

Ten replicates of 100 viable seeds (viability was ascertained for a given seed- 
lot on the basis of preliminary germination tests) of each species/population were 
sown at a depth of 5 cm at all the 4 study sites both in the open and under 
forested situations, in May 1978. The depth of 5 cm for sowing was chosen in order 
to avoid washout of seeds under heavy rainfall. Observations on the seedling 
emergence and establishment were taken at monthly intervals. Seedlings were 
harvested at the end of one year period and after noting plant height and leaf 
area using a planimeter, the root and shoot portions were separated and dried to 
a constant weight at 85 2 C. 



4. Results 

4.1. .Fruit and seed characters 

The capsules and seeds of S. khasiana were heavier than that of the populations 
of S. wallichii. While the fruit weight of the populations of S. wattichii were not 
very different, significantly higher seed weight was noticed for the Burnihat popu- 
lation of this species compared to that of the two other populations (table 2) t 

4.2. Germination studies 

4.2a. Effect of storage : When seeds were stored in the soil or in the laboratory 
at a temperature of 20 5 C, both viability and germinability of the seeds of all 



400 



Ram Boojh and P S Ramakrishnan 



the populations decreased markedly with passage of time so that after one year, 
seeds were totally non-viable or gave very poor germination. However, storage 
at C maintained better viability and germinability after 1 year storage (figure 1). 

4.2b. Depth of burial : As seen from figure 2, the depth of burial affected both 
the time and the final percentage of germination. Maximum germination was 
found to occur at 2 cm depth and it decreased at the depths greater than this for 
all the species and populations. At soil surface though faster germination 
occurred the total percentage was lesser than at 2 cm depth. 



Table 2. Fruit and seed weight of Schima species/populations. 



Fruit weight 
(g) 



S. khasiana 



1-6T0-09 



( S,E. of the mean) 



Seed weight 
(mg) 



M80-03 



S. wattichil 






Shillong 


M30-07 


0-460-06 


Umsaw 


1-040-03 


0-44002 


Burnihat 


1-070-05 


0-530'01 



80 


. khasiana 


6 












i 










1 


{ 


I 








J7 


r i 








L 
2 

o 




1 




\ 






1 


I 


y 
^ 

/ 
/ 
/ 
/ 

s 


1 
m 


?7 




1 


I 


1 


I 


I 








S 


.wallkhiJ 1 
















(Umsaw) 




'/. 


'/ 


y, 










% 


^ 


% 




^ 








'//, 


% 


y, 




/^ 


















1 




1 


y 

'/ 
i 
'/ 

V 


J 

II 

j 


I 




1 


I 


h 


\ 


. 







I 

1 


\ 


I 




( 

i 


I- 

Sh 

I 


2L- 
il 

I 


long 


} 

1 


I 


^3 


! 


I 


09 

60 

V* 

40 

20 










1 










1 

! 




I 




5 <e 

1 


ur 

1 


nihut) 


j 

1 


i 


b 


I 


a. 








[ 







9 12 3 

STORAGE PERIOD(MONTHS) 



Figure 1. Viability (open columns) and germinability (hatched columns) of Schinm 
seeds after different storage periods. First column, storage at C ; second column 
storage at 20 5 C ; and third column, storage under soil. 



Germination and establishment of Schima species 



401 



4.2c. The effect of light and darkness : There was germination both in the dark 
and light and the results obtained were not significantly different under these two 
conditions (table 3). 

4.2d. The effect of temperature : Table 4 reveals the effect of various tempera- 
ture regimes on the germination of the seeds of Schima species and populations 



S. knastana 
o 5- walli 

Shillong 

Urn saw and 
Burnihat 
Populations 




6 8 

DEPTH OF BURIAL (cm) 

Figure 2. The relationship between seed depth, germination and emergence period 
of seedlings of Schima species/populations over a period of 35 days after sowing. 
Continuous lines represent % emergence of seedlings and broken lines represent 
number of days taken for emergence. 



Table 3. The effect of light and dark treatment on seed germination of Schima 
species. 



Continuous light Continuous dark 
20 C 20 C 



S. khasiana 


50 


48 




5-4 


3"4 


S. wallichii 


62 


57 




6-4 


dz 1-3 



(db S.E. of the mean) 

The seeds of populations of 5. wallichii were pooled. 



402 Ram Boojh and P S Ramakrishnan 

Table 4. Germination (%) of S. khasiana and *S. wallichii seeds at various 
constant and alternating temperatures. 



Constant temperatures Alternating temperatures 

( C) ( C) 

15 20 25 30 35 25/15 25/20 



S. khasiana 55 46 44 42 46 45 

2-7 3-6 2*9 2'2 2-3 5-5 

S. wallichti 

Shillong 37 48 4S 33 20 48 50 

4-3 6-0 6-8 4-1 7-5 4-1 4-6 

Umsaw 42 65 58 40 ^1 61 70 

5-3 3-7 3-7 6-8 i 2*9 3-7 -H9-9 

Burnihat 38 69 56 50 33 59 64 

2-2 3-7 5-4 4-8 7'6 3-4 3'6 



( S.E. of the mean) 

S. khasiana showed maximum germination at constant 15C, with a gradual 
decrease with increase in temperature, so that at 35 C no seeds of this species 
germinated. Populations of S. wallfchii showed maximum germination at 20 
and 25 C with decrease in germination on either side. At 30 C, the lower alti- 
tude population of S. wallichii from Burnihat gave higher germination than its 
higher altitude population from Shillong. Two alternating temperature regimes 
tried were favourable for germination for all the species/populations. 

The rate of germination was faster at 15C for S. khasiana and 20 and 25 C 
or alternating (25/20 C) for S. wallichii populations (figure 3). 

4.3 Seedling establishment 

4. 3 a. Seedling emergence : Only a small proportion of seedlings could emerge 
under field conditions at all the study sites. Further the differences in emergence 
were not significant (at 5% level) between species/populations (table 5). 

4.3b. Survivorship : No seedlings could survive under forested situations beyond 
a period of 2 months. Under open grown situations, mortality was generally very 
high resulting in a steep decline in population upto January-February, at all the 
sites. At Upper Shillong, however, the rate of decline in population was slower 
for S. khasiana with an ultimately large population size compared to the popula- 
tions of S. wallichii. At the other 3 experimental sites, however, the pattern of 
survivorship was not very different for the populations of Schima, though the local 
populations showed better survivorship than the introduced ones. S. khasiana 
gave the lowest final survival at these three sites (figure 4). 



Germination and establishment of Schima species 



403 



70 

60 
50 
40 
3 

: 20 



1 




S.wa! iichii 
(Shillong) 





10 



15 20 .. 5 10 

DAYS OF GERMINATION 



IS 



Figure 3. Percentage germination of Schima species/population at different periods, 
at constant and alternating temperature of 15C(&); 20C(O); 25C(H); 
30C (Q); 35C (A,); 25/15 C (A) and 25/20 C (x). 

Table 5. Seedling emergence (%) of S. khasiana and S. waltichii at different alti- 
tudinal sites. 



Field stations 


S. khasiana 


Species/Populations 
S. wallichii 


Shillong 


Umsaw 


Burnihat 


Jpper Shillong 


20 
4-6 


10 

d= 2-3 


11 
1-8 


14 
1-8 


^hillong 


19 

2-4 


21 

3-9 


10 

3-3 


10 

2-1 


Jmsaw 


16 
1-7 


13 

2-7 


13 
1'2 


13 

2-5 


lurnihat 


16 

3'9 


12 

1-7 


11 

2-3 


11 
0-6 



'.(B) 



404 Ram Boojh and P 5 Htnic?!s'inan 

UPPER SHILLOKG 



SHtlLCNG 



J ASONDJ F W A M J JASOND J FMAMJ 




Figure 4. Survivorship of Schima seedlings under field conditions (in open). No 
seedlings could survive beyond 2 months under forested situations. S. khasiana 
(); S. wallichii, Shillong (O), Urnsaw () and Burnihat (D) populations. 



4 . 3c. Plant performance : The growth characteristics of the different species/ 
populations at different sites, given in figure 5, show that the naturalized popu- 
lation for a given site was superior to the other introduced populations. Thus 

5. khasiana gave better growth yield under Upper Shillong site, while the popula- 
tions of S. wallichti from 3 different sites did better in their respective natural 
habitats. 



5. Discussion 

Schima species being early successional colonizers depend for regeneration on the 
availability of open sites which favour their seed germination, establishment and 
growth. The most important factor limiting the ability of such species to colonize 
disturbed sites is the availability of seed, which must come either from a stand 
in close proximity or from storage in the soil. The latter is not possible for 
Schima as seeds do not remain viable in the soil for an extended period of time 
as seen from the present study where the viability of seeds is completely lost 
after storage in soil for one year. Thus, the species is fugitive in nature 
(Hutchinson 1951), where good dispersal mechanism would play an important role 
permitting the species to colonize new habitats (Salisbury 1942). Schima due to 



urerminunon ana 



s 



2 
S 

I 









7 ... 

2 

6 < 



x 
3 2 



2 5: 

5- 

ce 
o 

Jo 




Figure 5. Growth performance of Schima species/populations at different field 
conditions, a = Upper Shillong ; b = Shillong ; c = Umsaw and d = Burnihat 
site ; filled bars for 5. kjiasiana, and hollow bars for Shillong ; hatched bars for 
Umsaw and stippled bars for Burnihat populations of S. wallichti. 



its light, mobile (winged) seeds often invades highly disturbed areas after slash 
and burn agriculture in the region. Similarly in temperate forests it has been 
reported that light seeded species Fraxinus and Betula play an important role 
in revegetation after clearcutting (Bormann and Likens 1979). The variation 
in seed weight in between species/populations may partly be related to climate 



406 Ram Boojh and P S Ramakrishnan 

(Baker 1972 : Wearstler and Barnes 1977) and partly to ecotypic differences 
related to altitude which is supported by growth studies of the different Schima 
populations done at different altitudes discussed below. 

The differences in germination behaviour in response to temperature as seen 
in the present case where S. khasiana germinated at a comparatively lower tempe- 
rature compared to S. wallichii populations, have often been correlated with 
climatic conditions and seed source (Callaham 1970 ; Thomson 1973), whereby 
seeds from colder areas germinate better at lower temperature than those from 
warmer regions. Grose (1957) has demonstrated that montane species of Euca- 
lyptus germinated best at a lower temperature of 16 C, in contrast to somewhat 
higher temperatures for species of warmer areas. Though the total number of 
seeds of a species which ultimately germinates at a given temperature is a good 
indicator of that species potential, however, the time taken to germinate is of much 
significance since the early germinating individuals enjoy a considerable competi- 
tive advantage (Ross and Harper 1972). The germination rate which was higher 
at 15 C for S. khasiana and at 20 or 25 C for S. wallichii populations is consis- 
tent with temperature optima for their germination. The rapidity of germination 
in this species without a dormancy mechanism is advantageous in colonizing new 
areas by producing a profusion of seedlings and this has been reported for a 
majority of tropical trees which has been termed as biological nomads (Ng 1978). 

Schima seeds come under microbiotic category (Crocker and Barton 1953) as 
they normally lose viability and germinability within a year. Small and light 
seeds are reported to lose their viability faster (Quick 1961) and this has been 
reported in species of Salix, Populus and Ulmus (Wareing 1963) and Alnus (Boojh 
and Ramakrishnan 198 Ib). The better retention of viability and germinability 
under lower temperature storage may be attributed to slow biological and bio- 
chemical processes at such temperatures (Kamra 1967). 

There exists a large gap between seeding potential of a species and the number 
of seedlings emerged at a given site. The failure of survival of seedlings under a 
forest canopy may be attributed to the shade intolerance of the seedlings. The 
differences in survival pattern for different species/populations under field condi- 
tions are suggestive of the adaptation of a given population to the natural clima- 
tic conditions in which they grow. This is suggested by the relatively better survi- 
val and performance of local species/populations to that habitat compared to 
the introduced ones. Thus, the lower altitude population of S. wallichii which 
is adapted to longer growing season, higher temperature and frost-free winter is 
adversely affected at higher altitude. 



Acknowledgements 

This study was supported by a research grant from the Department of Science 
and Technology, Government of India. RB acknowledges the Council of 
Scientific and Industrial Research (CSIR), New Delhi also, for the partial support 
in form of a Junior Research Fellowship during the preparation of this paper. 



Germination and establishment of Schima species 407 

References 

Baker H G 1972 Seed weight in relation to environmental conditions in California ; Ecology 

53 997-1010 
Boojh R and Raimkrishnan P S 198 la Temperature responses to seed germination in two 

closely related tree species of Schima Reinw. ; Curr. Sci. 50 416-418 
Boojh R and Rarnikrishnan P S 1981b Germination behaviour of seeds of Alnus nepalensis 

Don. ; Natl. Acad. Sci. Lett. 4 53-56 
Bormann F H and Likens G E 1919 Pattern and process in a forested ecosystem (New 

York : Springer-Verlag) pp. 253 
Callaham R Z 1970 Geographic variation in forest trees ; In Genetic resources in plants 

Their exploration and conservation (eds) D H Frankel, E Bennett (Edinburgh : Oxford 

Blackwell Scientific Publications) pp 43-48 

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lation may exist between the conditions at the time a choice has to be made and the 

subsequent outcome ; /. Theor. Biol. 16 1-14 
Crocker W and Barton L V 1953 Physiology of seeds (Waltham Mass : Chronica Bota n ica) 

pp. 267 
Grose R J 1957 Notes On dormancy and effect of stratification on germination of some 

eucalypt seeds ; Victoria Bull. For. Cotnmn. 3 1 
Harper J L 1965 Establishment, agression and cohabitation in weedy species ; In The genetics 

of colonizing species (eds.) H G Bake r and G L Stebbins (New York : Academic Pi ess) 

pp. 243-268 
Harper J L, Lovel P H and Moore K G 1970 The shapes and sizes of seeds ; Ann. Rev. 

Ecol. Syst. I 327-356 
Kamra S 1C 1967 Studies on storage of mechanically damaged seeds of Scots pine (Pinus 

sylvestris L.) ; Stadia Forestalia Suecica 42 1-19 

Hutchinson G E 1951 Copepodology for the Ornithologist ; Ecology 32 571-577 
Kapocr P and Ramakrishnan P S 1973 Differential temperature optima for seed germination 

and seasonal distribution of two populations of Chenopodium album L. ; Curr. Sci. 42 

838-839 
Koller D, Mayer A M, Poljakoff-Mayber A and Klein S 1962 Seed Germination ; Annual 

Review of Plant Physiology 13 437-464 
Kozlowki T T 1971 Growth and development of trees Vol. 1 (New York : Academic Press) 

pp. 444 
Kozlowski T T 1979 Tree growth and environmental stresses (Seattle : Washington Univeisity 

Press) pp. 192 
Ng F S P 1978 Strategies of establishment in Malayan forest trees ; In Tropical trees as living 

systems (eds.) P B Tomlinson and M H Zimmermann (Cambridge Univ. Press) pp. 129-162 
Quick C R 1961 How long can a seed remain alive ? ; In Seeds, The Yearbook of Agri- 
culture (Washington D.C. : U.S. Govt. Print. Off.) pp. 94-99 
Rarnakrishnan P S 1972 Individual adaptation and its significance in population dynamics ; 

In Biology of land plants (eds.) V Puri, Y S Murti, P K Gupta and D Banerji (India ; 

Meerut : Sarita Prakashan) pp. 344-355 

Ross M A and Harper J L 1972 Occuptation of biological space during seedling establish- 
ment ; /. Ecol. 60 77-88 
Salisbury E J 1942 The reproductive capacity of plants (London : G Bell and Sons Ltd.) 

pp. 224 
Thompson P A 1973 Geographical adaptation of seeds ; In Seed ecology (eds.) W Heydecker 

(Penn. Univ. Park : Penn. State Univ. Press) pp. 31-58 
Wearstler K A and Barnes B V 1977 Genetic diversity of yellow birch seedlings ; Can. J. Rot. 

55 2778-2788 
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and C Taylor (New York : The MacMillan Co.) pp. 195-227 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 5, October 1982, pp. 409-416. 
printed in India. 



Anther and pollen development in cotton haploids and their parents 

S S MEHETRE 

Department of Botany, Mahatma Phule Agricultural University, Ralmri, District, 
Ahmednagar (MS) 413722, India 

MS received 29 October 1981 ; revised 29 July 1982 

Abstract. Development of anther tapetum from premeiotic stages to pollen 
formation was studied in six x-ray induced h a ploids of Gossypium hirsutum, three 
interspecific F 2 haploids, and one natural haploid of each of G. hirsutum and 
G. barbadense, and the observations were compared with those of their respective 
parents, a genetic male sterile, a male fertile and a cytoplasmic male sterile line of 
G. hirsutum. Significant diffeiences were recorded for number of anthers per 
flower, pollen size, pollen viability and number of microspores produced by PMC. 
Anther development in haploids was normal. Anther dehiscence was also normal 
in some haploids. Non-dehiscent anthers could be mostly attributed to the forma- 
tion of immature pollen grains. Normal development of anthers and degeneration 
of tapetum occurred in the parents and m the genetic fertile line. Contrastingly 
no degeneration of tapetum was noticed in the cytoplasmic male sterile line. 

Keywords, Gossypium spp. ; haploids ; anther tapetum ; male sterile. 

1. Introduction 

Haploids are characterized by significant decrease in size of vegetative plarrt 
parts, vigour and fertility (Kostoff 1943) and in diameter of pollen mother cells 
(Belling and Blakeslee 1923) by half of that in the diploid (Ivanov 1938), and by 
smaller guard cells (Lamm 1938). Kimber and Riley (1963) indicated a 
relationship between haploid and diploid guard cells of G. hirsutum and 
G. barbadense by a factor of 1-26. 

Partial or complete sterility due to halving of chromosome number and several 
meiotic irregularities in haploids of G. hirsutum, G. barbadense, their F 2 inter- 
specific crosses and x-ray induced haploids have been reported (Mehetre and 
Thombre 1981b, c, d). Studies were undertaken to investigate the development 
of anther tapetum and its role in pollen sterility observed in these haploids. The 
comparative observations made on haploids, their respective parents, genetic male 
sterile and fertile lines and one cytoplasmic male sterile line have been reported 

409 



S S Mehetre 





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2. Material and methods 

The length of flower bud at various meiotic stages was determined by studying 
meiosis in the fertile counterparts and haploids. Flower buds of different 
haploids., their respective parents, genetic male sterile and fertile lines and 
cytoplasmic male sterile line (table 1) were collected from premeiotic to 
pollen formation stages and fixed in Randolph's Graf ; after dehydra- 
tion, the anthers were embedded in paraffin. Sections of 12 JLCTR were 
cut and stained with iron alum hematoxylin (Johansen 1940). Pollen viability 
(fertility /sterility) was tested by differential staining with a solution comprising 
among other organic components malachite green, acid fuschin, and orange G. 
(Alexander 1969). The data collected from 25 observations for each of the 
parameters mentioned in table 1 were analysed statistically (Panse and Sukhatme 
1953) and standard deviations and significance of differences between means were 
calculated. 



3. Results and discussion 

Significant differences were noticed between haploid and diploid ^plants in size of 
flowers and and roecia, in number of anthers per flower (figure 1), tetrads per micro- 
spore (figure 2) and in pollen size and pollen sterility (figure 3). In haploids 
the average number of microspores resulting from a PMC ranged from 3 '11 to 
4*78. The large variation observed in pollen size (figure 3) in all the haploids 
indicated that the pollen grains contained varying number of chromosomes. 
Although well-developed exine and spines were noticed on some pollen grains, 
probably microspore mitosis and starch formation had not occurred in them, 
thus resulting in pollen sterility. 

In the- cytoplasmic male sterile line the tapetum was. well developed and its 
cells were enlarged. There was normal differentiation of anther wall, but the 
sporogenous tissue collapsed early during meiosis ; meiosis did not proceed 
beyond prophase and hence the tapetum remained intact and enlarged (figure 6). 
Similar observations were recorded by Murthi and Weaver (1974) and Mehetre 
and Thombre (198 la) for the cytoplasmic and genetic male sterile (MS 5 and MS 6 ) 
stocks of G. hirsutum. In the present study on the genetic male sterile stock 
normal development of anther tapetum was noticed, but the microspores aborted 
due to development of vacuoles in them (figure 7). In the male fertile counter- 
parts (figure 8), in all the tetraploid parents and in all the haploids (figures 4, 5) 
the tapetal cells begin to digenerate at the time of separation of microspores from 
the tetrads. 

In the fertile lines the microspores develop a thick exine and a thin intine. Spines 
develop on the exine and the germ pores become distinct. The microspore 
nucleus divides to produce the generative nucleus and the vegetative nucleus ; 
at this stage the pollen grain is considered to be mature and ready for shedding. 
Similar observations were reported by Murthi and Weaver (1974) and Mehetre 
and Thombre (1981a) for male fertile anthers and Mehetre (1981) for triploid 
(3x = 39) and tetraploid (4x = 52) anthers. In the haploids, however, after separa- 
tion of the microspores from tetrads the exine may become well developed but the 



Anther and pollen development 



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Anther and pollen development 415 

spines are not of uniform size, the germpores are not distinct and the pollen grains 
do not mature because the mircospore nucleus does not undergo division, as was 
also observed in the parents of the haploids, such underdeveloped immature pollen 
does not contain sufficient starch grains. 

Decrease in the radial dimensions of tapetal cells occurred in the fertile materials 
between anaphase I and pollen formation, while in the haploid plants the decrease 
vas observed between anaphase I and tetrad stage only. The extent of decrease 
vas variable from anther to anther, flower to flower and plant to plant. 

The data on measurements of radial width of anther tapetum in parents of 
laploids and in the genetic fertile line indicated that in parents of haploids the 
legeneration of anther tapetum continues progressively from meiotic anaphase 
;o pollen stages and it ranged from 3 to 6 microns at pollen stage, while in the 
genetic male sterile line the tapetum remained intact even after microspore tetrad 
itage. Pollen abortion occurred due to vacuolation of pollen caused presumably 
yy nutritional differences, while in the cytoplasmic male sterile line meiosis did 
lot proceed and hence the tapetum remained intact. A similar behaviour of 
mther tapetum was also reported by Brooks et al (1966) in anthers of different 
genetic and cytoplasmic male sterile, make fertile and fertility restorer lines of 
jorghum by Murthi and Weaver (1974) and Mehetre and Thombre (1981a) in 
cotton. 

In all the three groups of haploids a marked variation in the width of tapetal 
;ells from pre-prophase to pollen stage was observed. In some individuals, the 
legeneration of tapetum was rapid while in some individuals it was slow. Although 
dgniflcant differences in tapetal cell width were noticed in tetraploid and diploid 
)lants, it was not in a 1 : 2 ratio. The pollen abortion and sterility was mainly 
iue to microspores containing variable number of chromosomes and pollen with 
ligh variation in size and probably not due to the abnormal development of 
;apetum. 



References 

Vlexander M P 1969 Differential staining of aborted and non-aborted pollens ; Stain Techno!. 

44 117-122 
Jelling J and Balakeslee A F 1923 The reduction division in haploid, diploid and tetraploid 

Daturas ; Proc. Natl. Acad. Set. 60 106-111 
Jrooks M H, Brooks J S and Chien I 1966 The anther tapetum in cytoplasmic genetic male 

sterile sorghum ; Am. J. Bot. 53 902-908 
vanov M A 1938 Experimental production of haploids in Nicotiana rustica L. ; Genetica 20 

295-397 

bhdnsen D A 1940 Plant Microtechnique, Tata McGraw-Hill Co. 2nd ed. 
Cimber G and Riley R 1963 Haploid angiosperms ; Bot. Rev. 29 480-531 
iCostoff D 1943 Haploicie Triticum vulgare and die Variabiliabat inrer diploiden Nachkommens 

schaften ; Zuchter. 15 121-125 

R 1938 Note on haploid potato hybrid ; Herediatas, 24 39 
S S 1981 Anther and pollen development in traploid (3* = 39) and tetraploid (4* = 52) 

plants in cotton (Gossypium spp.) Phytomorphology (in press) 
vfehetre S S and Thombre M V 1981a Stages of pollen abortion in male sterile stocks of 

Gossypium hirsutum L.; /. Maharashtra Agril. Universities 6 159-161 
lehetre S S and Thombre M V 1981b Cytomorphological studies in x-ray induced glandless 

haploids in Gossypium hirsutum L. cotton ; Proc. Indian Acad. Sci. (Plant Sci.) 90 313-322 



416 S S Mehetre 

Mehetrc S S and TJiombre M V 19Slc Meiotic studies in the haploids (2n = 2x[= 26) of tetra- 

ploid cottons (2n = 4x = 52) ; Proc. Nat 1. Sci. Acad. B47 516-518 
Mehetre S S and ThombreM V 1981 d Microsporogenesis in interspecific F 2 haploids of cotton ; 

Phytomorphology (in press) 
Murthi A N and Weaver J B 1974 Histological studies on the five male stcrik strains of 

upland cotton ; Crop Sci. 14 658-662 
Panse V G and Sukhatme P V 1953 Statistical methods for agricultural workers ICAR, New 

Delhi 



Proc. Indian Acad. Sci. (plant Sc/.), Vol. 9l, Number 5, October 1982, pp. 417-426. 
Printed in India. 



Changes in proteins, amino and keto acids in different seedling 
parts of Cyamopsis tetragonolobus Linn, during growth in light and 
darkness 

PREM GUPTA and D MUKHERJEE 

Department of Botany, Kurukshetra University, Kurukshetra 132119, India 

MS received 23 May 1981 ; revised 21 April 1932 

Abstract. Comparative changes in protein, free amino and keto acids have been 
studied in different seedling parts of Cyamopsis tetragonolobus plants in light and 
dark. Endosperm recorded higher level of free amino acid? in darkness than in 
light, while a low concentration of protein was exhibited both in light and dark. 
The breakdown of soluble protein was more in darkened cotyledon due to higher 
protease activity. The large increase in the free amino acids in the hypocotyl during 
seedling growth in the dark may be due to its restricted capacity to incorporate all 
the amino acids into proteins. Root samples from light recorded higher soluble 
protein as well as a higher free amino acid pool. a-Oxoglutaric acid (a-OGA) was 
recorded in low levels and at few growth stages in both light and dark. In light 
raised cotyledon samples, the dominating keto acids are phosphoenolpyruvate and 
pyruvic acid. Low levels of oxaloacetate in light, like a-OGA, indicate its rapid 
utilization during growth, but its accumulation in the dark may suggest sluggish 
protein synthesis thus sparing the utilization towards the synthesis of amino acids. 
Utilization of asparagine and glutamine was also affected in dark. 

Keywords. Seedling parts ; protein ; amino acids ; keto acids ; protease activity ; 
Cyamopsis tetragonolobus. 



1. Introduction 

The correlative changes in amino and keto acids have been studied during seed 
germination and seedling growth (Fowden and Webb 1955 ; Webb and Fowden 
1955 ; Krupka and Towers 1958a, b; Mukherjee 1972 ; .Mukherjee and Laloraya 
1974, 1979, 1980). Recently, studies have been carried out in our laboratory 
(Gupta 1981 ; Afria and Mukherjee 1980, 1981) of the comparative changes in 
aforesaid metabolites along with organic acids and soluble protein in different 
seedling parts of various plants so that proper assessment could be made of their 
mobilization and/or breakdown at various growth stages. Leguminous plants 
can be divided into endospermic and non-endospermic ones depending upon 
whether the endosperm has been retained into maturity or not. Metabolic 

417 
P.(B)-6 



4i8 Prem Gupta and D Mukherjee 

changes during development of endospermic legumes have received less attention 
in comparison to the other group during seedling growth. For this reason, 
various biochemical changes with growth in Cyamopsis tetragonolobus, an endo- 
spermic legume, have been studied here. In this paper the comparative changes 
in soluble proteins, free amino acids, keto acids and protease activity have been 
described in endosperm, cotyledon, hypocotyl and root of this endospermic 
legume, during the early stages of seedling growth in light and dark. 



2. Material and methods 

Seeds of Cyamopsis tetragonolobus Linn, were surface sterilized with 0- 1 % mercuric 
chloride for 2 to 3 min followed by thorough washing. Acid treatment was given 
thereafter and washed thoroughly again with sterilized distilled water. Seeds were 
germinated on filter paper discs moistened with distilled water in Petri dishes and 
grown in darkness or light (2910 lux provided by fluorescent tubes) in a growth 
chamber maintained at 30 1 C. Three replicates of 30 seeds each were taken for 
each experiment. Every care was taken to select morphologically uniform seeds 
and to ascertain least variability, experiments on growth in light and darkness 
(table 1) were repeated thrice. Protein, free amino and keto acids were determined 
quantitatively in endosperm, cotyledon, hypocotyl and root 48 hr after sowing 
(termed " initial ") as well as 48, 72, 96 and 120 hr after * initial ' of both light 
and dark grown seedlings. 

Soluble proteins from fresh plant material were measured according to the 
method of Lowry et al (1951) using Folin phenol reagent. The plant material 
was boiled in 80% ethanol for 2 rnin on a water bath. It was allowed to stand 
for 15 rnin at room temperature, ground in the same ethanol and centrifuged at 
6000-7000 rpm for 5 min. Supernatant was discarded and the residue was again 
extracted with 80% ethanol. Supernatant was discarded again and the residue 
was extracted with 5 % perchloric acid, followed by centrifugation at 6000-7000 
rpm for 5 min. Supernatant was discarded and the residue was taken out in a 
test tube containing IN NaOH and kept for 30 min. in warm water (40-50 C) f 
0-5 to 1*0 ml of this clear solution was taken and 10 ml of reagent C, which was 
prepared by adding reagent A (2% sodium carbonate in O'lN NaOH) and 
reagent B (0-5% copper sulphate in 1% sodium-potassium tartarate) in the 
ratio of 50 : 1 (v/v), was added to it and allowed to stand for 10 min at room 
temperature. Then added 1 ml of Folin's reagent (diluted twice) rapidly with im- 
mediate mixing and allowed to stand for 30 min. The OD was measured at 540 nm 
in a Bausch and Lomb Spectronic-20 colorimeter. The amount of protein was 
determined in terms of Bovine Serum Albumin. 

The extraction procedure used for amino acids, their chromatographic separa- 
tion and estimations were the same as recommended by Steward et al (1954). 
For keto acids the extraction procedure of Kaushik (1966) which is a slight modi- 
fication of the method described by Towers and Steward (1954) has been followed. 
Free amino and keto acids were quantified as glycine and a-oxoglutaric acid equi- 
valents, respectively. 



Changes in C. tetragonolobus Linn, during growth 



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-H -H 41 41 



O rH rH OS 

CO O *O <NI 



-H 41 41 41 

vo oo oo o4 



: o o o o 
-H 41 41 41 



: o o -H o 

' 41 -H -H 41 

OS rH OS OS 

co co VO Xo 

r- p* o *<* 

rH rH 04 rH 



^u o o o o 
g 41 "H HH 41 
oo vo o t~- 



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xo o *o o4 r- 

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41 4141 4141 
o vo vo co o 
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420 



Prem Gupta and D Mukherjee 



Protease activity The method of extraction of the enzyme was a slight modi- 
fication of that described by Yomo and Varner (1973) and Ihnen (1976). 1% 
casein solution was prepared in 0-1 N NaOH. lOOmg of each seedling part 
(at least in 3 replicates) was homogenized in 10 ml of 100 mM phosphate buffer 
(pH 6-0) and centrifuged at 5000 rpm for 15 min. After filtration the pellet was 
homogenized with the 5 ml of buffer and the process repeated thrice for maximum 
recovery. All supernatants were combined so as to make the final volume to 
25 ml. Each reaction set received 1 ml of the enzyme extract and 1 ml of casein 
solution and the pH was 10. The blank set received 1 ml each of enzyme extract, 
casein solution and 10 % Trichloro acetic acid (TCAL) (cold). These sets were incu- 
bated at a temperature of 37 2 C for 2.5 hr. 1 ml of 10% TCA (cold) was added 
to each reaction set after the incubation period was over and both the sets centri- 
fuged. After discarding the residue 1 ml of filtrate was taken from each set and 
2ml of 0-5 N NaOH and 1 ml of 1 N Phenol Folin's reagent were added with 
immediate mixing. These sets were allowed to stand for 30 min and OD was 
taken at 540 nm in a ECI Junior Spectrophotometer. Protease activity was expressed 
in n mol of tyrosine equivalent lur* g~~* tissue. 



3. Results and discussion 

Results have been summarized in tables 1-2 and figures 1-3. 
3.1. Seedling growth in light and darkness 

Table 1 shows that the growing axis did not differentiate 48 hr after soaking (' Ini- 
tial ' stage) but at 48 hr seedling stage roots and hypocotyls were noticed and the 

Table 2. C. tetragonolobus showing protease activity (n mol) Tyrosine x 10 8 hr"" 1 g"* 1 
(fr. wt.) in different Seedling parts in light and darkness. 



Stagos (hr) Endosperm 



Cotyledon 



Hypocotyl 



Root 







Light 






Initial 


2'7720-036 


2-592-249 


*l-726 -106 




48 


0-684OOOO 


2- 880 -252 


2-558 -249 


4-464-200 


72 


900 i 0-253 


7-668-165 


2-520 -259 


3*096'l57 


96 


Tnce 


l-332-259 


l-296 -165 


0-576-072 


120 


Trace 


1-54S-190 


1-440 -252 


2'9l6-225 



Dark 



Initial 


34-128i -374 


59-148-655 


*16'488 1-590 




48 


H-592 -53S 


11 -988 -124 


35'1000-561 


40-068-533 


72 


8-100 -272 


8-532-409 


2'7720'036 


4- 068 -252 


96 


0-792 -060 


l'224-252 


0'6480-000 


3-060-095 


120 


0'432 -000 


l'728zb'286 


1-8360'000 


1'404-124 



Data represents growing axis as hypocotyl and root did not differentiate. 



Changes in C. tetragonolobus Linn, during growth 



421 



200 
180 
160 
UO 



100 

f 80 
60 : 



55 



6 



Soluble Proteins Total Free AmJno Acids 
o COTYLEDON a * 

o a ENDOSPERM ^ a 

a e HYPOCOTYL o o 

, ROOT 



LIGHT 



Cyamopsis tetrogonotobus 




Initial 48 72 9$ 120 hr* 




Initial 



Figure 1. Cyamopsis tetragonolobus : showing levels of soluble protein N and 
total free amino acid pool in light and darkness in different seedling parts at various 
growth stages. 



length of hypocotyls was greater in darkness than in light. At 120 hr stage the 
hypocotyls length of dark grown seedlings were 1ST 60% more than those raised 
in light. Root growth also exhibited the same pattern as recorded for hypocotyls 
but the increase ranged between 15 to 25% in dark as compared with light. 

As regards the fresh weight changes, the endosperm was of greater weight in 
darkness than in light at ' initial ' stage. But with further seedling growth, at 
120 hr stage, the value in dark was lower than in light. Changes in percent dry- 
mass showed a different pattern in that the endosperm from seedlings raised in 
dark had always a higher value than those in light irrespective of the seedling growth 
stages (table 1). Cotyledons at 120hr stage also had more fresh weight in light 
than in dark. Moreover, percent dry mass although initially more or less of the 
same value in light and dark decreased much less with further growth in the dark. 

Hypocotyls, after differentiation, show more than three-fold increase in fresh 
weight in dark compared with those in light. However, the dry weight values 
were slightly lower in the former. Roots exhibited a small decrease in their 
fresh weight both in light and dark. 

Growth data presented here illustrate the two common phenomena of photo- 
morphogenesis and etiolation in light and dark. Dark-grown seedlings having 
stimulatory effects on hypocotyl lengthening recorded a linear increase and direct 
relationship with the fresh weight but percent dry mass exhibited inverse relation- 
ship indicating the failure of translocation of the products of reserve hydrolysis 
to keep pace with the extension growth, 



Prem Gupta and D Mukherjee 






P. 

W) 







3 

d 



1 
g 
1 







E 



, M 

AJp 6/601 JSOI3V ONJW 



Changes in C. tetragonolobus Linn, during growth 



423 



A PEP 
8 Pyr 
CX-OGA 
D OAA 
E Urea 
F GLY 




Initial 48 hr 72 hr 96 hr 120 hr 



Figure 3. C. tetragonolobus : Keto acid changes in different seedling parts at 
various growth stages. Abbreviations: PEP, phosphaenolpyruvate ; PYR, pyruvic 
acid ; GLY, glyoxylic acid ; -OGA, a-oxoglutaric acid ; OAA, oxaloacetic acid. 



3-2. Biochemical studies 

The studies with this endospermic legume revealed that the endosperm had low 
soluble protein values initially which further decreased with seedling growth in 
both light and dark (figure 1). Endosperm samples from dark treatments recorded 
a higher number and amount of free amino acids, when compared to corresponding 
light samples. In light, levels of free amino acids remained low during early 
stage but dark-raised endosperm samples recorded increasing values up to the 
72 hi stage, whereafter they declined (figure 1). Glutamic acid, a-alanine, leucine- 
phenylalanine, serine-glycine, glutamine and histidine dominated quantitatively 
in dark-raised endosperm samples (figure 2). 

The breakdown of proteins could also be detected in cotyledons of both light 
and dark grown seedlings. However, the depletion was more in dark since light 
causes a retention of proteins as mentioned earlier (Rai and Laloraya 1967 ; 
Mukherjee and Laloraya 1979). Along with the protein depletion although an 
enhancement in the free aminoacid pool was expected in endosperm and cotyledons, 
their marked difference and a large increase in the latter suggests different rates 
and pattern of accumulation in light and dark and a rapid translocation of these 



424 frem Gupta and D Mukherjee 

metabolites (figures 1-2). The hydrolysis of endosperm reserves by enzymes 
released from the aleurone layer and their absorption by cotyledons followed by 
the translocation to the growing axis has also been noted by Bewley and Black 
(1978). Moreover, unequal rate of protein breakdown and free amino acid forma- 
tion in relation to light and darkness will also influence the transport of amino 
acids to the growing axes. Further, many-fold increase in the free amino acids 
in the hypocotyl during seedling growth in dark (figures 1-2) may be due to restric- 
ted capacity of dark grown seedlings to convert all the amino acids into proteins 
as also noticed by Srivastava and Kooner (1972) in Phaseolus aureus L. Oota et al 
(1953) while studying the changes in the content of various primary metabolites 
in germinating Vigna sesquipedalis beans showed a decline of these metabolites in 
the cotyledons while the hypocotyls and roots recorded an increase during early 
germination for six days. It is also proposed that the growing axes of light grown 
seedlings are capable of amino acid biosynthesis by animation of carbon skeleton 
produced in photosynthesis and this process accounts for increase in amino acid 
levels (Bewley and Black 1978). There is a differential concentration of amino 
acids in the hypocotyls under the two situations (figures 1-2). 

Root samples in the light showed higher protein content in comparison to dark 
raised samples at all stages and a very marked increase was noticed in the total free 
amino acid pool at 48 hr stage. Further growth gave a sharp increase in the free 
amino acid pool in light whereas in dark the decline was more marked and could be 
observerd from the beginning (figure 2). 

Proline and methionine were not widely distributed in different seedling parts of 
C. tetragonolobus. Proline was recorded in cotyledons in both light and dark 
while endosperm and root contained the same at only few growth stages. Proline 
may be converted to glutamic acid thus increasing the pool size of this amino acid 
as reported by Bewley and Black (1978). Cysteic acid was not traced in endosperm 
and root at any growth stage of light and dark while cotyledon and hypocotyl 
recorded the same in very low concentrations at few growth stages. Methionine 
was also traced at few stages. y-Aminobutyric acid was found in higher amounts 
in cotyledons of C. tetragonolobus in comparison to other seedling parts although 
it was of widespread occurrence in different organs. Its higher amount had been 
reported earlier also (Altschul 1958). 

Changes in protease activity in various seedling parts in the light and darkness 
are shown in table 2. Dark-grown seedlings showed a significantly higher activity 
of this enzyme than those in light in all parts during growth. Initial stages of all 
seedling parts were unique in having maximum protease activity. The maximum 
decline in the activity was noticed in the endosperm and cotyledon which corre- 
lated with their protein depletion. 

a-Oxoglutaric acid, the predominant keto acid, was recorded only at few growth 
stages mostly in low levels in both light and dark seedling parts of C. tetragonolobus. 
In cotyledons of light grown seedlings, the dominant keto acids were phosphoenol- 
pyruvate and pyruvic acid (figure 3), the levels of which were maintained during 
seedling growth. Dark raised cotyledon samples had low values which declined 
further (figure 3). A characteristic feature of the keto acids was a rapid increase 
in their concentrations followed by later decline. Hypocotyls of light-raised seed- 



Changes in tetragonolobus Linn, during growth 425 

liags maintained higher levels of phosphoenolpyruvate, pyruvic acid, oxaloacetate 
and urea up to 72 hr stage followed by a decline and then a small increase, while 
in dark although above-mentioned keto acids could be detected in higher concentra- 
tions, levels of hydrazones recorded a gradual decline after 72 hr stage (figure 3). 
Higher levels of keto acids could also be found in roots. Low levels, in most 
of the samples of oxaloacetate, the keto analogue and precursor of aspartic acid 
like a-oxoglutaric acid, can be explained by their rapid utilization during seedling 
growth. The tendency for accumulation of oxaloacetate especially at 120 hr stage 
in endosperm, cotyledon and hypocotyl of dark-raised samples may suggest that 
this keto acid was rapidly utilized in the light-induced growth of seedlings. It has 
been suggested by Webb and Fowden (1955) that accumulation of keto acids is 
related to sluggish rate of protein synthesis thus sparing the utilization of keto 
acids for the synthesis of amino acids. 

Higher amounts of keto acid hydrazones of urea in light and dark in C. tetra- 
gonolobus account for its active role in nitrogen metabolism during seedling 
growth (figure 3). Asparagine and glutamine, the two common amides, which 
store excess ammonia to get rid of the toxic compound, recorded higher amounts 
from hypocotyl of dark grown seedlings in comparison to light while root samples 
from dark contained no detectable glutamine and asparagine content declined with 
seedling growth (figure 2). Cotyledons of 120 hr seedling stage were unique in 
exhibiting accumulation of asparagine and glutamine in dark in comparison to 
those in light thus sparing the utilization of these compounds in protein synthesis 
which is affected by dark. 



Acknowledgements 

We are grateful to Dr H S Choudhary of the Chemistry Department for help 
and many valuable suggestions during the protease assay. Thanks are also due to 
Prof. R S Mehrotra for laboratory facilities and to csm, New Delhi, for giving a 
SRF to one of us (Prcm Gupta) during the tenure of this work. 



References 

Afria B S and Mukhcrjee D 1980 Biochemical studies during seedling growth of sweet pea 

(Lathyrus odoratus L-) ; Proc. Indian Natl. Acad. Sci. 46 490-494 
Atria B S and Mukherjee D 1981 Metabolic studies in Sorghum vulgare Pers. and Zea mays L. 

during seedling growth ; Proc. Indian Acad. Sci. (plant scl) 90 71-78 

Altschul A M 1958 Processed plant protein food stuffs (New York : Academic Press Inc.) 

Bewley J D and Black M 1978 Physiology and biochemistry of seeds in relation to germination. 

I. Development, germination and growth (New York : Springei-Verlag, Berlin Heidelberg) 

pp. 193, 221 
Fowden L and Webb J A 1955 Evidence for the occurrence of y-methylene a-oxoglutaric acid 

in groundnut plants ; Biochem. J. 59 228-234 
Gupta P 1981 Ecophysiology of germination in certain plants ; Ph.D. Thesis, Kurukshetra Univ., 

Kurukshetra (India) 
Ihjaen, J L 1976 Lung proteases and protease inhibitors ; M.S. Thesis, University of Iowa, Iowa 

aty (USA) 
Kaushik D D 1966 Studies on certain aspects of plant metabolism ; Thesis for D.Phil. Degree 

of University of Allahabad (India) 



426 Prem Gupta and D Mukherjet* 

Krupka R M and Towers G H N 195Sa Studies on the keto acids of wheat. I. Behaviour during 

growth ; Can. J. Bot. 36 165-177 
Krupka R M and Towers G H N 1958b Studies on the keto acids of wheat- It. Glyaxylic acid 

and its relation to allantoin ; Can. J. Bot. 36 179-186 
Lo.wry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with 

the folin phenol reagent ; /. Biol. Chern. 193 265-275 
Muklierjee D 1972 Keto acid metabolism lit certain plants ; D.Phil. Thesis, University of 

Allahabad (India) 
Mukherjee D and Laloraya M M 1974 Metabolism of y-methyl-a-ketoglutaric acid, 7-methyleae- 

a-ketoglutaric acid and other keto acids during the seedling growth in Tamarindus indica ; 

Biochem. Physiol Pflanz. 166 429-436 
Mukherjee D and Laloraya M M 1979 Nitrogen and free amino acid changes during seedling 

growth in Banhinia purpurea ; /. Indian hot. Soc. 58 75-82 
Mukherjee D and Laloraya M M 1980 Changes in the levels of keto acids in different parts 

of seedlings of Bauhinia purpurea L. during growth ; Plant. Biochem. J. 7 120-125 
Oota Y, Fuzii R and Osawa S 1953 Changes in content of protein nitrogen of embryonic 

organs in Vigna sesquipedalis during germination ; /. Biochem. Tokyo 40 649 
Rai V K and Laloraya MM 1967 Correlative studies an plant growth and metabolism. 

II. Effect of light and gibberellic acid on the changes in protein and soluble nitrogen 

in lettuce seedlings ; Plant Physiol. 42 440-444 
Srivastava A K and Kooner N K 1972 Physio-logical and biochemical studies in seed germination 

of moong (Phaseolus aureus Roxb.) ; Indian J. Exp. Biol. 10 304-306 
Steward F C, Wetmore R H, Thompson J F and Nitsch J P 1954 A quantitative chromato- 

graphic study of nitrogenous components of shoot apices ; Am. J. Bot. 41 123-134 
Towers G H N and Steward F C 1954 The keto acids of Tulip (Tulipa gesneriana) with special 

reference to the keto analog of y-methylene glutamic acid ; /. Am. Chcm. Soc. 76 1959- 

1961 
Webb J A and Fowden L 1955 Changes in oxo-acid concentrations during the growth of 

groundnut seedlings ; Biochem. J. 61 1-4 
Yomo Harugo-ro and Varner J E 1973 Control of the formation of amylases and proteases in 

the cotyledons of germinating peas ; Plant Physiol 51 708-713 



Proc. Indian Acad. Sci. (Plant Sd,) 3 Vl* 91, Number 5, October 1982, pp. 427-431. 
Printed in India. 



Effect of ridge gourd pollen on zoospore germination of 
Pseudoperonospora cubemis and its significance in epidemiology 



AMARNATHA SHETTY, H S SHETTY and K M SAFEEULLA 
Downy Mildew Research Laboratory, University of Mysore, Mysore 570006, India 

MS received 29 October 1981 ; revised 3 June 1982 

Abstract. Ridge gourd pollen has a stimulatory effect on the germination of 
Pseudoperonospora cubensis. The rate and percentage germination of zoospores 
increased in the presence of pollen leachates. Spraying of leaves with a mixture 
of pollen and sporangial suspension enhanced the development cf lesions. Early 
germination of zoospores in the presence of pollen proved advantageous for infec- 
tion as it provided a prolonged favourable infection period. The results are 
discussed in relation to the epiphytotics o.f the disease during flowering period. 

Keywords. Pseudoperonospora cubensis ; ridge gourd; pollen effect ; zoospore 
germination ; epidemiology. 



1. Introduction 

Pseudoperonospora cubensis (Berk, and Curt.) Rostow, the incitant of cucurbit 
downy mildew is one of the serious and production limiting diseases of Luffa 
acittangula Roxb. in India. The disease attains serious proportions when the 
plants start flowering and as a result the susceptible varieties of plants fail to 
produce fruits. Bains and Jhooty (1975) reported that in Cantaloupes downy 
mildew appeared during 1972-74, under field conditions only during the flowering 
and fruiting stage. The effect of host pollen on stimulation of spore germination 
of fungal pathogens has been worked out in different crops (Chou and Preece 
1968 ; Fokkema 1976 ; Preece 1976 ; Meenakshi and Ramalingam 1979 ; 
Suryanarayana and Ramalingam 1979). So far no report on the effect of host 
pollen on germination of downy mildew pathogens has been made. 



2. Materials and methods 

2.1. In vitro effect of pollen on zoospore germination 

One of the susceptible varieties of ridge gourd (Pusa Nasdar) was .grown in the 
downy mildew experimental plots to obtain the sporangial inoculum and the host 



428 Arnaniatha Shetty, H S Shetty and K M Safeeulla 

pollen. Sporangial suspension was prepared by the following method : downy mildew 
infected leaves were collected at 6 p.m. and the remnants of the downy growth 
was washed off with moist cotton under running tap water. The leaves were air 
dried and small bits of leaves with lesions were cut and placed inside petri plates 
containing a wet blotter with the adaxial surface of the leaf in contact with the 
blotter. A good crop of sporangia was obtained after incubating the leaf bits 
for 12 hrs at 22 C in dark. The sporangia were scraped with a blade into a dish 
containing distilled water. The concentration of sporangial suspension was 
measured using a haemocytometer and was adjusted to about 10,000/ml. Host 
pollen was collected and stored at 5 C. Five mg of the pollen was mixed in 
10ml of the sporangial suspension. A suspension of the mixture was placed on 
glass slides and incubated in moist chambers at room temperature (22-26 C). 
In controls no pollen was added. Observations for zoospore germination were 
made under binocular microscope and results were recorded at hourly intervals 
after fourth hour. 

2.2. Effect of pollen on infection of host leaves and lesion development 

Pollen plus sporangial suspension was sprayed on the lower surface of the leaf 
of 20-30 day old plants and retained inside a glass house. The leaves were covered 
with moist polythene bags for about 24 hrs and observations for number and size 
of lesions were made. The effect of pollen in reducing the infection threshold was 
tested for moisture requirement by covering the leaves with polythene bags for 
3, 4, 5 and 6 hrs. After removing the polythene bags the leaf was air dried and 
left inside the glass house. 

2.3. Disease development in the field in relation to age of the crop 

Two varieties of ridge gourd viz. Pusa Nasdar, a highly susceptible variety and 
long variety which is moderately resistant ware sown in the month of August 
1980 in plots. Disease rating was made at weekly intervals using a to 5 scale 
as described by Thomas (1977). Fertiliser (NPK 17 : 17 : 17) was applied twice 
at the age of 20 days and 45 days. 



3. Observations 

3.1. In vitro effect of pollen on zoospore germination 

Sporangial suspension when incubated at room temperature (22-26 C) released 
zoospores within H 2 hrs. Maximum number of zoospores were observed after 
2 hrs of incubation. Zoospores remained active in water for 90 to 120 min and 
then encysted. The data with regard to percentage of zoospore germination and 
germ tube length with and without pollen are recorded in figures. 1 and 2 respec- 
tively. Zoospore germination started an hour earlier in the presence of pollen. 
The percentage of zoospore germination and vigour of the germ tubes in the 
presence of pollen was greater compared to the zoospore which germinated in the 
absence of pollen. 



Pseudoperonospora cubensis and its significance 



429 



80- 



' - 



40- 



D Treatment 
E3 Control 




678 
Time (hr) 



10 



Figure 1. Effect of host pollen on the germination of zoospores of P. cubensis. 



50 



Z 30 
o 



10 



Treatment 
Control 




20 



60 

Length (yxm) 



100 



140 



Figure 2. Comparison of the germ tube length of zoospores in presence of pollen 
and in distilled water and their relative frequency of occurrence. 



3.2. Effect of pollen on infection of host leaves and lesion development 

Difference in disease reaction was apparent in plants sprayed with the mixture 
and the sporangial suspension alone. The number and size of lesions were more 
on leaves sprayed with the pollen mixture. Leaves inoculated with sporangial 
suspension required a minimum of four hrs of leaf wetness for successful infec- 
tion under glass house conditions. Only 3 hrs of leaf wetness was needed for 
infection in the presence of pollen under the same conditions of temperature 
and inoculum concentration. 

3.3. Disease development in the field in relation to age of the crop 

Downy mildew of ridge gourd makes its appearance at the seedling stage. The 
young true leaves are resistant to downy mildew. When the seedlings attain the 



430 



Amamatha Shetty, H S Shetty and K U Safeeiilla 



age of 20 days (3-5 true leaf stage) symptoms appear on the true leaves as 
greenish to yellow lesions. Disease appearance in " Long ' is delayed by a week 
in comparison with Pusa Nasdar. Severity of disease in relation to age is plotted 
in figure 3. Flowering in both the varieties start 35-40 days after planting. Till 
then disease severity is less than stage 3 of the 0-5 scale. Soon after flowering 
the disease reaches severe proportions and it ultimately results in the death of 
vines in c Pusa Nasdar 9 whereas in c Long ' it is not very severe and the 
vines continue to grow but the yield is significantly reduced. 



4. Discussion 

In saprophytic fungi and facultative pathogens the stimulating effect of pollen on 
germination of conidia is attributed to carbohydrates (Suryanarayana and Rama- 
lingam 1979 ; Fokkema 1976) but the aggressiveness of such fungi depends on the 
pollen leachates, rather than the nutrients (Chou and Preece 1968). In P. cubensis 
the zoospores germinate in distilled water thereby showing that it is not nutrient 
dependent. Hence it is quite probable that pollen leachates provide a stimulatory 
effect on zoospore germination. 

Preece (1976) stated that the effect of 'pollen on leaf infection may be due to 
(a) increase in the speed and rate of spore germination, (b) restoring the germina- 
bility and infectivity of old spores and (c) reducing the infection threshold by 
enhancing the speed and virulence of the pathogen. From the present study, it 
is evident that there is an increase in the number of zoospores germinating and the 
vigour of germination is enhanced. Under field conditions, sporangial liberation 
occurs during morning hoars (Cohen and Rotem 1971 ; Thomas 1977 ; Bains 
and Jhooty 1978). It starts at around 6 ajn. and reaches a peak at 8 a.m. For the 
successful infection of a fresh host leaf a minimum of 4 hrs of leaf wetness is 
needed. But when the zoospore germinates in presence of host pollen it needs 
only a period of 3 hrs for infection. 



cu 

I 



P 3 



o 

O) 



5 



\/eqetatlve phase 



Reproductive phase 



Pusonasdar 
Long var 




20 40 60 

Age of plant (days) 



80 



100' 



Figure 3. Disease progress in two varieties of ridge gourd in relation to a$e of 
the plant, " -....- 



Pseudoperonospora cubensis and its significance 43 i 

The sporangia liberated during morning hrs are subjected to a period of 
unfavourable conditions which last until the next dew fall and infection of host 
plant occurs during night hrs (Cohen and Rotem 1971 ; Cohen and Eyal 1980). 
From our experiments under Mysore conditions, it is quite probable that those 
sporangia liberated early in the morning get deposited on the leaves and they 
start germinating by the production of zoospores. As dew persists at Mysore 
condition till 9*30 to 10 a.m., infection of the leaf tissue in presence of host pollen 
can occur successfully during the daytime. In addition, those sporangia which 
are deposited in later hours of the day, i.e., those sporangia which fail to infect 
during the morning hours due to the advent of unfavourable period for infection, 
survive till the next dew fall with a considerable loss in viability of sufficient 
number of sporangia. Those viable sporangia germinate and cause infection 
during night hours. As a result, severity of the disease increases significantly 
during flowering period. This is supported by the results of studies of lesion 
development and zoospore infection under different leaf wetness periods. 



Acknowledgements 

The senior author is grateful to the Council of Scientific and Industrial Research, 
New Delhi, for the award of a Junior Research Fellowship. 



References 

Bains S S and Jhoofcy J S 1975 Studies on the epidemiology of downy mildew of muskjnelon 

caused by Pseudoperonospora cubensis (abstract) ; Indian J. of Mycol. and PL PathoL 5 

46-47 
Bains S S and Jhoaty J S 1978 Epidemiological studies on downy mildew of nmskmelon 

caused by Pseudoperonospora cubensis ; Indian PhytopathoL 31 42-46 
Chou M and Preece T F 1968 The effect of pollen grains on infection caused by Botrytis 

cineria ; Ann. Appl. BhL 62 11-22 
Cohen y and Rotem J 1971 Dispersal and viability of sporangia of Pseudoperonospora cubensis ; 

Trans. Br. Mycol. Soc. 57 67-74 
Cohen. Y and Eyal H 1980 Effects of light during infection on the incidence of downy mildew 

(Pseudoperonospora cubensis) on cucumbers ; Physiol. PL PathoL 17 53-62 
FokJkema N J 1976 Antagonism between fungal saprophytes and pathogens on aerial plant 

surfaces in Microbiology of aerial plant surfaces (ed.) C H Dickinson and T F Preece 

(London : Academic Press) pp. 487-506 
Meenakshi M S and Ramalingam A 1979 The effect of sorghum pollen on the germination 

of Drechslem turdca (Pass.) Subram. and Jain ; Curr. Sci. 48 447-448 
Preece T F 1976 Some observations of leaf surfaces duiing the early stages of infection by fungi 

in biochemical aspects of plant parasite relationships, (ed.) J Friend and D R Threlfall 

(London : Academic. Press) pp. 1-10 
Suryanarayana K and Ramalingam A 1979 Influence of pollen on the germination of conidia 

of Drechslera turdca (Pass.) Subram. and Jain ; Curr. Sci. 48 1045-1047 
Thomas C E 1977 Influence of dew on the downy mildew of cantaloupes i n South Texas ; 

Phytopathology 67 1368-1369 



Proc. Indian AcadL Sci. (Plant Sci.), Vol. 91, Number 5, October 1982, pp. 433-441. 
Printed in India. 



Leaf proteiuase and nitrate reductase activities in relation to grain 
protein levels and grain yield in four species of grain amaranth 



K RAMAMURTHY NAIDU, Y SEETHAMBARAM and 
V S RAMA DAS* 

School of Life Sciences, University of Hyderabad, Hyderabad 500 134, India 
* Department of Botany, Sr-i Venkateswara University, Tirupati 517502, India 

MS received 24 November 1981 ; revised 19 August 1982 

Abstract. The relationship of leaf nitrate reductase (NR) and proteinase acti- 
vities to the grain protein level and grain yield was investigated in four species of 
grain amaranth (Amaranthus hypochondriacus, A. caudatus, A. cruentus and A. edulis). 
A strikingly positive correlation between the leaf proteinase activity and the grain 
protein content was found. A. edulis with higher gr?in protein level possessed 
high leaf proteinase activity, while A. hypochondriacus, with relatively lower grain 
protein content had lower leaf proteinase levels. Although there was no definite 
correlation between the leaf proteinase levels and the grain yield, the integrated 
leaf NR activity was positively correlated with the grain yield. The total nitrogen 
content per plant seems to be dependent on the extent of rcot growth and the 
levels of NR activity in leaves. 

Keywords. Grian amaranth ; leaf proteinase activity ; leaf nitrate reductase acti- 
vity ; grain protein ; grain yield. 



1. Introduction 

Grain amaranth is presently one of the under-exploited crop plants with a consi- 
derable economic potential. The grain is regarded to be unique for its high 
protein content. It is also known to be rich in lysine and sulfur containing amino 
acids and can therefore be considered superior to the proteins of wheat, corn and 
rice (Senfit 1980). 

Significant correlations were found in the past between the integrated leaf 
nitrate reductase (NR) activity and grain yield, reduced nitrogen levels of grain 
and of whole plant in the case of wheat and maize (Abrol and Nair 1978 ; 
Brunetti and Hageman 1976 ; Hageman 1979). On the other hand Dalling et al 
(1975) found that wheat cultivars with similar levels of NR activity could accumulate 
variable amounts of reduced nitrogen. Deckard et al (1973) identified one maize 
genotype with relatively low NR activity but a high capacity to accumulate reduced 

; 433 

P.(B)-8 



434 K Ramamurthy Naidu, Y Seethambaram and V S Rama >as 

nitrogen. However, several studies have indicated that NR assays could be a 
useful predictive selection criterion for grain yield and grain protein levels (Croy 
and Hageman 1970 ; Fakorede and Mock 1978). Not much work was done on 
the role of proteinases of leaf in relation to the protein content on grain. That 
leaf proteinase activities were correlated with grain nitrogen was evident in wheat, 
rice and maize (Bailing et al 1976 ; Perez et al 1973 ; Reed et al 1980). 

In view of meagre work on grain amaranths in general and its nitrogen meta- 
bolism in particular, the present study was carried out, leading to an under- 
standing of the relationship between the leaf proteinase activity, grain protein, 
grain yield and secondly between the leaf NR activity, root growth and the accu- 
mulation of reduced nitrogen in four species of grain amaranth. 



2. Materials and methods 

Seeds of Amaranthus hypochondriacus, L., Arnaranthus caudatus L., Amaranthus 
cruentus and Amaranthus edulis L. were obtained from National Botanical 
Research Institute, Lucknow. Plants were grown in 30cm diameter earthenware 
pots on soil supplemented with manure (3 parts of red soil 4- 1 part of farm 
yard manure) under natural (approximately 12 hr) photoperiod (temperature 
about 35 C day and 22 C night). Three plants were retained in each pot. 

The plants were harvested at three different stages, viz., vegetative stage (30 days 
after sowing), flowering stage (45 days after sowing) and grain filling stage (60 
days after sowing). At each stage, plants from 3 pots were collected and were 
subdivided into leaf, stem, root and panicle. Fresh and dry weights of these 
plant parts were determined and the shoot/root ratio was calculated. 

Reduced nitrogen content in the dried samples was determined by kjeldahl method 
using Tecator digestion and distilling systems (Tecator Manual). After anthesis 
(90 days after sowing) plants were finally harvested to the ground level and the 
dry weights of the panicle and stover were determined. Grain protein content 
was calculated by multiplying the grain nitrogen by a factor of 6*25. 

2.1. Nitrate reductase (NR) assay 

Leaf NR activity was measured at the vegetative, flowering and grain filling stages. 
Fully expanded young leaves from each pot were collected (at 10 a.m.) into a poly- 
ethylene bag placed on an ice bath and were carried into the laboratory. The 
leaves were deribbed, weighed and were then chopped into pieces. The in vitro 
NR assay was as described by Hageman and Hucklesby (1971). 

2.2. Proteinase activity 

The proteinase activity was measured thrice at 15 day intervals after anthesis 
(60, 75 and 90 days after sowing). The proteinase activity in the middle leaf of 
the plant was assayed by the modified procedure of Peoples and Dalling (1978). 
Leaves were homogenised by grinding in a mortar and with a pestle for 90 sec 
with 5 ml/g extracting medium containing 23 mM sodium citrate; 155mM 



Leaf proteinase and nitrate reductase activities 



435 



sodium phosphate ; 5mM L-cysteine; 5mM EDTA and 1% PVP, pH 6-8. After 
straining through cheese cloth the homogenate was centrifuged at 25000 g for 
10 min. The supernatant was dialysed at 4 C for 48 hr against 50 mM potassium 
phosphate buffer, pH 7*0. 

1% Bovine serum albumin solution was prepared in 0-05M Tris-HCl 
(pH 7 -8). 0- 1ml of extract was incubated with 0"5ml of substrate for 2 hrs 
at 37 C. The reaction was terminated by adding 0'7 ml of 15% trichloroacetic 
acid and the soluble nitrogen in the mixture was determined by ninhydrin 
(Spices 1957). Leucine was used as the amino acid standard. 



3. Results 

3.1. Shoot dry weight 

There was a wide variation in the dry weight of shoot at the vegetative stage 
between the four species of grain amaranth studied (table 1). Maximum dry 
matter production during the vegetative stage was in A. hypochondriacus followed 
by A. edulis, A. caudatus and A. cruentus. Though a similar trend was observed 
at flowering stage, the variation in dry matter content was not significant suggesting 
that the growth rate during different stages of growth period varied among four 
species (table 2). The higher dry matter accumulation noticed in A. caudatus 
than that in A. hypochondriacus during filling stage may be due to the faster 
growth rate in A. caudatus from flowering stage (table 3). The greater dry matter 
accumulation in shoots of A. hypochondriacus (table 4) than the other three 
species, might be due to the bigger panicles in the former species. 



Table I. Dry weights and leaf reduced nitrogen during vegetative stage (30 days 
after sowing) of four species of grain amaranth. 



Species 


Total dry wt/plant 


- S/R 


Leaf Reduced N 
reduced of stover 










Shoot 


Root 


Total 




N% 


g/plant 




8i 


g 


g 








A. hypochondriacus L. 


9-0 


0-80 


9*80 


11-25 


3-85 


0-2S 




0-4 


o-i 


0-45 


0-25 


0-12 


0-02 


A. caudatus L. 


3-5 


0-25 


3-75 


14-00 


4-27 


0-13 




0-2 


0-05 


0-26 


0-36 


0-15 


0-02 


A. cruentus L. 


3-1 


0-40 


3*5 


7-75 


4-13 


0-11 




0-3 


0-04 


0-32 


0-15 


0-08 


o-oi 


A. edulis L. 


5-8 


0-45 


6-25 


12-89 


3-99 


0-19 




i 0-4 


0-03 


0-42 


0-28 


0*07 


0-03 



436 K Ramamurthy Naidu, Y Seethambamm and V S Rama Das 

Table 2. Dry weights and leaf reduced nitrogen during flowering (45 days after 
sowing) of four species of grain amaranth. 



Total dry wt/plant 


Leaf Reduced N 
reduced of stover 
N% g/plant 




kjpcwaca 

Shoot 


Root 


Total 


kJ/iV 




g 


g 


g 








A. 


hypochondriacus L. 44*0 


8-2 


52-2 


5*36 


3-92 ' 


1-27 




1-8 


0-5 


2-1 


0-25 


0-06 


0-07 


A. 


caudatus L. 41 '5 


9-5 


51-0 


4'37 


4-47 


1-39 




2-4 


0-7 


2-0 


i 0-28 


0-08 


0-06 


A. 


emeritus L. 35*5 


8-0 


43-5 


4'44 


3-71 


0-09 




1-6 


0-3 


1-8 


0-16 


0-04 


0'05 


A. 


eduKsL. 37-5 


7-5 


45-0 


5-00 


3'64 


1-02 




1*5 


0-2 


1'4 


0-32 


0-05 


0-05 


Table 3. Dry weights and leaf reduced 


nitrogen 


during grain 


filling (60 


days after 




sowing) of four species 


of grain amaranth. 


Total dry wt/plant 


Leaf 


Reduced N 










- S/R 


reduced of stover 










Shoot 


Root 


Total 




N% 


g/plant 




g 


g 


g 








A. 


hypochondriacus L. 82 '0 


12-0 


94-0 


6-80 


3-50 


2-86 




4-2 


0-8 


4-5 


0-4 


0-06 


0-21 


A. 


caudatus L. 87-0 


15-0 


102-0 


5-80 


3-57 


3'29 




3'6 


1-2 


4-8 


0'26 


0*04 


0-18 


A. 


emeritus L. 66*5 


12-0 


78-5 


5-54 


3-44 


2-41 




3-8 


1-4 


4-2 


0-32 


0-07 


0-18 


A. 


edulisl*. 69*5 


11-5 


81-0 


6-04 


2-94 


2-65 




' 2-3 


0-7 


3-1 


0-22 


0-08 


d= 0-14 



3.2. Shootf Root (SfR) ratio 

The S/R ratio in all the four species was maximum at vegetative stage and decreased 
gradually during flowering and grain filling stages (tables 1, 2 and 3). A. caudatus 
had the highest S/R ratio among the four species during vegetative phase of 
growth, but the ratio decreased during flowering and grain filling stages due to 
in rpot dry weight at later stages of growth* .... 



Leaf proteinase and nitrate reductase activities 



437 



Table 4. Mean NR activity (calculated from figure i) 9 dry matter at final harvest 
(90 days after sawing) grain yield, grain protein and mean proteinase activity 
(calculated from figure 2) of four species of grain amaranth. 



Species 



NR 
activity 



Dry matter 



Grain 



# mol NOJ Panicle Stover Total g/plant 
g/plant 



Grain Proteinase Total N 
protein activity g/plant 



A. hypochon- 
driacus L. 



26'2 75-0 51'0 126'0 23'6 12*5 21*6 2-42 

1-4 2-5 1-0 3-4 1-8 0-4 0*8 0-12 



A. caudatus L. 23'43 45-0 60" 

0-8 2-2 1-8 



105-0 16-8 14-0 31-5 2'96 

4-0 0-7 0*3 0-6 0-18 



A. cruentus L. 20*07 44*0 58'0 102*0 15'6 12'6 28-4 2-15 

1-2 1-9 2-1 3-8 0-6 0-2 0-7 0-11 

A. edulis L. 24' 17 69*0 46'0 115'0 21*8 15*0 35*0 224 

0-9 1-7 1-4 3-1 0-7 0-3 1-2 0-09 



3.3, Levels of leaf reduced nitrogen (%) and grain protein (%) 

Variation in the concentration of leaf reduced nitrogen was observed among the 
four species of grain amaranth. Maximum protein content was in grain of 
A. edulis while the maximum content of leaf reduced nitrogen was recorded in 
A. edulis (table 4). 

3.4. NR activity and grain yield 

The greatest NR activity was at the flowering stage of all the four species of grain 
amaranth and the activity decreased at grain filling stage (figure 1). The level 
of NR in A. hypochondriacus was more than that in the other three species during 
vegetative and flowering stages whereas, at grain filling stage, A. caudatus had 
the greatest NR activity. There was a positive correlation between the mean NR 
activity the three stages and grain yield (r = + 0-89) in all the four species of 
grain amaranth. 



3.5. Proteinase activity 

The results of the mean proteinase activity calculated from figure 2 are given in 
table 4. The level of leaf proteinase activity was in the decreasing order of 
A. edulis followed by A. caudatus ; A. cruentus and A. hypochondriacus. The 
leaf proteinase Activity was positively correlate^ with the percentage of proteiu 



438 



K Ramamurthy Naidu, Y Seethambaram and V S Rama Das 



20 




o A.hypochondriacus 

A. caudatus 

A A.cruentus 

A A- edulis 



Vegetative Flowering Grain fillinq 

Figure 1. The leaf in vitro nitrate reductase activity of four species of grain 
amaranth during different stages of growth. 



60 



50 



40 



30 



o 



20 



10 



O A. hypochondriacs 

A. caudatus 

A A. cruentus 

A A. edulis 




15 30 

Days after anthesis 



45 



Figure 2. The leaf proteinase activity of four species of grain amaranth. 



in grain (r = + 0'85) in all the four species. Leaf proteinase activity increased 
during grain development (figure 2). 



4. Discussion 

The total reduced N per plant was positively correlated with leaf NR activity 
(r= +0-86) during the vegetative phase. The leaf NR activity was positively 
correlated with the total reduced N per plant (r = +0-86) but not with leaf 
reduced nitrogen (r = + 0'19). There was also significant correlation between 
root growth and the total reduced N per plant (r = + 0'87) and NR activity 
(r = + 0-65). These data in4ic^ tfeat total reduced nitrogen per plant 



Leaf proteinase and nitrate reductase activities 439 

influenced by NR activity as well as by the root growth. Therefore the higher NR 
activity and root growth in A. hypochondriacus and A. edulis than those in 
A. caudatus and A. cruentus resulted in more total reduced nitrogen per plant 
in the former two species. 

During the flowering stage also no positive correlations could be traced between 
NR activity and leaf reduced nitrogen or (r = + 0*16) or total reduced nitrogen 
per plant (r = +0*42) (table 2). A. caudatus had high N levels in leaf and in 
total plant during flowering stage, but its NR activity was less than that of the 
other three species. Reed and Hageman (1980) observed that accumulation of 
reduced nitrogen was dependent more on nitrate uptake than on the relative 
levels of NR activity per plant. Raper et al (1977a, b) suggested that 
nitrogen uptake was positively related to the rate of root growth in tobacco, 
cotton and soybean. The remarkable increase in root growth of A. caudatus 
during the flowering stage might have resulted in its comparatively high reduced 
nitrogen per plant (table 2). 

A. caudatus retained more reduced nitrogen per plant during grain filling 
stage. Again the NR activity was not related with either leaf reduced nitrogen 
(r = +0-14) or total reduced nitrogen per plant (r = -f 0'34) in all the four 
species (table 3). The high root growth of A. caudatus could have facilitated the 
accumulation of more reduced nitrogen per plant either through mobilisation of 
reduced N or by augmenting the uptake of nitrate and efflux. While there was 
always a positive correlation between the root growth and total reduced nitrogen 
per plant (r = -f 0*78), the NR activity was not correlated with the total reduced 
nitrogen per plant even at the harvest (table 4). Thus, our observations confirm 
that at any stage of growth period in the grain amaranths studied, the leaf NR 
activity alone is not an index of total reduced nitrogen per plant but the extent 
of root growth along with the NR levels would together influence the nitrogen 
content per plant. 

The average nitrate reductase activity in the leaf was positively correlated with 
the grain yield (r = +0-89) as well as total dry matter accumulation at harvest 
(r = 0'86) in all the four species of grain amaranth (table 4). These findings 
confirm the observations of earlier workers where NR activity was correlated with 
grain yield (Blackwood and Hallam 1979 ; Deckard et al 1977 ; Bailing and 
Loyn 1977 ; Johnson et al 1976). 

On the other hand, the protein levels in the grain were not related with the NR 
activities (r = 0*37), but were strongly correlated with proteinase activity 
(r = -f 0*85) in all the four species (table 4). This agrees with the results of 
Deckard et al (1977) who concluded that the NR activities were not correlated 
with grain protein. Differences in nitrogen translocation efficiency could reduce 
the correlation between NR activity and grain protein (Croy and Hageman 1970 ; 
Eilrich and Hageman 1973). Leaf proteinase activities were related more closely 
to accumulation of grain nitrogen than leaf NR activity (Reed et al 1980). In 
the present study, A. edulis contained greater proteolytic activity in the leaf and 
accumulated more protein in its grain than that in A. hypochondriacus in spite of 
high NR activity in the latter species. 

Although the number of observations are limited, the present investigation 
nevertheless suggests that in grain amaranths the level of leaf proteinase activity 



446 K Ramamurthy Naidu, Y Seethambaram and V S Rama Das 

is an important determinant of the grain protein content whereas the leaf NR 
activity and root growth modulate the total reduced nitrogen per plant. 

Acknowledgements 

K Ramamurthy Naidu acknowledges the award of Teacher Fellowship from 
the University Grants Commission, New Delhi, and Y Seethambaram acknowledges 
AP Agricultural University, Hyderabad, for providing study leave facility. 
The authors thank Dr A S Raghavendra for reading the manuscript. 



References 

Abrol Y P and Nair T V R 1973 In Proceedings of National Symposium on Nitrogen assimila- 
tion and crop productivity Hissar, India ; (eds.) S P Sen, Y P Abrol and S K Sinna 

(New Delhi: Assoc. Publishing Co.) Pp. 113 
Blackwood G C and Hallam R 1979 Nitrate reductase activity in wheat (Triticum aestivum L.) 

II. The correlation with yield ; New PhytoL 82 417-425 
Bmnetti N and Hageman R H 1976 Comparison of in vivo and in vitro assays of nitrate 

reduction in wheat (Triticum aestivum) seedlings ; PL Physiol. 5 583-587 
Croy L I and Hageman R H 1970 Relationship of nitrate reductase activity to grain protein 

production in wheat ; Crop. $ci. 10 280-286 
Dalling M J, Halloran G M and Wilson J H 1975 The relationship between nitrate reductase 

activity and grain nitrogen productivity in wheat ; Aust. J. Agri. Res. 26 1-10 
Dalling M J, Balland G and Wilson J H 1976 Relation between acid protejnase activity and 

redistribution of nitrogen during grain development in wheat; Aust. J. PL Physiol. 3 721- 

730 
Dalling M J and Loyn R H 1977 Levels of activity of nitrate redutcase at the seedling stage as a 

predictor of grain yield in wheat (Triticum aestivum L.) ; Aust. J. Agri. Res. 28 1-4 
Deckard E L, Lambert R J and Hageman R H 1973 Nitrate reductase activity in corn leaves 

as related to >ields of grain and grain protein : Crop. Sci. 13 343-350 
Deckard E L, Lucken K A, Joppa L R and Hammond J J 1977 Nitrate reductase activity, 

nitrogen distribution, grain yield and giain protein cf Tall and Semidwarf. Near-isogenic 

lines of Triticum aestivum and T. turgidum ; Crop. Sci. 17 293-296 

Eilrich G L and Hageman R H 1973 Nitrate reductase activity and its relationship to accumu- 
lation of vegetative and grain nitrogen in wheat (Triticum aestivum L.) ; Crop. Sci. 13 

59-66 
Fakorede M A B and Mock J J 1978 Nitrate reductase activity and grain yield of maize 

culiivar hybrids; Crop. Sci. 18680-682 
Hageman R H and Hucklesby D P 1971 Nitrate icductase. In Methods in enzymology (ed.) 

A San Pietro, Vol. XXIII, Part A (New York: Academic Press) pp. 491-503. 
Hageman R H 1979 Integration of nitrogen assimilation in relation to yield. ID Nitrogen 

assimilation of plants (eds) E J Hewitt, and C V Cutting (New York: Academic Press) 

pp. 591-611 
Jhonson C B, Wittingtan W T and Blackwood G C 1976 Nitrate reductase as a possible 

predictive test of crop yield ; Nature (London) 262 132-134 
Peoples M B and Dalling M J 1978 Degradation of ribulose 1, 5-aiphosphate carboxylase by 

proteolytic enzymes from crude extracts of wheat leaves ; Planta 138 153-160 
Perez C M, Cagampang G B, Esmana B V, Monserrate R U and Julliano B O 1973 Protein 

metabolism in leaves and developing grains of rices differing in grain protein content ; 

PL Physiol. 51 537-542 
Raper CD, Patterson D T, Parsons L R and Kramer P J 1977a Relative growth in nutrient 

accumulation rates for tobacco ; Plant Soil. 46 743-787 



Leaf proteinase and nitrate reductase activities 441 

Raper C D, Patterson D T, Parsons L R and Kramer P J I977b Relationship between growth 
and nitrogen accumulation for vegetative cotton and soybean plants ; Bet. Gaz. 138 
129-149 

Reed A J and Hageman R H 1980 Relationship between nitrate uptake, flux and reduction 
and the accumulation of reduced nitrogen in maize (Zea mays L.). II. Effect of nutrient 
nitrate concentration; PL Physiol. pp. 1184-1189 

Reed A J, Below F E and Hageman R H 1980 Grain protein accumulation and the relation- 
ship between leaf nitrate reductase and protease activities during grain development of 
maize (Zea mays L.) I. Variation between genotypes ; PL Physiol. 66 164-170 

Senfit P J 1980 Protein quality of amaranth grain. Proc. 2nd Amaranth Conference ; Rod ale 
Press Inc. 

Spices J R 1957 Colorimetric procedures for amino acids ; Methods in EnzymoL 3 467-477 



Proc. Indian Acatf. Sci. (Plant Sci.), Vol. 91 , Number 5, October 1982, pp. 443-447, 
Printed in India. 



Cell division in Staumstrum gradle Ralfs. under the scanning 
electron microscope 

VIDYAVATI 

Botany Department, Kakatiya University, Warangal 506 009, India 

MS received 3 December 1981 ; revised 14 August 1982 

Abstract. Staurastmm gradle Ralfs. was grown in Chu's No. 10 culture medium, 
in a culture cabinet at 18-20 C with 16 hrs light and 8 hrs dark period. The 
cells exhibited polymorphism. The cells were fixed and their division and growth 
was examined under the scanning electron microscope. 

Keywords. Desmids ; Staumstrum gracile Ralfs. 



1, Introduction 

The process of cell division is unique in desmid biology, with special reference 
to placoderms, and differs from the other algal groups. Some of the problems 
it solved, may help in providing a better understanding of some of the principles 
of morphogenesis and the control of the shape of cells in general. With each 
division of these, often elaborately-shaped desmids two daughter semicells are 
produced, which then acquire the typical complex and symmetrical shape of the 
parent semicells. 

Having studied the morphological features, surface ornamentation and 
polymorphism under the scanning electron microscope (Vidyavati 1981), it was 
thought desirable to study the division of the cells also. 

Previous work on cell division, under TEM and SEM, were mainly contributed 
by Dodge (1963), Drawert and Kalden (1967), Drawert and Mix (1961), Pickett- 
Heaps and Fowke (1969, 1970), Pickett-Heaps (1973, 1974, 1975), Schiille (1975) 
and Brook (1981). 

2. Material and methods 

Staurastmm gracile Ralfs. 679/3 was obtained from the culture collection of 
Algae and Protozoa, Cambridge, U K. The work was carried out at the Botany 
Department, Royal Holloway College, University of London, U K. 

From the cultures thus obtained, unialgal isolations were made following 
Pringsheim's method and these cultures were maintained in Chu's (1942) 10 
medium, at 1 8-20 C temperature, subjected to alternate light and dark conditions 
for 16 and 8 hrs, respectively, 

443 
P.(B)-4Q 



444 Vidyavati 

Fixation from healthy cultures in exponential growth were made at hourly 
intervals in order to study the cells at various stages of division and follow the 
change in shape of the new semicells. The cells were fixed in 1% glutaraldehyde, 
made up in the culture medium (Chu's 10) for about 1 hr at room temperature, 
after washing in culture medium, they were then post-fixed for about 1 hr in 1% 
Osmium tetroxide also made up in the culture medium. They were then washed 
3 times in culture medium. The cells were dehydrated in acetone of 30%, 50%, 
70%, 90% and 100 %. Fixation, washing and dehydration were all carried out 
in the centrifuge tubes and each time the cells were centrifuged discarding the 
supernatant. The cells were then passed through critical point drying procedure. 
The dried specimens were moved from the CPD apparatus and were mounted on 
specimen stubs, using transfer on double-sided sticky tape. These were then 
coated quite heavily with carbon and gold. Specimens were examined at 15 KV 
in a jeel-JSM-25 S scanning electron microscope. 



3. Observations and results 

Staurastrum gracile Ralfs. is known for its polymorphic -form, cells are variable, 
medium in size (length 27-60 ji ; breadth, including processes, 44-1 10 /* ; breadth 
of isthmus 5'5-13/f), constriction slight, usually an acute notch; .semicells 
variable, upper angles produced to from long, slender processes of variable lengtl 
each with ,3 or 4 minute spines and provided with denticulations. The vertica 
view is usually triangular, sometimes quadrangular, angles are produced to font 
long processes, chloroplasts are axile with a central pyrenoid in each semiceli 
(West and West 1923). 

For many placoderm desmids cell division seems to be the only means of repro 
duction. Sexual reproduction is rarely observed in nature or under laboratory 
conditions. During division, the cell symmetry is completely destroyed by 2 
wall that grows around the narrow isthmus joining semicells. During the process 
of division, the cell enlarges at the isthmus region, and elongates, as a result the 
semicells are pushed farther apart. The median septum then forms and the wall! 
push out to produce the new semicells. As the semiceli enlarges lobe formatioi 
proceeds and finally the arms of the typical species will be formed by further wal 
elongation. 

When the primary wall is almost fully expanded, the outer secondary wall begin* 
to be laid down with its pattern of ornamentation matching that already esta- 
blished, in the primary wall. The secondary wall also acquires its system o: 
mucilage pores, the position of which are indicated very early in wall," deposition 
which penetrate the entire secondary wall. 

The daughter cells remain joined to one another, with their apices, until thi 
shedding of the primary wall. These newly formed daughter cells move apart 
probably due to the extrusion of mucilage. 

It was found that cell division always occurred at a definite time after the beginning 
of the light period, when the light and dark periods were alternated regularly 
This suggests that the onset of illumination triggers the events, which set eel 
division in motion. Schiille (1975) reported that the total period of development 



Cell division in Staurastrum gracile Ralfs. 



445 




Figure 1. Staurastrum gracile Ralfs. showing triradiate form ( X 980). 




Figures 2-13. Staurastmjn gracile Ralfs. cell division. 2. Isthmus regie r 
becoming elongated. 3 and 4. Semi cells becoming separated. 5, 6 and 7. The 
young serai cell showing bulged and lobed condition. 8. The lobes elongating 

9. The development of the typical ornamentation and shape of the semi cell 

10. Mature cell, in side-view. 11 and 12. Development of the semicell in i 
triradiate form. 13. Mature cell viewed from above. [(2, 3, 11 end 13 (x 840) 
4-10 and 12 ( x 1120)] 



Cell division in Staurastrwn gracile Ralfs. 447 

!" newly-formed semicells was from 2-3 hrs. Their development was complete 
"ter this interval of time, but the actual separation of the newly formed cells usually 
>ok another 3 hrs. Under these conditions, cells divide only once every 24 hrs. 
Scannnig electron microphotographs were taken at various stages of division, 
igure 1 illustrates cells in a population, mostly, in a triradiate form. Figures 2, 
and 4 show enlargement of the isthmus region. Figure 5 shows one smooth 
>ung semicell. Figures 6, 7 and 8 show young semicells bulged and lobe forma- 
on. Figure 9 shows the development of the typical ornamentation and shape 
F the semicell. Figure 10 shows mature cell inside-view. Figures 11 and 12 
iow development of the semicell in a triradiate form and figure 13 shows mature 
>11 viewed from above. Thus figures 2-13 illustrate the various stages in the 
;11 division of the species, under investigation. 



cknowledgements 

he author expresses her deep sense of gratitude and thankfulness to Prof, Jafar 
[izam, Vice-Chancellor, Kakatiya University, for his constant encouragement, 
hanks are also due to Prof. John D Dodge, Head, Department of Botany, Royal 
[olloway College, London, for the guidance, where the present investigation was 
irried out. The author is also thankful to the UGC and the British Council for 
le award of a Commonwealth academic staff fellowship. 



efereuces 

rook and Alan J 1981 The Biology of Desmids, Botanical Monograph, Vol. 16, Blackwell 

Scientific Publications 
hu S P 1942 The influence of the mineral composition of the medium on the growth of 

planktonic algae. I. Methods and culture media ; /. EcoL 30 284-325 
odge J D 1963 The nucleus and nuclear division in the Dinophyceae ; Arch, Protistenk 106 

442-452 
irawert H and Kalden G 1967 Licht und elektron mikroskopische Untersuchungen an Desmidia- 

ceen. XTK. Mitteilung : Der Gestaltwandel des Nucleolus in interphasekero von Micras- 

terias rotata Mitt. ; Staatsinst. Allg. Bot. Hamburgl2 21-27 
irawert H and Mix M 1961 Licht und elektron mikroskopische Untersuchungen an Desmidia- 

ceen. HI. Der Nucleolus in Interphasekern von Micrasterias rotata Flora', 150 185-190 
ickett-Heaps J D and Fowke L C 1969 Cell division in Oedogomum. I. Mitosis, cytokinesis 

and cell elongation ; Aust. J. Biol Sci. 22 857-894 
ickett-Heaps J D and Fowke L C 1970 Mitosis, cytokinesis and cell elongation in the 

desmid Closterium littorale ; /. PhycoL 6 189-215 

ickett-Heaps J D 1973 Cell division in Cosmarium Botrytis ; /. PhycoL 8 343-360 
ickett-Heaps J D 1974 Scanning electron microscopy of some cultured desmids ; Trans. Am. 

Microsc. Soc. 93 1-23 

ickett-Heaps J D 1975 The green algae, (Sunderland, Mass Sinaver Assoc.) 
:hiille H M 1975 Untersuchungen zum Synchronen zellwachstum von Stauratrum pingue Telling 

(Desmidiaceae) in Licht-Dunkel-Wechsel ; Heih. Nova Hedwigia 42 275-281 
idyavati 1981 Polymorphism in Staurastrwn gracile Ralfs. under Scanning election microscope 

Paper read at the IV Botanical Conference, from 28-30th December, 1981 at Calicut, 
test W and West G S 1923 A Mouograph of the British Desmidiaceae Vol. V, 96-97 



. Indian Acad. Sci. (Plant Sci.) Vol. 91, Number 5, October 1982, pp. 449-461 
g) Printed in India. 



Leaf surface studies of some medicinal salvias* 



H P SHARMA and US HA SHOME 

Pharmacagnosy Laboratory, National Botanical Research Institute, Lucknow 226001, 

India 

MS received 18 May 1982 ; revised 18 August 1982 

Abstract. Scanning election microscopic studies on the leaves of 8 medicinal salvias 
comprising mainly surface ornamentation of the various epidermal cells and the 
appendages, provide useful parameters to distinguish one species from another. 
Some of the distinguishing features of the species studied arc : Sohia cabidica 
Benth. Striated lower epidermis, stomatal ledges "broad and smooth ; 5. Icnata 
Roxb. Ab axial side completely covered over by a thick coat of trichomes ; 
S. macrosiphon Boiss. verrucose trichomes with constricted joints ; S- moorcroftiana 
Wall- longitudinal folds an basal cells of trichomes ; S. officlnalis Linn. curved 
cylindrical trichomos, cells over veins with characteristic longitudinal ridges, gland 
stalk very long ; S. plebeia R. Br. basal cells of trichomes transversely striated ; 
S. pratensis Linn. verrucose trichomes and series of irregular folds on lower 
epidermis ; S. spinosa Linn. smooth collapsible hairs, folds on general surface 
similar to S. pratensis. 

Keywords. Salvia ; Lamiaceae ; leaves ; SEM studies. 



.. Introduction 

{ large number of species of the Lamiaceae are presently in medicinal use, parti- 
ularly in the Indian Systems of Medicine mainly for their essential oils,. However, 
ike most crude drugs these are subject to substitution and adulteration. Deter- 
aination of reliable criteria for distinguishing the genuine drugs is, therefore, 
;reatly important. 

Very often the marketed drugs consist of small broken pieces of different 
rgans which are, therefore, difficult to identify. Ultra morphology of the surfaces 
>f diffferent plant organs offers a useful, simple and reliable procedure for 
Authentication and standardization of herbal drugs where often only minute 
urfaces are available for study (Cappelletti and Casadoro 1977). 

Salvia (commonly known as Sage in European countries) is one of the most 
mportant genera in this respect, of which 9 species are medicinal (Chopra 'et a! 



NBRI Research Publication No. 148 (New series). 

449 



450 H P Sharma and Usha Shoirtd 

1956). The genus, comprising of ornamental herbs and shrubs, is distribute 
mostly in the temperate regions. Some 24 species of Salvia are reported fror 
the Indian subcontinent (Anonymous 1972). 



2. Materials and methods 

The present study deals with scanning electron microscopy of the leaf surface 
of 8 Salvia species, viz., S. cabulica Benth. (Afganistan-Kunar Prov. 
S. lanata Roxb. (Himachal Pradesh-Panjain), S. macrosiphon Boiss. (Afganistan 
Kandhar), S. moorcroftiana Wall. (Afganistan-Maidan), S. officinalis Lini 
(W. Germany-Garmisch), S. pkbeia R. Br. (Afganistan-Kunar Prov.), S. pratens 
Linn. (West Germany-Bavaria), and S. spinosa Linn. (Iraq-Basra) collected froj 
the Herbarium of the Institute for Systematic Botany and Botanical Garden 
University of Munich, West Germany. Material of S. lanata Roxb. froj 
Herbarium, NBRT, was also used for confirmation. 
The dried leaves were first soaked in hot water and after thorough washing 

1 cm square strips were cut from the middle portions of the lamina midws 
between the midrib and the margin and dehydrated through ethyl alcohol serif 
followed by critical point drying procedure using liquid CO^. This was part 
cularly necessary as the oil glands presented a distorted picture on simple dry in 

2 mm square pieces were cut from the dried material and one piece each of tl 
adaxial and abaxial surfaces were mounted on to the specimen stubs usir 
double sided adhesive tapes. 

Gold coating of the specimens, about 200 A thick, was carried out in an ic 
sputter coater (JFC-1100). The specimens were examined under a JEOL-JSM-35 
scanning electron microscope at an accelerating voltage of 10 kV and tilt of 3< 
incident to the electron beam at an aperture 100 pm. The image was observed 
magnifications ranging from x 200- x 5000 and photographs recorded on ORVS 
120 films. 



3. Observations 

3.1- Salvia cabulica Benth. (figures 1-3) 

3. la. Adaxial surface ; Sparsely hairy, trichomes small, 1-2 celled, extremely thi 
walled having narrow lumina, basal cells bulbous showing a distinct girdle 
disc at the base and at the joint ; upper cell swollen just above the joint, curv 
and pointed, more warty than the basal one, warts mammilate, irregularly arrang 
(figure 1). Epidermal cells polygonal, mostly straight walled or smoothly curve 
without epicuticular extrusions. Stomata few, mostly diacytic or with four su 
sidiary cells and flush with the surface. Guard cells longitudinally striat< 
parallel to the pore (figure 1). Glandular hairs very few. 

3.1i. Abaxial surface : Hairs more profuse, up to 4-celled, otherwise simil 
to the upper ones, stomata also profuse, raised above the surface, lips ridg 



Leaf surface studies of some medicinal sahias 



451 




a stoma on the 

2. Lower epKiernus skowmg dtaot 



striations art of a 
the right (x 600 ). 



epicuticular folds 



cell (x 



Lcaj swjace miaies oj some meaicmai saivias 4o3 

ipward forming a rim round the pore. Epidermal cells wavy in outline and 
urface rough and profusely striated; striations on each cell sparse except for 
lose over the veins (figures 2 and 3). Glandular hairs frequent, all of the same 
fpe, thin walled, heads very large (figure 2). 

.2. Salvia Janata Roxb. (figures 4-7) 

.20. Aclaxlal surface : Thickly hairy, hairs of three types (besides the glandular 
airs) : (i) in some of the 2-celled hairs the basal cells taper upward and appear 
istinctly cuticularised with broad epicuticular folds. The apical cell is also 
lickly cuticularised, has short longitudinal wavy wrinkles and pointed curved 
r beaked tips (figure 4). (ii) Short, 2-3 celled, basal cells broad at the base 
nd flattened at the top with edges forming a broad rim. From the centre of 
lis arises the next upper cell which is distinctly narrower and gradually tapers 
pward (figure 5). (iii) The other types are very long, thin, flattened, woolly and 
ollapsible forming a thick mat which has to be removed to permit a glimpse 
f the epidermal characters (figure 4). 

The epidermal cells are wavy in outline and have raised cuticular rims all along 
le periphery. The central part of the cell surface is again convex upwards* 
'urther, parallel cuticular striations are also found on the epidermal surface 
igure 6). The glands are stalked as usual and the heads are very small, one- 
slled and capitate. 

The lower epidermis is completely hidden under the woolly hairs (figure 7). 

. 3 Salvia macrosiphon Boiss. (figures 8-9) 

.30. Adaxial surface : Hairs upto 6- celled, long, thin-walled, cylindrical but 
ollapsible on drying, closely tuberculate, tubercles laterally flattened, arranged 
i continuous longitudinal ridges (figure 8), ground cell of the hair swollen and 
used above the epidermal surface, basal cell shorter and broader than the 
ibsequent one. Joints constricted with annulus type thickening on either side, 
pidermal cells smaller than those of other species, outline wavy; intercellular 
artitions depressed but the general surface raised and striated; striations longi- 
idinal and discontinuous. Stomata on both surfaces equally profuse, distinctly 
lised above in a broad dome-like manner. Stomatal ledges wavy but compara- 
vely narrow (figure 8). Glandular hairs infrequent, 

.36. Abaxlal surface : Hairs similar to those of the upper surface. Cell out- 
nes marked by longitudinal folds; surface thrown into fine wrinkles and folds 
11 over. Stomata on the lower side similar to those of the upper surface except 
lat the subsidiary cells have broad longitudinal folds along their outer periphery 
igure 9). Glandular hairs of two types, 1 and 8 celled. 

A Salvia moorcroftiana Wall (figures 10-11) 

Aa. Adaxial surface : Hairs sparse, verrucose, 2-6 celled, comparatively thinner; 
parts minute, sparse, arranged on longitudinally flattened ridges ; surface wrinkled 



454 H P Sharma and Usha Shame 

with loose cuticular folds ; joints -constricted with upper and lower cells swollen 
just above and just below the constriction respectively; tips narrow and pointed, 
sometimes . curved (figure 10). Long, thin-walled collapsible type hairs also 
present. Anticlinal walls of epidermal cells sinuous,, but sinuosities obscured by 
irregular loose cuticular folds all over surface of cells (figure 10) ; stoniata scanty. 
Glandular hairs scarce. 

3.46. Abaxial surface : Hairs similar to the upper ones. Long thin collap- 
sible hairs also present. Cell margins appear ridged: stomata almost similar to 
5. plebeia but distinctly smaller and flush with the surface (figures 10 and 15); 
ledges narrow, margins of the ledges wavy (figure 11). Glandular hairs are 
of two types: (i) small, one-celled with one-celled stalk and (ii) larger 8-celled, 
sessile. 

3.5 Salvia officinalis Linn, (figures 12 and 13) 

3.5a. Adaxial surface : Hairy, hairs 2- to several-celled, thin and cylindrical, 
apex obtuse, usually psilate; basal cell bulbous at the lower end and psilate with 
very faint transverse markings, upper ones with longitudinal ridges ; joints slightly 
swollen. Upper cells of some hairs over midrib sometimes having a few sparsely 
arranged warts. Two-celled glandular hairs having long stalks present. Anticlinal 
walls of epidermal cells sinuate, sometimes sharply so; intercellular partitions 
depressed; cell surface with broad depressions and epicuticular folds (figure 12). 
Cells over the midrib elongated having characteristic prominent longitudinal ridges 
involving even the stalk cells of the secretory hairs. Stomata diacytic, cuticular 
ridges of subsidiary cells converging on stomata, outer rim of guard cells raised 
but their central portion depressed ; inner ledges wavy ; surface with a series 
of wavy cuticular folds parallel to the opening. Transverse cuticular folds also 
prominent (figure 12). 

3 . 5b. Abaxial surface : Densely hairy, hair ; similar to those of the upper epidermis, 
those over the veins turned backward; characteristic minute, club-shaped hairs 
also present in this region. Their basal cells are depressed laterally and the 
upper cells are broader, blunt and flattened (figure 13). Epidermal cells over 
the veins elongated and very deeply furrowed longitudinally (figure 13). Large 
8 celled glandular hairs with small stalks present. 

3.6. Salvia plebeia R.Br. (figures 14-15) 

3.60. Adaxial surface : Hairs sparse, evenly distributed, stout, thick- walled and 
verrucose, basal cell broad at the distal end and tapering upward; lower cells 
polygonal; the apical one cylindrical, very narrow and elongated with pointed 
tip ; joints greatly constricted ; tubercles sparse on the two lower cells but much 
denser on the apical one. Horizontal striations on the basal cell of trichomes 
noticeable (figure 14 A, B). Stomatal guard cells raised upward, ledges quite 
prominent . and sinuous. Epidermal cells also striated; striations longitudinal. 
Glandular hairs occur .on both surfaces, heads usually small, 1- and 4-celled; stalk 
also 2-celled. 



Leaf surface studies of some medicinal salvias 



455 




Figures 5-8. 5. Salvia to/w/o Trichomes from the upper epidermis with a broad 
rounded basal cell and much narrower upper'cell (X 600). 6. Upper epidermis 
showing epicuticular folds and glandular hairs. Cell outlines indistinct (x 1000), 
7. Lower epidermis covered with a thick mat of collapsible multicellular trichomei 
(x 600). 8. Salvia macrosiphon Upper epidermis showing cells with distinct 
cell outlines, verrucose trichomes showing a constricted joint with annular type 
thickening and stomata with wavy ledges (x 600). 



456 



H P Sharma and Usha Shome 




Figures 9-12. 9. Salvia macrosiphonLowQr epidermal cells showing broad 
longitudinal folds and stomata with wavy ledges (x 2000). 10. Salvia moorcrof liana 
Upper epidermis showing wrinkled surface with loose epicuticular folds, trichome 
with longitudinal folds on the basal cell and a glandular hair on the left (x 600). 
11. Lower epidermal cells showing a stomawith wavy ledges (x 2000). 12. Salvia 
officinalis -Upper epidermis showing trichomes and a stoma (x 1000). 



Leaf surface studies of some medicinal sahias 



457 




JfV 

Figures 13-16. 13. Salvia offidnalisLwei epidermis showing cylindrical 
trichomes over the vein cells having characteristic epiculicular ridges (x 260). 
14 A Salvia plebia Upper epidermis showing a trichome with its basal cell 
having fine transverse stations (x 400). 14 B. A portion of trichome fiom 
figure Ha enlarged to show transverse striations (x 960). 15. Lower epidermis 
showing collapsible trichomes and astoma with wavy ledges (x 2000). 16. Salvia 
pratensisUppu epidermis with distinct cell outlines, small glandular hairs, 
stornata and a trichome (x 600), 



458 



H P Sharma and Usha Shome 




Figures 17-20. 17. Salvia pratemis- -Lower surface showing wrinkled epidermis, 
a stoma and glandular hair (x 2000). 18. Salvia spinosa Upper surface of the 
leaf showing wrinkles all over (x 660). 19. Lower epidermis showing a large 
number of trichomes (x 600). 20. Lower epidermis showing stoma with fungal 
mycelium coming out of one of the stomatal pores (x 860). 



Leaf surface studies of some madicinal salvias 



459 



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Leaf surface studies of some medicinal saivias 46i 

3 . 66. Abaxial surface : Beside the stout hairs, thin several-celled collapsible hairs 
also present, particularly over the veins. Epidermal cells wavy and thick walled. 
Stomata raised and characterised by cuticular ledges which are wavy and over- 
hang the stoma on either side. Beside these the cuticle over the guard cells thrown 
into two sets of folds, one set wavy and arranged more or less parallel to the 
stomatal opening while the other set is represented by a fine series of wrinkles 
at right angles to the long axis of the opening (figure 15). The cuticle of sub- 
sidiary cells is also thrown into a series of folds perpendicular to and conver- 
ging towards the guard cells. 

3.7. Salvia pratensis Linn, (figures 16 and 17) 

3.7#. Adaxial surface : Hairs on the lamina very sparse, short, cylindrical 
thick-walled and sharply pointed at the tip, 1-3-celled ; basal cell very broad at the 
lower end forming a disc ; upper one swollen just above the joint, verrucose, warts 
denser on the upper cell and arranged in vertical rows (figure 16). Hairs on 
midrib and petiole very long, more dense, thin walled and collapsible in dried 
material. Ordinary epidermal cells sinuate and striated, striations very fine; anti- 
clinal partitions depressed and forming a groove with edges of the adjacent cells 
raised (figure 16). Stomata slightly raised, lips with raised longitudinal ledges. 
Glandular hairs very few. 

3.76. Abaxial surface : Hairs of two types : (i) longer collapsible type over 
the midrib and (ii) thick cylindrical types elsewhere. The latter 1-4 celled, but 
otherwise similar to those on upper surface, faint horizontal wrinkles present on 
the hairs. Stomata diacytic, slightly raised, ledges on stomatal lips quite 
prominent (figure 17). Epicuticular ornamentation in the form of irregular 
wrinkles present particularly along the edges of the guard cells (figure 17). 
Glandular hairs frequent, stalk 1-3 celled, gradually expanding upward, head 
globular and composed of eight cells. 

3.8. Salvia spinosa Linn, (figures 18-20) 

3.8fl. Adaxial surface: General surface undulating and greatly wrinkled all over, 
wrinkles short, irregular and wavy (figure 18). Peripheral margins of epidermal 
cells raised, stomata diacytic, similar on both surfaces, outer and inner margins 
of the guard cells raised. Outer margin broadly ridged, central part concave 
and wrinkled, one set at right angles to the stomatal pore and the other dis- 
continuous but more or less parallel to it (figure 19). Stomatal ledges prominent 
(figures 18 and 19). Glandular hairs with large, smooth, 8-celled heads. 

3. 86. Abaxial surface : Surface similar to the upper one but hidden under a thick 
mat of multicellular hair. Hairs long, thin, smooth with faint longitudinal 
cuticular markings and a few transverse wrinkles (figure 20); joints constricted. 
Stomata similar to those on the upper surface but smaller and more dense. 
A large number of fungal mycelia observed entering through storaatal pores 
(figure 20). 



46 H P Shanna and Usha Shome 

4. Discttssiqii 

Comparative morphology of epidermal surfaces using SEM has proved valuable 
in elucidating taxonomic problems (Atwood and Willams 1979; Ayensu 1974; 
Cutler and Brandliam 1977; Dehgan 1980; Ehler 1974; Rollins and Banerjee, 
1975). 

Cappelletti and Casadoro (1977) distinguished Atropa bella-donna (family 
Solanaceae) from its adulterants Ailanthus altissima (Miller) Swingle (family 
Simurubaceae) and Phytolacca amerlcana L. (family Phytolaccaceae) with the 
help of ultra morphology of leaf surfaces. 

The present study was undertaken with a view to determine such parameters 
as could help characterise the different medicinal Sahia spp. even if the surfaces 
available are too small to be of much diagnostic value otherwise. 

The study has shown that ultra morphology of the leaf surface provides a useful 
tool in authentication of vegetable drugs, at least in the case of salvias. The 
epidermal surface on both sides have epicuticular ornamentation, in the form of 
striations or loose folds in all species except for the adaxial surface in S. cabulica 
and the abaxial one in S. qfficinalis. Of the remaining, S. plebeia has striations 
on both surfaces while loose folds occur on both sides in S. moorcroftiana and 

5. spinosa. In all others the two surfaces are dissimilar. Similarly, there are 
differences in the stomatal characters. Another important feature which helps 
to characterise the different species studied is the type of trichomes and surface 
ornamentation (table 1). For instance, whereas only a single type of hair 
(besides the glandular hair) were observed in three species, S. cabulica, 
S. macrosiphon and 5. qfficinalis, all others have long, psilate, thin-walled collap- 
sible type of hair as well- In case of S. lanata even the short hairs are of 
two types. Again, except S. cabulica and S. spinosa all others have verrucose 
to tuberculate ornamentation all over the trichome surface. However, of the 
latter ones S. moorcroftiana has tubercles on the uppermost cell and S. officinalis 
on the middle one. 

Scanning electron microscopic studies have, thus., proved to be extremely 
useful in characterisation of the different species of the same genus Salvia and 
a positive advantage in standardization of herbal drugs. 



Acknowledgements 

The authors express their thanks to Dr T N Khoshoo, Director, National Botani- 
cal Research Institute, for encouragement and keen interest in the progress of 
the work, to Mr S L Kapoor, Incharge, Herbarium Section, NBRI, for sparing 
the herbarium sheet of S. lanata Roxb. and to Dr M Yunus, for taking the 
SEM photographs. 

The senior author is particularly indebted to Prof- H Merxmuller, Director, 
Institute for Systematic Botany, University of .Munich, for the excellent labora- 
tory and herbarium facilities made available to him during his visit to the said 
Institute and for permission to use herbarium material of the Staatsherbarium 



Leaf surface studies of some medicinal salvias 46 3 

for the present studies. He is also thankful to Prof. J Grau of the same 
Institute for familiarising him with some techniques of Scanning electron micro- 
scopy and to DAAD and the CSIR for arranging his visit to West Germany under 
the exchange of Scientists Programme. 



References 

Anonymous 1972 The wealth of India. A dictionary of Indian raw materials and industrial 

products. IX, Rh-So (New Delhi : CSIR) 
Atwood J T and Williams N H 1979 Surface features of the adaxial epidermis in the con- 

duplicatc leaved Cypripcdioideae (Orchidaceae) ; Bot. J. Linnean Soc. 78 141-156 
*Ayejv>u E E 1974 Leaf anatomy and Systematic^ of New World Vellaziaceae. In Smithsonian 

contributions 1o botany (15 City of Washington : Smithsonian Institution Press). 
Cappelletti E M and Casadoro G 1977 Leaf surface morphology of Atropa hello donna and of 

some adulterant species by Scanning electron microscopy ; Planta Medica 31 356-366 
Chopra R N, Nayar S L and Chopra I C 1956 Glossary of Indian medicinal plants (New 

Delhi : CSIR) 
Cutler D F and Brandham P E 1977 Experimental evidence for genetic control of leaf surface 

characters in hybrid Aloineae (Liliaceae) ; Kew Bull. 32 23-32 
Dehgan B 1980 Application of epideimal morphology to taxonomic delimitations in the genus 

Jatropha L. (Euphorbiaceae) ; Bot. J. Linnean Soc. 80 257-278 
*Ehler N 1974 Die Feinskulpluren Madagassischer Euphorbien-Hockblatter und ihre taxonomiche 

vertigkeit ; Feddes Repertorium 85 345-351 
Rollins R C and Banerjee U C 1975 Atlas of the Trichomes of Lesquerella (Cruciferae) : 

The gussy Institute, (Harvard University) pp. 1-48 

* Not seen in original. 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 5, October -1982, pp. 465-472. 
C) Printed in India. 



Morphological and histochemical changes in the egg and zygote of 
Lagerstroemia speciosa. I. Cell size, vacuole and insoluble 
polysaccharides* 



P RAGHAVAN and V J PHILIP** 

Department of Botany, University of Calicut 673 635, Kerala, India 
** P.O. Box 43844, Nairobi, Kenya 

MS received 1 February 1980 ; revised 16 December 1980 

Abstract. In Lagerstroemia speciosa, the decrease in size of the egg and its 
micropylar vacuole immediately after fertilization is followed by a progressive 
and marked expansion of the cell. The PAS-positive cell wall material in egg is 
confined to the micropylar half. Soon after fertilization, but before completion 
of decrease in size of the zygote, its cell wall grows in thickness. A complete wall 
is not formed around the zygote. The bulk of the insoluble polysaccharides in the 
cytoplasm is legalized at the chalazal pole of the egg and zygote. Following ferti- 
lization, the size and number of starch gianules in the egg cytoplasm significantly 
increased followed by a decrease and again an increase during zygote develop- 
ment. The morphological changes in the egg following fertilization are probably 
related to the osmolarity of the cell and of the vacuole which would account for 
the change in cell size. 

Keywords. Lagerstroemia speciosa ; variation in vacuolar size ; cell wall ; insoluble 
pclysaccharides. 



1. Introduction 

The zygote is the fundamental structural and functional unit which constitutes 
a new beginning and affords opportunities for investigating growth, development, 
differentiation, assumption of form and functional activities. Ultrastructura* 
changes have been described in the fertilized egg of a few plants such as cotton 
(Jensen 1968), Capsella bursa-pastoris (Schulz and Jensen 1968), Zeamays (Diboll 
1968), Petunia hybrida (van Went 1970b), flax (D'Alascio-Deschamps 1972), 
barley (Norstog 1972) and Quercus gambelii (Mogensen 1972 ; Singh and 
Mogensen 1975). But little is known about the metabolic changes accompanying 
the formation of the zygote. The present study deals with the structural and 
certain histochemical changes that result on fertilization of the egg of Lagerstroemia 
speciosa and its subsequent development up to the first division of the zygote. 



This paper is dedicated to. late Professor B. G. L. Swamy. 

* part of tfye thesis of PR approved by the University of Calicut for the Ph.D. degree. 

465 



466 P Raghavan and V J Philip 

2. Material and methods 

Ovules and seeds of Lagerstroemia specicsa (Linn.) Pars, at different stages in 
development were collected at weekly interval from areas in and around Calicut 
University campus, fixed instantaneously in the field in formaim-acetic-alcoho 
or Carnoy's fluid. Conventional method of dehydration through tertiary butyl 
alcohol series was employed and serial microtome sections (10-1 5 /on) were 
prepared. Insoluble polysaccharides were demonstrated by pAS-reaction (Jensen 
1962). Tissue oxidation was carried out in 0'5% periodic acid in distilled water 
for 20-30 min. Response to the stain was the same by sections fixed by either 
fixatives. Treatment of tissues fixed in Carney's fluid with 1% aqueous mercuric 
chloride solution gave a negative reaction with Schiffs reagent. Confirmatory 
test carried out with potassium iodide-iodine stain indicates that the materials 
stained with PAS is constituted of starch grains and that these occur in plastids. 
Cellular, nuclear and vacuolar areas were calculated from camera lucida drawings 
of the stained preparations. 



3. Results and discussion 

3.L Change in size of the cell and its vacuole 

The pear-shaped egg cell with its broad chalazal and tapering micropylar poles 
ranges from 457 ^m to 632 /tm in length. It contains a thin layer of cytoplasm 
surrounding a large vacuole which reaches almost the cell wall at the micropylar 
pole. The cytoplasm is concentrated in the chalazai half. The nucleus is confined 
to either the chalazal pole (figure 1) or to a lateral position in the chalazal half 
(figure 2). 

Immediately after fertilization the cell shrinks to about 240 ^m and the vacuolc 
also decreases (figure 3). This decrease in size is followed by a progressive and 
marked expansion of the cell (figures 4-8), so that the maximal size attained is 
about five-fold the initial size of the [zygote and about two and a half-fold the 
size of egg cell (figure 8). Furthermore, a progressive flattening of the micropylar 
region of the zygote occurs. In few preparations the nucleus was seen displaced 
towards the centre of the cell due to the appearance of two smaller vacuoles at the 
chalazal pole (figure 3). Subsequently these two apical vacuoles expanded 
disproportionately to the remainder of the cell and displaced the nucleus to a 
more central position (figure 7). In 75% of the zygotes examined, only the 
micropylar vacuole was present which enlarged retaining the nucleus at the chalazal 
pole itself. 

The observed size changes in the egg cell and zygote of Lagerstroemia cannot 
be accounted for merely by biological variation among the preparations examined. 
In cotton also a prominent shrinkage of the zygote occurs (Jensen 1964, |1968), 
but not in Capsella (Schulz and Jensen 1968) and barley (Norstog 1972). Cell 
enlargement and cell elongation are considered as general features of the zygote 
preparing for division. Assuming that cell shrinkage is due to loss of water, 
the process is reversible because water from the exterior can be abstracted by 
the maturing zygote. The underlying osmotic changes in the zygote and the 



Morphological and hlstochemical changes in zygote 



467 










30JU 




Figures 1*8. Lagerstroemia speci&sa- changes in size of egg, zygote and their 
vacuole. All are longi sections ; micropylar pole towards bottom of page. 1. Egg 
cell ; the major part of cytoplasm is confined to chalazal pok . 2. Egg cell showing 
lateral disposition of nucleus and cytoplasm at chalazal half. 3. Zygote ; note 
decrease in cell volume, and formation of two smaller vacuoles at the chalazal 
pole. 4-8. Zygote at later stages in development. Note the progressive increase 
in size and the flattening of micropylar region. 



surrounding milieu result from metabolic changes at these two sites. One of 
these changes is the reversible soluble carbohydrate/polysaccharide transformation 
in zygote to be discussed in 3.3. 

3.2. Cell wall 

Both in the egg and in the zygote the extent of cell wall present is variable. 
Electron microscopic studies have shown that in mature eggs of cotton (Jensen 



468 P Raghavan and V J Philip 

1964, 1965), Torenia fournieri (van der Pluijm 1964), maize (Diboll and Larson 
.1966) and Petunia hybrida (van Went I970a, b) the cell wall extends to only half 
way up the micropylar pole. However, in the egg cell of Capsella bursci-pastoris 
the wall extends almost over the entire cell ; at the chalazal pole the structure is 
honey-combed with large gaps (Schulz and Jensen 1968). Thus, in all the species 
investigated the egg cell shows regions of the plasma membrane at the chalazal 
pole in direct contact with the embryosac, a feature which possibly enables the 
egg to derive nutrition directly from the central cell. In general, in angiosperms 
the micropylar region of the zygote is anchored to the wall of the embryosac, and 
during development wall formation extends over the open chalazal region and 
envelops the zygote all round. In Capsella bursa-pastorls simultaneously with 
the deposition of the wall material in the gaps in the chalazal region, the wall in 
the micropylar portion of the zygote becomes thickened (Schulz and Jensen 1968). 
In barley the wall of the zygote is thicker at the micropylar region than elsewhere 
(Norstog 1972). 

In Lagerstroemia speciosa a positive periodic acid-Schiff reaction was obtained 
both in the cell periphery and in the cytoplasm of the developing embryo. But 
in the egg the reaction was confined to the micropylar half, resembling in ' this 
respect, cotton, maize, Torenia and Petunia. But, unlike in cotton, in Lager- 
stroemia a complete cell wall is not formed around the young zygote. In the egg 
the reaction product is visible as a fine film extending to 60% of the perimeter 
of the cell from the micropylar pole (figure 9). Soon after fertilization, but 
before completion of decrease in size of the zygote, the cell, wall grows further in 
length covering 70% of the cell perimeter measuring 56/wn (figure 10). Subse- 
quently, however, the addition of wall material does not appreciably increase the 
thickness, and the percentage of wall material to the perimeter of zygote remains 
unchanged (figure 11). In a nearly mature zygote the wall extends to 112/xm 
from the micropylar pole covering 80% of the cell perimeter (figure 12), and its 
thickness is slightly less than that in the two earlier stages but more than that in 
the egg. 

Street and Opik (1970) have pointed out that during cell expansion (elongation), 
the cell wall does not thin out, but there 'occurs a proportionate increase in cell 
wall synthesis. It is not clear how a cell would respond to a decrease in cell size. 
Because in both the egg and the zygote of Lagerstroemia speciosa the cell wall is 
present only at the micropylar half, a decrease in size of the young zygote will not 
presumably impose as much strain as it would have been if the zygote had an 
entire cell wall. 

3.3. Insoluble polysaccharides (starch granules) 

Histochemical staining with periodic acid-Schiff reagent showed, besides cell wall, 
numerous granules of insoluble polysaccharides in the cytoplasm of both egg and 
zygote, especially localized in their chalazal pole. In all stages of development 
beginning from egg, the starch granules are heterogeneous in size and varied 
in number. The egg cell contains about 50 tiny starch granules (figure 9). 
Following fertilization, there is a significant increase in their size and number. In 
the young zygote, which showed a decrease in size, the number of starch granules 
increased to 90-100 (figure 10). As the zygote enlarged, but before attaining 



Morphological and hlstochemlcal changes In zygote 



469 




Figures 9-12. Lagerstroemia speciosamstian longisections of egg and zygote after 
treatment with periodic acid-Schiff reagent. 9. Egg with PAS-positive wall covering 
60% of its perimeter from the micropylar pole. Note the size and number of 
starch granules at the chalazal pole. 10. Zygote as seen immediately after forma- 
tion ; note further growth of wall in thickness, decrease in cell size, and number 
of starch granules. 11. Zygote at a later stage, showing decrease in size and 
number of starch granules but with no appreciable change in wall mateiial deposi- 
tion. 12. Nearly mature zygote with the wall covering SO % of the cell peri- 
meter. X 850. 



Morphological and liistochemical changes in zygote 471 

naturity, the number of granules decreased to about 60 ; their size also decreased 
figure 11). In the nearly mature zygote, a second increase in number to about 
100 granules can be noticed (figure 12), accompanied by an increase in size. 

In cotton the egg cell contains one or two small starch granules per plastid and 
:here is no specific association of the plastids with other organelles in the cell. 
Following fertilization, when cell size is decreasing, the plastids accumulate along 
with mitochondria, around the nucleus (Jensen 1968). At this stage, starch begins 
to accumulate in the plastids. The close association of the nucleus, plastids and 
ribosomes presumably facilitates the ready elaboration of the biosynthetic system (s) 
.ind the transfer of the enzyme concerned to the granule or the cytoplasm, as the 
:ase may be. During maturation of the zygote, when additional wall material 
is being formed so as to complete the wall around the entire cell, the number of 
plastids remains unaltered, but their size increases. Also, the number of starch 
grains per plastid increases so that the plastids become filled with starch. 

The occurrence of starch grains in the egg cell of Lagerstroemia speciosa, the 
increase in their number soon after fertilization and their presence in large amounts 
in the mature zygote, are in accord with the findings in cotton (Jensen 1968), but 
the decrease in number and size of starch granules which occurs in the intermediate 
stage is distinctive of Lagerstroemia. The increase in number and/or size of the 
starch granules following fertilization is -accompanied by the decrease in the 
vaculoar volume. Similarly, a decrease in the number and size of starch granules 
is sometimes associated with increased vacuolar size, as when the early zygote 
reaches the intermediate stage of development. Such a formation of starch 
granules at the expense of soluble sugars stored in the vacuole would reduce the 
osmotic pressure and result in a diminution of cell and vacuolar size. This would 
account for the morphological changes, namely, a marked decrease in size, on 
fertilization of the egg. The reverse process, namely starch degradation and 
transfer of soluble sugars to the vacuoles, would increase the osmolarity of the 
cell and vacuole. This would account for the increase in size of the early zygote 
and its vacuole. Contrary to expectation, the second increase in number and 
size of starch granules, which occurs in the mature zygote is associated with an 
actual increase both in cell and vacuolar size. 

A significant point is that cell wall synthesis in the zygote occurs at the micro- 
pylar half, even though the protoplasm is concentrated at the chalazal half. 
Evidently, the polarity of the cell does not extend to the metabolic activity of the 
cell relative to wall synthesis. 

Acknowledgements 

The authors are grateful to Prof. P S Krishnan, Emeritus Professor of Bio- 
chemistry, University of Calicut, for help in the preparation of this paper. One 
of us (PR) thanks the University of Calicut and the University Grants Commission 
for financial assistance in the form of fellowships. 

References 

D'Alascio-Deschamps R 3972 Le sac cmbryoniiaire du lin apr^s la fecondation ; Bctaniste 50 
273-288 



P Raghavan and V J Philip 

Diboll A G 1968 Fine structural development of the mega-gametophyte in 2ea mays following 

fertilization ; Am. J. Bet. 55 7S7-806 
Diboll A G and Larson D A 1966 An electron microscopic study of the mature mega-gamato- 

phyte in Zea mays \ Am. J. Sot. 53 391-402 

Jensen W A 1962 Botanical histo chemistry (San Francisco ; W H Freeman) pp. 198-199 
Jensen W A 1964 Cell development during plant embryogenesis in Meristems and differentiation 

Brookhaven Symp. BioL 16 179-202 
Jensen W A 1965 The ultrastructure and composition of the egg and central cell of cotton ; 

Am. J. Bat. 52 781-797 

Jensen W A 1968 Cotton embryogenesis : the zygote ; Planta 19 346-366 
Mogensen H L 1972 Fine structure and composition of the egg apparatus before and after 

fertilization in Quercus gambeUl : the functional ovule ; Am. J. Bot. 59 931-941 
Norstog K 1972 Early development of the barley embryo : fine structure ; Am. J. Boi. 59 

123-132 
Schulz S R and Jensen W A 1968 Capsella embryogenesis : the egg, zygote and young embryo ; 

Am. /, Bot. 55 807-819 
Singh A P and Mogensen H L 1975 Fine structure of the zygote atfd early embryo in Quercus 

gambelii; Am. J. Bot. 62 105-115 
Street H E and Opik H 1970 The physiology of flowering plants : Their growth and development 

(London : Edward Arnold) p. 16o 
van der Plmjm J E 1964 An electron microscopic investigation of the filiform apparatus in the 

embryosac of Torenia fournieri in Pollen physiology and fertilization (ed.) H E Linskens 

(Amsterdam : North-Holland PubL Co.) pp. 8-16 
van Went J L 1970a The ultrastructure of the egg and central cell of Petunia ; Acta Bot. Neerl. 

19 313-322 
van Went J L 1970b The ultrastructure of the fertilized embryo sac of Petunia ; Acta Bot. 

NeerL 19 468-480 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 6, December 1982, pp. 473-478, 
Printed in India. 



The floral anatomy of Puya spathacea Mez. (Bromeliaceae) with 
special reference to nectaries 



R A KULKARNI and R M PAI 

Plant Morphology Laboratory, Department of Botany, Marathwada University, 
Aurangabad 431 004, India 

MS received 25 May 1981 

Abstract. The floral anatomy of Puya spathacea Mez. is dcsciibed in detail. The 
outer floral whorls are united to develop a short hypanthium which is adnate 
to the base of the ovary. The sepals are five-traced and the petals, three-traced. 
The placentation is axile. The occurrence of numerous ovules in more than two 
rows as well as the extension of the carpellary ventrals into the style are less 
advanced features. The ovarian nectary is extensively developed and shows a 
transition between typical septal and epigynous nectaries of certain monocotv* 
ledonous taxa. 

Keywords, Puya spathacea ; floral anatomy ; nectaries. 



1. Introduction 

The Bromeliaoeae are a fairly large family with about sixty genera and about 
2000 species. Hutchinson (1959) considers the family to be a homogeneous taxon 
representing the * climax of a line of descent wherein the calyx and corolla have 
remained distinct or fairly distinct from each other '. He treats it as related to, 
but more advanced than the Commelinales. 

Smith's extensive studies (1934) point out that the family has strongest affinities 
with the Rapateaceae and that both families probably arose from a common 
ancestral stock. Within the family, Puya is treated as probably the source of 
ancestral types from which the other sub-families developed. According to 
Pittendrigh (1948), Puya is the most primitive of living bromeliad genera. 

The genus Puya is a native of thei Andes with unique habit and habitat. Some 
species of the genus are considered to be the largest and most interesting of the 
bromeliads, e.g., Puya raimondii Harms, a rare monument of the Peruvian Andes 
threatened with extinction. Plants of this species attain a height of 9*5 m and 
bear thousands of flowers with sugar-poor honey and pollinated by birds. The 
tiny, winged seeds may number even a billion per plant. The plants are mono- 

473 
P. (B)-l 



474 ... R A Kulkami and R M Pal . . 

carpic and hapaxanthic (blooming once in their life and dying out thereafter, 
and propagated exclusively by seed). Puya spathacea Mez. is a much smaller 
plant attaining a height of 1 m. 

Studies in the floral anatomy and morphology of this interesting group of 
plants are meagre. An extensive investigation on the vascular anatomy of the 
flower of the group is, therefore, undertaken in this laboratory, and the results 
on one of the most .unique genera are presented in this paper. Amongst the 
noteworthy earlier contributions on the family is a paper by Budnowski (1922) who 
is of the opinion that probably no Bromeliaceae lack septal glands. Some work 
on the group is in progress at Professor Rauh's laboratory in West Germany. 



2. Materials and methods 

The fixed flowering material was received from Prof. H Merxmuller, West 
Germany, collected from his University Botanic Gardens. The usual paraffin 
method has been followed. Serial transections (10-12 p, in thickness) and longi- 
sections were stained with crystal violet using erythrosin as a counter stain. 



3. Observations 

The pedicel contains a ring of six to eight prominent bundles surrounded by 
many discrete smaller strands (figure 1). All these bundles divide and form nume- 
rous strands which resolve into a large number of centrally placed placental 
bundles and six outer groups of strands from which the principal bundles of the 
floral whorls emerge out" (figure 2), From each of the posterior and antero-lateral 
groups of strands three LS strands a*e derived. While the laterals amongst the 
three branch and extend into the sepals on either side, the median one bifur- 
cates and branches into the margins of both the sepals (figure 3). The remainder 
of these groups resolve into an inner is bundle and an outei? strand which splits 
into an MP bundle and two LP strands (figure 4). The three groups in the postero- 
lateral and anterior positions resolve into the MS, os and D strands (figure 4) 

The nectary is developed from the very base of the ovary beneath the 
level of the loculi (figure 4). It is extensively developed with many canal- 
like passage ways and surrounded by the repeatedly dividing placental bundles 
(figures 3, 5). This gives the appearance of ' processes ' of axile tissue of the 
ovary lined by glandular cells. Upwards, the nectary is closed in the centre to 
result in. three glandular clefts and some of the * processes ' persist in the form 
of lobes of- carpellary tissue enclosed in septal cavities or canals. These open to 
the outside towards the middle of the length of the ovary (figure 6). 

The ovary is teilpcular with the placentation axile (figure 6). Upwards, the 
margins of the carpels meet only in the centre to continue syncarpy (figures 6, 7). 
Some of the placental bundles arrange themselves opposite to the loculi and these 
bear traces into the placentae and the ovules (figures 6, 7). The ovules are 
numerous and borne in many rows on each placenta. Most of the placental 
bundles end in bearing traces to the ovules and towards the nectary. Six placental 



Floral anatomy of Puya spathacea Mez. 



475 




Figures 1-5. Puya spathacea, serial transections of the flower from the base 
upwards ; D, carpellary dorsal ; is, inner staminal strand ; LP, lateral bundle of 
petal ; LS, lateral bundle of sepal ; MP, median bundle of petal ; MS, median bundle 
of sepal ; N, nectary ; P, petal ; PL, placental bundles ; os, outer staminal strand ; 
s, sepal. 




Figures 6-11. Puya spathacea, serial transections of the upper part of the flower ; 
QL, gland ; PL, placental bundles ; sc, stylar canal ; sx, stamen ; STY, style. 



476 R A Kulkarni and R M pai 

bundles (caipellaty ventrals) continue upwards into the base of the style (figure 8). 
The ovarian loculi continue in the form of three canals which merge into a 
triradiate canal in the style (figure 9). The carpellary dorsals continue up to the 
tip of the style and into the three shallow stigmatic lobes. 

The outer floral whorls form a short hypanthium which is adnate to the base 
of the ovary. The sepals, petals and stamens separate out simultaneously 
(figure 6). The sepals are twisted to the left and the petals to the right (figure 7). 
At the level of insertion, the sepals and the petals receive 8 or 9 traces (figure 5). 
The median bundles of the sepals and the petals bear lateral branches, some of 
which may divide further. 

The six stamens are one-traced. The staminal bundle extends upwards into 
the connective and ends at the base of the short crest of the anther (figure 10). 
It bears a lateral branch towards either anther lobe (figure 9). The anther is 
two-celled. The outer stamens are longer (figure 10). 



4. Discussion 

The vascular anatomy of the flower of Puya presents several, features of interest 
and importance. The outer floral whorls are united into a short hypanthium which 
is adnate to the ovary for some length. This is a trend in the development of 
an inferior ovary which is characteristic of the allegedly advanced bromeliads, 
e.g., Aechmea, Billbergia, Cryptanthus, Neoregelia. The adnation is, however, 
not of a marked degree which is also reflected by the absence of fusion of the 
principal strands of the floral whorls. The placentation is axile. The occurrence 
of numerous ovules borne in many rows is a primitive feature. The extension 
of the carpellary ventrals into the style is also a less specialised condition. 

The development and the structural details of the nectary are very significant. 
Initially, the nectary is in the form of a central crater with canal-like passageways 
which proliferate profusely. The placenta! bundles repeatedly divide and are lodged 
in what appear as * processes ' of carpellary tissue lined by glandular cells. The 
placental bundles have to be associated with the nectary in its function (Agthe 
1951 ; Budnowski 1922 ; Frei 1955 ; Pai and Tilak 1965 ; Tilak and Pai 1974). 
Upwards, the nectary is closed in the centre and the * processes' of carpellary 
tissue appear in the form of distinct glandular outgrowths lodged in septal cavi- 
ties. These are vascularised by the placental bundles. The glandular lobes 
open to the outside. In Costus and Tapeinochilus of the Costaceae a more or 
less similar initial condition is observed which probably prompted Brown (1938) 
to describe the nectaries in Costus as septal. Rao etal (1954) describe them as 
not septal nectaries but as vascularised outgrowths of carpellary tissue \vMch 
extend upwards in ovarian canals along the septal radii and open at the top of 
the ovary. In Kaempferia rosea (Pai 1966) these glandular lobes appear on. the 
septal radii in similar but epiovatian canals at about the level where those in 
Costus end, and extend above the ovary. In the majority of zingibers, the 
nectaries are epigynous. 

It may be noted that in monocotyledonous taxa with extensively developed 
septal nectaries with canal-like passageways, vascularised * processes ' of carpellary 
tissue dp occur, e.g., Muw and Ensete (Tilak and Pai 19?4). However, these 



Floral anatomy of Puya spathacea Mez. 477 

do not develop into glandular outgrowths. This would seem to indicate that 
these ' processes ' may persist and extend upwards as glandular lobes or outgrowths 
in some taxa as in Puya, Costus and Tapelnochilus. While they are short in Puya 
and open to the outside, they proliferate and extend upwards and above the 
ovary in the latter two genera. The function of nectar secretion is taken over 
by the lobes or outgrowths so that the canals in which they appear lodged do 
not have the secretory lining layer. The lining layer in Puya is secretory in the 
basal half and the function of secretion is taken over by the glandular outgrowths 
upwards. 

Anatomical evidence is also significant in this context. The glandular out- 
growths in Puya, Costus.. Tapelnochilus and Kaempferia rosea 2 re vasculaiised by 
the ptacental bundles. As the glands ate elevated to an epigynous position, the 
placental bundles are replaced by the vascular tissue derived from an anastomosing 
vascular plexus which is generally developed at the top of the ovary in the zingibers 
(rf. Pai 1966). 

The condition in Puya may, therefore, be considered as transitory ; rather the 
origin of epigynous nectaries of most zingiberaceous taxa may have to be sought 
from extensively developed septal glands. 

The sepals receive basically five traces while the petals are three-traced. The 
differential twisting of the two whorls of the perianth is characteristic of many 
bromeliads. 

The stamens are one-traced and the outer whorl of the stamens is longer than 
the inner. This is a feature observed in many petaloid monocotyledonous taxa 
(Kulkarni 1973 ; Markandeya 1978 ; Vaikos 1974 ; Vaikos etal 1978) and demon- 
strates the trend in differentiation of the two androecial whorls and the ultimate 
reduction of either whorl. In the bromeliads, this has not made much headway 
as is revealed by a study of many genera of the family (Kulkarni, unpublished 
data). There is a short crest for the anther which is not described in most 
taxonomic accounts. However, it does not appear to be of pertinent phylogenetic 
significance. 



Acknowledgements 

The authors are grateful to Dr Merxmuller for his generous help with the flowering 
material. Thanks are due to the University Grants Commission, New Delhi, 
and Marathwada University, Aurangabad, for the award of a teacher fellowship 
to one of them (RAK). RAK also thanks the Government of Maharashtra 
and the Principal, Government College of Arts and Science, Aurangabad, for 
permission to accept the same. 



References 

*Agthe C 1951 Uber die Physiologische Herkunft des Pflanzennektars ; Ber. Schweiz. Bot. Ges. 

61 240-277 
Brown W H 1938 The bearing of nectaries an the phytogeny of flowering plants ; Proc. Am, 

Phil. Soc. 79 549-59? 



478 



R A Kulkami and R M fai 



J V 1973 



, 

Pittendrigh C S 19 k 48 H T1 ^^Q^ e K "954 The floral anatomy of some Scitatmneae I .; 

^7. Indian bat. Soc. 33 118 ^ 47 the Unes of evolution in the Bromeliaceae ; 

Smith L B 1934 Geographical evidence on m 

""-* <R " WCMam ' i 



M^rathwada Univ. I97g The flora , anatomy of tlie Liliaceae-the 

s N P, Markandeya a *. ana. ." . 
*** /- *' 1 61 - 168 



; *** 
Not consulted in the original 



Proc. Indian Adad. Sci. (Plant Sci.)> Vol. 91, Number 6, December 1982, pp. 479- 486. 
Printed in India. 



Cytological studies on certain Acanthaceae from Central India 



M I S SAGGOO and S S BIR 

Department of Botany, Punjabi University, Patiala 147002, India 

MS received 17 September 1981 ; revised 31 August 1982 

Abstract Cytological studies have been made on 19 species of Acanthaceae from 
Pachmarhi hills in Central India. Present studies reveal the first count of chromo- 
some numbers for four species, namely, Dyschoriste depressa Nees, n = 30 ; 
Lepidagathis fasciculata Nees, n = 10 ; L. hyalina Nees, n = 10 and Justkia diffusa 
Willd. var. prostrata Roxb., n = 9. New cytotypes have been located in three 
species as Hemi%mphis latebrosa Nees, n = 28 (4x) ; Rungia parviflora Nees, n = 13 
(2x) and jR. pectinata Nees, n = 13 (2x). Diploid cytotypes of three speder, v/z., 
Blepharis maderaspaiensis (Linn.) Roth, n 15 ; Justida betonica Linn., n = 17 
and Thunbergia alata Bojer ex. Sims, n 9 have been detected for the first time 
from India. An analysis of the worked out species reveals the existence of only 
10-53% polyploid species. 

Keywords. Cytology ; polyploidy ; cytotypes ; Acanthaceae. 



1. Introduction 

Acanthaceae is a large pantropical family of about 250 genera and 2,500 species 
(Airy Shaw 1973). The members are chiefly distributed in tropics and sub-tropics 
but are also found in the Mediterranean regions. There is great diversity in 
habit and habitat of the members of the family which are usually herbs or shrubs 
and very rarely small sized trees. Economically, the family is important due 
to the presence of large numbers of ornamentals and some medicinal plants. 

In India the family is represented by about 427 species falling in 81 genera 
(Santapau and Henry 1973). Although the members are widely represented in 
our flora and are of economic importance yet very little attention has been paid 
towards their cytological analysis. The only contributions are by Narayanan 
(1951); De (1964; 1966); Joseph (1964) ; Kaur (1965a,b, 1966, 1970; Kaur and 
Nizam (1970); Vermaand Dhillon(1967); Datta and Maiti (1968, 1970), Sareen 
and Sanjogta (1976) and Krishnaswami and Menon (1974). In spite of these, 
the members of the family from Pachmarhi hills, Central India by and large 
have not been worked out. Therefore, as a part of our project of cytological 
studies on the flora of Central India, the present work was taken up in 1978. 

479 



480 MIS Saggoo and S S Sir 

Table 1. Ciuromosome numbers in members of Acanthaceae from Central India. 



Name of Taxan 



Locality PUN Chromosome Ploidy 

Accession number level 

number/s 



1 


23 4 


5 


Adhatoda vasica Nees 


Pachmarhi (M.P.) 24240 n - 17 
Jata Shankar, 
1,000 m. 


Diploid 



Blepharis mademspatensis (Linn.) Pachmarhi 
Roth (= B. boerhaaviaefolia Pers) Little Fall, 

950m. 

Crossandra infundibuliformis Nees Pachmarhi 

Raj Bhawan, 
1,000m. 



Didiptera bupleuroides Nees 



D. roxburghiana Nees 



*Dyschoriste depressa Nees 



Gendarussa vulgaris Nees 
(= Justida gendarussa Linn.) 

**Hemigraphis latebrosa Nees 



Pachmarhi 
Jata Shankar, 
1,000m. 

Pachmarhi . 
Pathar Chata, 
900m. 

Pachmarhi : 
Raj Bhawan, 
1,000 m. 

Pipariya (M.P.) : 
200m. 

Pachmarhi : 
Jata Shankar, 
1,000m. 



Hygrophila auriculata (Schum.) Heine Pachmarhi : 
(=*H. spinosa T. And.) 1,050m. 

Justida betonica Linn. 



/. diffuse* Willd. 



*J. diffusa Willd. var. 
prostrata Roxb. 



Pachmarhi : 
Bee Fall, 
950 m. 

Pachmarhi : 
Mandadeo Caves, 
900m. 

Pachmarhi : 
Mandadea Caves, 
900m. 



20847 n 15 Diploici 
(figure 11) 

24235 n = 19 Diploid 



24207 /i = 13 Diploid 

(%ure 2) 



24208 n = 13 Diploid 

(figure 1) 



20841 TI = 30 Tetraploid 
(figure 12) 



24223 n 15 Dipioid 

(figuic 3) 

20832 n 28 Tetraploid 
(figure 13) 



24228 n 16 Diploid 

(figure 4) 

20840 TI 17 Diploid 
(rigure 5) 



24232 n - 9 Diploid 

(figure 7) 



20839 n * 9 Diploid 

(figure 6) 



Cytological Studies on Acanthaceae 



481 



Lepidagathis cuspidata Nees 


Pachmarhi : 
Dhupgarh, 
1,200m. 


24239 H = 11 
(figure 14) 


Diploid 


L. fasciculata Nees 


Pachmarhi : 
1,000m. 


20830 n = 10 
(figure 16) 


Diploid 


*L. hyalitiG Nees 


Pachmarhi : 
Jambu Dwip, 
950 m. 


20838 n = 10 
(figure 15) 


Diploid 


Petalidium bralerioides Nees 


Pachmarhi : 
Pagara, 
800m. 


24237 n = 16 
(figure 8) 


Diploid 


**Rungia parviflora Nees 


Pachmarhi : 
Jata Shankar, 
1,000m. 


20833 n = 13 
(figure 17) 


Diplcid 


*'*jR. pectlnata (Linn.) Nees 


Pachmarhi : 
Polo garden, 
950m. 


20842 n - 13 
(figure 9) 


Diplcia 


Thunbergia alata Bojer ex Sims 


Pachmarhi : 
1,050m. 


24226 n = 9 
(figure 10) 


Diplcid 



* Reported for the first time. 

** Reports of new chromosome number. For previous reports reference is made to Darlington 
and Wylie (1955), Love and Love (1961, 1964, 1975) Fedorov (1969). Index to Plant Chromo- 
>ome numbers (1956-1974) and IOPB chromosome number reports (1964 onwards). 

2. Material and methods 

Materials for the present investigations were collected from different populations 
>f wild as well as cultivated plants from Pachmarhi and its surroundings. The 
specific locality and altitude of each taxon are indicated in table 1. For meiotic 
itudies anthers from young flower buds were squashed in 1% acetocarmine after 
ixing for 24 hrs in carnoy's fluid. Slides were made permanent in the usual 
nanner and mounted in cuparal. Voucher specimens have been deposited in the 
rlerbarium, Punjabi University, Patiala (PUN). 

f. Observations 

nformation about chromosome numbers of the presently worked out 19 species 
>elonging to 13 genera of the family has been provided in table 1 (figures 1-17). 
Dourse of meiotic division was found to be normal in all the worked out taxa. 

L Discussion 

'iemigraphis latebrosa Nees with n = 28 is a new record for the species and is 
etraploid. Earlier Sareen and Sanjagta (1976) recorded a diploid species with 

P (B) 2 



482 MIS Saggoo and S S Sir 

n = 14 from North India. Both the presently worked out species of Rungia 
namely, R. parviflora Nees' and R. pectinata Nees are diploid with n = 13 
Record of n = 13 for R. parviflora Nees is new because the earlier reports are 
of 7i = 8 (Baquar 1967-68), n = 15 (Datta and Maiti 1970) and n = 26 (Mehra 
and Vasudevan 1972). Similarly, count of n = 13 for R. pectinata Nees is 
different from that of 2n = 50 by De (1966) for the same species. So far only 
3 species of Rungia are known cytologically. Datta and Maiti (1970) suggested 
x = 10 to be the base number for the genus, but the possibility of polybasic 
nature of genus with numbers x = 8, 10 13, 15, 25 cannot be ruled out although 
x = 15, 25 would naturally be secondary basic numbers. 

Cytotype of Blepharis maderaspatensis (Linn.) Roth with n = 15 has been 
worked out for the first time from India. Earlier, Kaur (1966) recorded n = 13 
from South India. From Africa, n = 15 had earlier been reported by Miege 
(1960). Apparently, the present species is diploid. Cytological information about 
Blepharis is meagre since only 5 out of a total of 100 species have been worked 
out so far. Somatic members reported for the genus are In = 24, 26, 30, 34, 
clearly indicating polybasic nature with base numbers x = 12, 13, 15, 17. 

Dyschoriste depressa Nees (n = 30) has presently been worked out for the first 
%ne and is a tetraploid. Base number x = 15 is well established in all the 
cytologically worked out species. Justida diffusa Willd. var. prostrata Roxb. 
with n = 9 has been worked out for the first time. Justida diffusa Willd. with 
n = 9 and /. betonica Linn, with/* = 17 confirms the earlier reports of n = 9 by 
Mehra and Vasudevan (1972) and Bir and Sidhu (1974) for the former and n = 17 
by Ellis (1962) for the latter. Narayanan (1951), however, recorded In = 28 fo r 
J. betonica Linn. A perusal of literature reveals the polybasic nature of the 
genus with x = 9, 13, 14, 15, 16, 17. Base numbers x = 9 and 14 are of 
common occurrence. Thus all the three presently worked out species of Justida 
are at diploid level. Lepidagathis fasdculata Nees (n = 10) and L. hyalina 
Nees (n = 10) have been worked out for the first time. The other species, 
L. cuspidata Nees with n = 11 confirms the earlier counts by Verma and Dhillon 
(1967). All the three present species are diploid. 

The basic numbers in the family range from x = 8-25 but the commonest 
numbers are jc = 9, 14, 16, 17. A number of genera show polybasic nature as 
Barleria (x = 15, 16, 19, 20), Blepharis (x = 12, 13, 15, 17), Thunbergia (x 9, 
10, 14, 16), Rungia (x = 8, 10, 13, 15, 25), Justida (x = 9, 13, 14, 15, 16, 17) 
and Strobilanthes (x = 8, 9, 10, 11, 13, 14, 15). The variability in the base 
numbers and polybasic nature of several genera clearly indicate that cytologically 
the family is a highly evolved one and all this could possibly be the result of 
aneuploidy operative at generic level. This has led to the evolution of morpho- 



Figures 1-10. Meiosis in pollen mother cells. 1. Didiptera roxburghiana, M-I 
with 1311. 2. D. bupleitroide.-, n = 13 at M-I. 3. Gendarussa vulgam, M-I 
with 1511. 4. Hygrophila auriculata, M-I with 1611. 5. Justida betonica, 1711 
at M~T. 6. J. diffusa, n = 9 at diakinesis. 7. /. diffusa var. prostrata, h =* 9. 
8. Patalidium barlenoides, 1611 at M-I. 9. Rungia pectinata, 13H at M-I. 
10. Tliunbergia date, M-TI with n = 9. 



Studies on Acanthaceae 




483 




FIGS. 3.8.l6 

20nm 
FIGS.1.2A.5.6.7.9 




Figures 1-10. 



Cytological studies on Acanthaceae 485 




Figures 11-1 ;. Meiasis in pcllen mother cells. 11. Blepharis niaderaspatensis, 
n 15 at diakinesis. 12. Dyschoriste depressa, 3011 at M-1. 13. Hemigraphis latebrosa, 
diakinesis showing 2811. 14. Lepidagathis cuspidata, n = 11. 15. JC. hyalina 
showing 10 + 10 chromosomes, at M-II. 16. L. fasciculate* with 1011 at M-I. 
17. Rungia parviflora, M-I showing n = 13. 

ogical variations. Out of presently investigated 19 species of Acanthaceae only 
wo (10*53%) are polyploids and both at tetraploid level only. Taking an overall 
dcture on the basis of the world- wide cumulative data, it is seen that only 17*81%* 
>f the worked out species are polyploids. Intraspecific polyploidy is so 
ar reported in only eight species, namely, Crossandra infundibuliformis (2x, 6x) ; 
7. nilotica (4#, 6#) ; Didiptera elagans (2x, 4x), Hemigraphis latebrosa (2x, 4x) ; 
lungia parviflora (2x, 4x) and Thunbergia grandiflora (2x> 4x). It appers that 
olyploidy has not been as potent a factor of cytological evolution in the family 
3 aneuploidy. 



Out of 2500 species in Acanthaceae, only 219 are worked out and amonsst these 180 soecies 
:e diploid and 39 polyploids (for details see footnote to table 1). 



486 MIS Saggoo and S S Bir 

Acknowledgement 

One of the authors (MISS) is thankful to CSIR, New Delhi, for the award of , i 

Research Fellowship. . j 

References ; 

Airy Shaw H K 1973 A dictionary of the flowering plants and ferns (ed.) J C Willis 8th ecL f 

(Cambridge : Univ. Press) pp -ixxii. 1-1245 I 

Baquar S R 1967-6$ Chromosome numbers in some vascular plants of East Pakistan ; Rev. J 

BioL 6 400-448 ( 

Bir S S and Sidhu M 1974 In Love A : IOPB chromosome number repoits XLIV ; Taxon 

23 373-380 j 

Darlington C D and Wylie A P 1955 Chromosome atlas of flowering plants (London : George ! 

Allen and Unwin Ltd.) pp. i-xx, 1-519 
Datta P C and Maiti R K 1968 Chromosomal biotypes of Adhatoda vasica Nees. growing in 

the Eastern Part of India ; Cytologia 33 220-229 
Datta P C and Maiti R K 1970 Relationships of Justicieae (Acanthaceae) based on cytology ; 

Genetica 41 431-450 
De A 1964 Cytological investigations in the family Acanthaceae and importance in the study J" 

of phylogeny ; Proc. 51st and 52nd Indian Sci. Cong. Session Sect. Bot. Abst. pp. 354 
De A 1966 Cytological investigations ir> the family Acanthaceae ; Sci. Cult. 32 198-199 
Ellis J L 1962 Chromosome numbers in some members of Acanthaceae ; Sci. Cult. 28 191-192 
Fedorov An A (ed) 1969 Chromosome numbers of the flowering plants ; Academy of Sciences 

of the USSR Komarov Botanical Institute Leningrad (1974 reprint) pp. 1-928. 
Joseph J 1964 Chromosome numbers and abnormalities observed in a few members of 

Acanthaceae ; Curr. Sci. 33 56-57 |* 

Kaur J 1965a Chromosome numbers in Acanthaceae J ; Curr. Sci. 34 295 i 

Kaur J 1965b Chromosome numbers in Acanthaceae II ; Sci. Cult. 31 D3 1-532 ; 

Kaur J 1966 Chromosome numbers in Acanthaceae III ; Sci. Cult. 32 142-143 

Kaur J 1970 Chromosome numbers in Acanthaceae V ; Sci. Cult. 36 103-106 ! 

Kaur J and Nizam JT 1970 Karyotype analysis of some members of Acanthaceae ; Nucleus j 

13 23-28 j 

Krishnaswami S and Menon P M 1974 Cytomorpho logical studies on some species and an 

interspecific hybrid of Barleria L. ; Cytologia 39 397-402 ' \ 

Love A anc L5ve D 1961 Chromosome number of Central and North-West European plant species ; I 

Opera Botanica (Stockholm : Ainquist and Wiksell) pp. 1-581 
Love A and Love D 1974 Cytotaxonomical Atlas of the Slovenian Flora ; J. Cramer. FL-3301 

Lehre pp. i-xx, 1-1241 , 

Love A and Love D 1975 Cytotaxonomical Atlas of the Arctic Flora ; /. Cramer. FL-9490 

Vaduz pp. i-xxiii, 1-598 
Mehra P N and Vasudevan K N 1972 In Love A : IOBP chromosome number reports 

XXXVI ; Taxon 21 333-346 
Miege J 1960 Troisieme lists de nombres chiamosomiques despecas d' Afrique o> cidentale ; 

Am. Fac. Sci. Univ. Daker 5 75-85 

Naiayan?n C R 1951 Somatic chromosomes in the Acanthaceae; J.Madras Univ. B21 220-231 ; 

Santapau H and Henry A N 1973 A Dictionary of the Flowering Plants in India (New Delhi CSIR) 

pp. i-viii, 1-198 
Sareen T S and Sanjogta, K 1976 Chromosome numbers in some species of Acanthaceae ; 

Cytologia 41 283-290 
Verma S C and DhUlon S S 1967 In Love A : IOPB chromosome number reports XI : Taxon : 

16 146-157 



Proc. Indian Acad. Sci. (Plant Sci.), Vol. 91, Number 6, December 1982, pp. 487-493. 
Printed in India. 



Heterotrophic bacteria associated with seaweed 



P LAKSHMANAPERUMALSAMY* and A PURUSHOTHAMAN 

Centre of Advanced Study in Marine Biology, Annamalai University, 
Parangipettai 608502, Tamil Nadu, India 

* Present address : Department of Marine Sciences, Univeisity of Cochin, 
Cochin 682016, India 

MS received 30 March 1982 ; revised 30 August 1982 

Abstract The heterotrophic bacterial population associated with seaweeds 
(Enteromorp/ta sp., Chaetomorpha sp. and Hypnea sp.) and water of the Vellar 
Estuary, Porto Novo, were estimated. Total heterotrophic bacteria associated 
with the seaweeds were found to be more abundant than in water samples. Repre- 
sentative cultures wore isolated and their morphological and biochemical charac- 
teristics were studied. In addition, pi eduction of amylase, lipase and protdnase 
of the isolates was also studied. Bacillus, Corynebacterium, Vibrio, Alcaligenes 
and Pseudomonas woro the genera commonly encountered. The role of these 
bacteria associated with seaweeds is discussed. 

Keywords. Heterotrophic bacteria; seaweed; Enteromorpha sp. ; Chaetomorpha sp. 
Hypnea sp.; Vellar estuary; Porto Novo. 



1. Introduction 

Available information, on mioro-o^gantsrm in an estuarins environment concerns 
water and sediment predominantly. The epiphytic bacterial flora of seaweed 
appears to have been neglected. Bacteria adhere to many types of solid surfaces, 
probably by means of several mechanisms and in some cases in a selective manner 
(Daniel 1972 ; Gibbons and van Houte 1975 ; Marshall 1976). Currently increasing 
attention has be0n paid to bacterial adhesion and its ecological significance. Some 
are adhesive to rocks in flowing streams (Geesey et al 1977), to suspended matter 
and solid surfaces in an estuary (Goulder 1976, 1977 ; Austin et al 1979a), to 
animal surfaces (Gibbons and van Houte 1975) and to sea weed (Sieburth 1962, 
1968 ; Kong and Chan 1979 ; Sjoblad and Mitchell 1979 ; Shiba and Taga 1980). 
Much interest has not been shown on the epiphytic bacteria associated with 
seaweed in Indian estuaries, except by Chandramohan (1971). The purpose of 
the present study is to describe hetepotrophic bacteria present in water and in 
association with seaweed (Enteromorpha sp. ; Chaetomorpha sp. ; and Hypnea sp.) 
at a particular location in a tropical estuary such as Vellar. 

487 
P. (Bh-3 



488 P Lakshmanaperumalsamy and A Purushothaman 

2. Material and methods 

Seaweed samples were collected and transferred to sterile polyethylene bags after 
draining completely. Water samples were collected in sterile glass bottles. The 
samples were stored in a small insulation container (5 C) and brought to the 
laboratory. All the samples were plated within an hour of collection. 

A portion of the seaweed was washed separately in sterile water and trans- 
ferred to 100 ml sterile estuarine water blank in a 500 ml flask shaken on a 
reciprocal shaker (120 strokes per min) for 30 min at room temperature. The 
bacterial population was estimated by serial dilution plate method using Estuarine 
Peptone Yeast Extract Agar (EPYA; Bacto Peptone 1 %, Yeast extract Difco 0-3%, 
Bacto Agar 2%) and the bacterial population was expressed as number s/g dry 
weight of the seaweed and per ml of water. Bacterial strains were randomly 
isolated, purified by repeated streaking and identified to various genera using 
the taxonomic key of Simidu and Also (1962). Amylolytic, proteolytic and 
lipolytic ability of the isolates were also tested (Harrigan and McCance 1972) 

3. Results 

The total aerobic heterotrophic bacterial population from all the samples were 
estimated and the results are presented in table 1. The epiphytic bacterial popu- 
lation associated with Chaetomorpha sp. varied from 1*16 to 9*22 x 10 8 /g, 
Enteromorpha sp. from LI 3. to 18-63 X 10 6 /g and Hypnea sp. from 1*14 to 
13-75 x 10 6 /g dry weight. Estimates of bacterial populations in the water sample 
ranged from 3*9 x 10 3 to 1 -53 x 10*/ml. The maximum bacterial population 
among the samples was recorded in October. Highet? (18-63 x 10 6 /g) number 
of epiphytic bacteria was found to be associated in Enteromorpha sp. followed 
by Hypnea sp. and Chaetomorpha sp. Since this year experienced a heavy rain- 
fall in monsoon season, no seaweed was found in the estuarine environ- 
ment in November 1978 through February 1979, and this might be due to 
heavy freshwater inflow and lower salinity. They appeared again in March. So, 
no data could be collected between November 1978 and February 1979. 

The total heterotrophic bacterial flora of water in Vellar estuary was found to 
consist of Bacillus (13-33%), Corynebacterium (20%,) Micrococcus (10%), Vibrio 
(16*67%), Pseudomonas (16*67%), Alcaligenes (10%), Flavobacterium-Cytophaga 
group (10%), and Enterobacteriaceae (3*33%) as shown in table 2. In general, 
gram-negative bacteria were more common (56*67%) than gram-positive. How- 
ever gram-positive groups (Bacillus, Corynebacterium and Micrococcus) were 
abundant in April than the gram-negative groups. Bacillus, Vibrio, Pseudomonas, 
Alcaligenes, Flavobacterium-Cytophaga were recorded in all the samples. The 
maximum number of Corynebacterium was recorded in March. The results 
suggest that the dominant epiphytic flora associated with seaweed, Chaetomorpha 
sp., Enteromorpha sp. and Hypnea sp., is Vibrio. Various genera associated were 
Akaligenes, Flavobacterium-Cytophaga group, Pseudomonas, Enteiobacteriaceae, 
Corynebacterium and Micrococcus. The domination of gram-negative bacteria 
was observed in all collections. The epiphytic 'Vibrio "peak was recorded in 
October in association with all three seaweeds and a reduction in number was 



Heterotrophic bacteria associated with seaweed 
Table 1. Total aerobic hetero trophic bacterial population. 



489 



Month Chacromorpha sp. Enteromorpha sp. 
(10 6 /g) (10 ft /g) 


Hypnea sp. 
(10 8 /g) 


Water 
(10* /ml) 


September 1978 
October 
March 1979 
April 
May 


3-51 
9-22 

3-81 
1-76 
1-16 


3-19 
18-63 
2-93 
1-36 
1-13 


4-32 
13-75 
4-10 
1-49 
1-14 


4-9 
15-3 
15-1 
3-9 
4-2 


Table 2. 


Total number of 


isolates assigned to 


various genrra. 




Choetomorpha sp. Enteromorpha sp. 


Hypnea sp. 


Water 


Total nurr-ber of 
isolates tested 


72 


74 


73 


60 


Bacillus 


... 


... 


... 


8 
(13-33) 


Corynebacterium 


2 
(2-78) 


3 
(4-05) 


1 
(1'37) 


YL 
(20-00) 


Micrococcus 


2 
(2-78) 


1 
(1*35) 


... 


6 
(10-0) 


Vibrio 


42 
08-33) 


38 
(51-35) 


40 
(54-8) 


10 
(16-67) 


Pseudomonas 


8 
(1M1) 


11 
(14-86) 


14 

(19-18) 


10 
(16-67) 


Alcallgenes 


4 
(5-56) 


3 
(4-05) 


5 
(6-85) 


6 
(10-0) 


Fkvobacterium-Cytophaga 8 
group (11*11) 


10 
(13-51) 


9 
(12-33) 


6 
(10-0) 


Enterobacteriaceae 


6 
(8-33) 


8 
(10-81) 


4 
(5-48) 


2 
(3"33) 



(Values in parentheses are percentages in total number of isolates tested). 

noticed in March. Members of genus Vibrio could be isolated throughout the 
period of study and formed major portion of the flora, followed by Flavobacterium- 
Cytophaga group, Pseudomonas and Alcaligenes, 



490 P Lakshmanaperumalsamy and A Purushothaman 

The bacterial flora in water and in association with seaweed were assigned to 
various physiological groups (table 3). Majority of heterotrophs in water appeared 
proteolytic (63*33%), lipolytic (61*67%) and a low percentage (43 '33%) amylo- 
lytic. Bacterial populations associated with Chaetomorpha sp., was dominated 
by proteolytic (56*16%) followed by lipolytic (48.61%). 



4. Discussion 

Results of this study on the bacterial populations in water are comparable to 
those reported for Long Island Sound, Chesapeake Bay and Tokyo Bay. 
Altschular and Riley (1967) and Murchelano and Brown (1970) reported 10 3 to 
10 4 bacteria pcsr ml in Long Island Sound and 8' 4 X 10 1 to 2*0 X 10 4 /ml in 
Chesapeake Bay and 1* 8 x 10 1 to 9" 1 X 10 4 /ml in Tokyo Bay (Austin et al 
1979b). 

The maximum bacterial populations were recorded in October and minimum 
in April, within the period of study. According to Velanker (1969), the numerical 
magnitude of heterotrophs broadly parallels the distribution of other living orga- 
nisms and the dissolved organic matter in the sea. Murchelano and Brown (1970) 
suggested that the annual bacterial cycle in Long Island Sound coincided with 
that of phytoplankton. Phytoplankton constitutes a locus for bacterial attach- 
ment and produces organic substrates for bacterial utilization. Ecological para- 
meters, viz., physical, chemical and biological can have a definite influence on the 
bacterial population in an estuary. The annual bacterial cycle can be studied 
by repetitive sampling at one geographic locus, along with environmental para- 
meters. Since this study was done for only five months, no definite conclusions 
could be reached regarding the annual cycle. The density of bacterial populations 
associated with seaweeds varied considerably, from 1*3 to 18*63 x 10 6 /g. The 
epiphytic populations may result from availability of vitamins, growth factors or 

Table 3. Physiological groupings of the bacterial isolates. 



Sample 


Total cumber 
of isolates 


Proteolytic 


Amylolytic 


Lipoly'ic 


Chaetomorpha sp. 


77 


39 
(54-16) 


12 
(16-67) 


35 
(45-61) 


Enteromorpha sp. 


74 


42 
(56-75) 


9 
(12-16) 


37 
(50-0) 


Hypnea sp. 


73 


35 
(47-94) 


13 
(17-81) 


35 
(47-94) 


Water 


60 


3g 
(63-33) 


26 

(43-33) 


37 
(61-67) 



(Values in parantheses are percentages of isolates tested). 



Heterotrophic bacteria associated with seaweed 491 

other external metabolites extruded by actively growing algal filaments which 
nourish the epiphytic flora (Fogg 1966 ; Sieburth 1968 ; Wetzel 1969 ; Wetzel 
and Allen 1972 ; Bell etal 1974; Morishita etal 1978). Chandramohan (1971) 
reported l"26xlO G /g bacterial population associated with Enteromorpha 
mtest inalls in the Vellar estuary. Actively growing algal filaments were reported 
to carry high numbers of epiphytic bacteria (Sieburth 1968 ; Ramsay and Fry 
1976). Mary (1977) recorded high bacterial counts when mullets wefte fed on 
large quantities of green filamentous alga Enteromorpha sp., which might account 
for the epiphytic flora associated with the algae in the same area. Quantitative 
data for bacteria were significantly high when there was an abundance of phyto- 
plankton, Navicula sp. 

The generic composition of the bacteria in water showed the presence of various 
groups in Vellar estuary. In general, gram-negative bacteria were predominant 
compared with gram-positives. On a generic basis, Corynebacteriwn were domi- 
nant (20%), followed by Vibrio (16' 67%), Pseudomonas (16' 67%) and Bacillus 
(13*33%). Murchelano and Brown (1970) reported that Pseudomonas was the 
dominant bacterial group in Long Island Sound and. Pseudomonas, Achromobacter 
and Flavobacterium composed of 92* 3% of the bacteria isolated. Vibrio, Bacillus, 
Micrococcus and Cytophaga accounted for only 7* 3%. The various studies carried 
out using water samples showed a domination of Vibrio and Achromobacter 
in Chesapeake Bay, Vibrio in Kanagawa Bay, Pseudomonas in Long Island Sound 
and Flavobacterium in Naragansett Bay (as cited by Murchelano and Brown 1970). 
Austin etal (1979b), in their comparative study, found that the prominent aerobic 
heterotrophic bacterial flora of the water column of Chesapeake Bay consisted of 
Vibrio, Achromobacter, Pseudomonas and Corynebacterium, but in Tokyo Bay 
predominantly Acinetobacter-Moraxella-tifo species, Caulobacter and Pseudo- 
monas. It is difficult to quantify the bacterial genera diversities unless considerable 
data are collected. 

It may be inferred from the study reported here that members of seven genera 
were found associated with seaweeds. Vibrio was found dominating over Pseudo- 
monas, Flavobacterium and Alcaligenes. Abundance of Vibrio and Alcaligenes 
during luxurious growth of algae (Enteromorpha sp.) suggested that these genera 
were probably associated with algal bloom. The recovery of large population 
of Vibrio throughout the study suggests that they form part of indigenous flora 
of seaweed. Presence of epiphytic flora of marine algae has been reported by 
Sieburth (1968) who examined species of Polysiphonia and Sargassum foi? possible 
generic specificity of epiphytic flora and noticed Vibrio as the dominant species in 
both. Kong and Chan (1979) reported seven genera to be associated with marine 
algae and similar study was conducted by Shiba and Taga (1980) who found that 
Flavobacterium was the dominant microflora. Vibrio were present in minimum 
numbers, suggesting metabolites being toxic for Vibrio sp., a phenomenon not 
observed in this investigation. No other taxonomic study has been carried out 
to date on epiphytic bacteria associated with seaweed in Indian estuaries. The 
generic composition, seasonal variation and interrelationships of associated hetero- 
trophic bacteria remain to be defined. 

The existence of bacterial populations on seaweed suggests that there exists a 
beneficial relationship between the seaweed and the epiphytic bacteria. The 
bacteria belonging to the genus Vibrio were predominant on the seaweed. It is 



492 p Lakshmanaperumalsamy and A fiimshothaman 

reported that the extract of seaweed acted as attractant for the marine bacterium, 
Vibrio algmolyticus, by Sjoblad and Mitchell (1979). Hence it may be suggested 
that a beneficial relationship exists between seaweed and their epiphytic bacteria 
Vibrio. 



Acknowledgements 

Thanks are due to Dr R Natarajan, Director, for his encouragement and to 
Dr D Chandramohan, Scientist, NIO, Goa, for his comments. One of the 
authors (PL) thanks CSIR and UGC for financial assistance. 



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Heterotrophic bacteria associated with seaweed 493 

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Wetzel R G and Allen H L 1972 Functions and interactions of dissolved organic matter 
and the littoral zone in lake metabolism and eutrophication ; In Productivity problems in 
freshwaters (eds) Z Kajak and A Hillbricht-IIkowaka (Warszawakrakowa) pp. 333-347 



Proc. Indian Acad. Sci. (plant Sci.), Vol. $1. lumber 6, fiecember 1^8:2, pp. 
Printed in India. 



Association of chlorophyll content, phyllotaxy, photosynthesis and 
B group vitamins in some C 3 and C { plants 

P GOPALA RAO and J KODANDARAMAIAH 

Department of Botany, Sri Venkateswara University, Tirupati 51 7 502, India 

MS received 17 November 1981 ; revised 21 October 1982 

Abstract. The phatosynthetically efficient C 4 plants viz Amamnthus viridis, Euphorbia 
hirta and a C 3 plant, Acalypha indica with mosaic leaf pattern showed the maximum 
amount of B vitamins when compared to the other C 3 plants. It is observed that 
photosynthesis and vitamin synthesis go hand-in-hand showing close correlation. 
The results also indicate that there is a close relation between chlorophyll content 
and vitamin content. However, there appears to be no relation between phyllotaxy 
and photosynthesis. Between the two C 3 plants, viz., Acalypha and Carica* the 
photosynthetic inefficiency of the latter might be due to more cf chlorophyll b and 
less of chlorophyll a as seen from chlorophyll a] chlorophyll b ratios. 

Keywords. Chlorophyll ; B vitamins ; phyllotaxy ; photosynthesis ; C% and C 4 
plants. 



1. Introduction 

The chlorophyll content of the cell must be closely associated with photosynthetic 
activity because the photosynthetic rate is proportional to the chlorophyll concen- 
tration (Maksymowych 1973), but there are contrary reports also. Black (1972) 
concluded that the high rate of photosynthesis/mg chlorophyll is directly related 
to the low chlorophyll content. The photosynthetic rates of different chlorophyll 
mutants of pea, soybean, cotton and tobacco were studied by Benedict (1972). 
The photosynthetic rate/mg of chlorophyll in mutants is 2-11 times faster than 
in the wild type leaves. This phenomenon is compared to the photosynthetic 
rate of the yellow sectors of variegated leaves. These yellow sectors although 
containing a reduced chloropyhll content, have a much higher photosynthetic 
rate/mg chlorophyll than the green sectors. Bonner and Bonner (1948) found 
that thiamine synthesis in seedlings is often light-dependent and mature leaves of 
full grown tomato plants are the centre of production. Gustafson (1948) sugges. 
ted that light stimulates thiamine biosynthesis. It was stated that the photosynthe- 
tic unit and not the chlorophyll content determines the rate of photosynthesis 
(Black 1972). 

495 
P. (B) 4 



496 P Gopala Rao and J Kodandaramalah 

The present investigation has been designed to understand the relationship 
between chlorophyll content, vitamin content, phyllotaxy and photosynthesis in 
some C 3 and C 4 plants. Work on the relationship between phyllotaxy and 
photosynthesis is scanty. Earlier work (Evans 1975) indicates that canopies 
with more vertically inclined leaves have a higher photosynthetic rate than those 
with horizontal leaves. 



2. Materials and methods 

Young and fully expanded leaves from various plant species, viz., (1) Acalypha 
indica, L., (2) Amaranthus viridis, L., (3) Carica papaya, L., (4) Commelina 
benghalensis, L., (5) Euphorbia hirta, L., (6) Euphorbia pulcherrima, Willd., (7) 
Ervatamia coronaria, Stapf., (8) Werium odorum, Soland., (9) Nyctanthus arbor, 
tristis, L., (10) Petunia hybrida, L., (11) Sida acuta, Burn, (12) Tridaxprocumbens, 
L., growing in the university Botanical Garden under natural photoperiod 
constitute the experimental material. 

Chlorophylls were extracted with 80% acetone and estimated according to the 
method of Arnon (1949). The chloroplasts were isolated using N/15 phosphate 
buffer (pH 7*3) containing sucrose (0-33M), disodiumsalt ofEDTA (2 X 10~ 3 M) 
dithiothreitol (5 X 10~ 3 M), MgCl 2 (1 X 10~ 3 M), MgSO 4 (1 ' 5 x 10~ 3 M) and 5% (w/v) 
polyvinylpyrolidene as isolation medium following the procedure of James and 
Das (1957). The method of Jagendorf and Evans (1957) was used to assay Hill 
reaction activity of chloroplast preparation. 14 CO 2 fixation studies were done 
using a technique similar to that described by Berry et al (1970), using 14 C 
sodium bicarbonate (specific activity, 27 mci/m mole) and the net photosynthesis 
was expressed as mg CO 2 fixed dm~ 2 hr~ 1 . 

The different vitamins of the B group, viz., thiamine (BJ 9 riboflavin (J? 2 ), 
pyridoxin (# 6 ), niacin and folic acids were extracted and estimated colorimetri- 
cally following the methods given by Manzur-ul-Haque Hashmi (1973) and the 
results were expressed as //g/g dry wt. All the results were averages of three 
individual experiments. 

3. Results and discussion 

The results in table 1 indicate that Amaranthus viridis, Euphorbia hirta which are 
C 4 photosynthetic plants and Acalypha indica, a C 3 plant with mosaic pattern 
of 'phyllotaxy have maximum Hill activity, net photosynthesis and chlorophyll 
coatent. Commelina benghalensis, Euphorbia pulcherrima, Ervatamia coronaria and 
Sida acuta occupy the next position in order in this respect. Nerium odorum 
shows the minimum activity. 

It is tempting to note that there is a close correlation between the photosyn- 
thetic parameters (table 1) and vitamins of the B group (table 2) viz., thiamine 
(Bj), riboflavin (5 2 ), pyridoxin (5 6 ), niacin and folic acid in the plants mentioned 
above. Nerium odorum shows the minimum amount of vitamins of the B group. 
The data thus gives a circumstantial evidence to show that photosynthesis and 
vitamin synthesis go hand-in-hand showing a close correlation (table 3). 
The photosynthetically efficient C 4 plants viz., Amaranthus veridis, Euphorbia hirta 



B group vitamins and photosynthesis 



497 



Table 1. The pattern of chlorophylls and the rate of photosynthesis in different 
plant species. 





Plant species Phyllotaxy 


Plant Total Chlorophyll Hill 
type chloro- alb ratio activity 
phylls 

# * _i_ 


Net 
photo 
synthesis 

** 


1. 


Acalypha indica L. mosaic 


C 3 4- 


62 


1- 


Ib 


163- 


5 


38- 


4 






0- 


'84 


o- 


04 


4- 6- 


21 


2-22 


2. 


Amaranthus viridis L. alternate 


C 4 3- 


58 


1- 


21 


186- 


2 


45- 


8 


0-22 


o- 


12 


10- 


6 


5- 


16 


3. 


Carica papaya L. mosaic 


C 8 1- 


90 


o- 


18 


121- 


1 


26- 


5 


0-31 


o- 


01 


7- 


81 


1- 


76 


4. 


Commelina alternate 


C 3 2- 


43 


1- 


17 


148- 


4 


31- 


6 




benghalensis L. 





14 





06 


3- 


42 


4- 


32 


5. 


Euphorbia hirta L. opp-super- 


C 4 3 


96 


1- 


25 


169- 


5 


42' 







imposed 





86 





22 


12- 


31 


1- 


78 


6. 


Euphorbia opp-decussate 


C 3 3 


47 


! 


28 


142' 


6 


36- 


8 




pWcherrima 





86 


0' 


08 


db 16- 


2 


6- 


52 




Willd. 


















7. 


Ervatamia opp-decussate 


C 3 2 


02 


1- 


25 


136- 





29- 


1 




coronoria Stapf. 


o 


10 





12 


d= 5- 


61 


I' 


34 


8. 


Neriwn odorum Whcrled 


C 8 1 


69 


1 


63 


84- 


3 


17- 


2 




So-land 





21 


o- 


34 


7- 


52 


2- 


88 


9. 


Nyctanthes opp-super- 


C 3 1 


98 


1 


26 


117- 


5 


21 


6 




arbortristis L. imposed 





28 





07 


11- 


6 


1- 


06 


10. 


Petunia hybrida L. alternate 


C 3 1 


76 


1 


31 


104- 


2 


19 


o 




- 





11 





26 


18 


2 


3 


82 


11. 


Sida acuta Burm. alternate 


C 3 3 


13 


1 


17 


126- 


1 


23 


2 









16 


0-34 


db 7- 


01 


2 


17 


12. 


Tridax procumbens alternate 


C 3 1 


65 


1 


24 


100 


8 


21 


8 




L. 





81 





12 


9 


44 


4 


61 



* mg gr 1 fresh wt. 
** mg CO 2 fixed d- 2 m hr" 1 . 
f p moles of DCPIP reduced mg"" 1 chl hr" 1 . 
^Values are means "S.E. of three individual experiments), 



498 P Gopala Rao and J Kodandaramaiah 

and Acalypha indica, a C 3 plant with mosaic leaf pattern showed the maximum 
amount of vitamins. Commelina benghalensis, Eurphorbia pulcherrima, Ervatamia 
coronaria and Sida acuta form the second group in their vitamin contents quite 
parallel to their photosynthetic parameters. 

Tin results categorically indicate that there is a close relation between chloro- 
phyll content and vitamin content (table 3). The maximum amount of chloro- 
phyll (total) is seen in Acalypha indica (4-62mg/g fresh wt.). Nevertheless, 



Table 2. Vitamin content 0*g g- 1 dry wt.) in the leaves of different plant species. 



Plant 
$PP* 


Thiamine 
(Bj) 


Riboflavin 
(B 3 ) 


Pvridoxin 
(B 6 ) 


Niacin 


Folic acid ' 


Total 


1. 


369-0 
12'1 


124-0 

8-9 


215-0 
16-2 


152-0 
20-4 


134-0 
6-2 


994 


2. 


280-0 
21-3 


136-0 
12-4 


198-0 

17-4 


169-0 
11-2 


149-0 
13-1 


932 


3. 


114-0 
16-2 


82-0 

3-2 


128-0 
db 15-8 


97-0 
8-6 


78-0 

4-4 


499 


4. 


232-0 
31-0 


64-0 

5-7 


176-0 

2M 


160-0 

1M 


52-0 

2-3 


684 


5. 


291-0 
18-6 


99-0 

2-8 


195-0 

8-8 


141-0 

6-1 


130-0 

7-6 


856 


6. 


207-0 
i 26-2 


76-0 

4-9 


180-0 
11-2 


122-0 
10-1 


104-0 
11-2 


689 


7. 


197-0 
16-6 


108-0 
db 13-1 


209-0 

6-8 


135-0 

14-2 


126-0 
13-6 


775 


8. 


74-0 
8-1 


42-0 

3-6 


96-0 

2-4 


28-0 

3-8 


49-0 

6-4 


289 


9. 


139-0 
11*2 


34-0 

2-7 


126-0 

db 7-6 


47-0 
1-2 


61-0 

db 3-4 


407 


10. 


98-0 

14-1 


51-0 

2-6 


113-0 

3-2 


62-0 

5-6 


93-0 

2-8 


417 


11. 


156-0 
21-7 


78-0 
1-7 


174-0 
14-6 


103-0 

2-8 


82-0 
4-1 


593 


12. 


124-0 

6-4 


46-0 

M 


101-0 
9-9 


94-0 
2-1 


76-0 

3-6 


441 



* Plant names as represented in table 1 serially. 
(Values are means S.E. of three replications), 



B group vitamins and photosynthesis 
Table 3. Statistical analysis. 



499 



1. Correlation coefficient (for tables 1 and 2) 
between a set of parameters 



correlation coefficient 



(a) Total chlorophylls x hill activity 


-h 0-822(4-564)* 


(b) 


x photosynthesis 


+ 0- 826 (4- 635)* 


(c) 


x thiamine (Bj) 


4-0-895(6* 346)* 


(d) 


x riboflavin (B 2 ) 


H- 0-730 (3 -378)* 


(e) 


x pyridoxin (B 6 ) 


+ 0-209 (0-676) 


(0 


x niacin 


+ 0-676(2-901)* 


(g) 


x folic acid 


+ 0-907(6-809)* 


(h) 


x total vitamins 


4- 0-849 (5- 083)* 



2. Analysis of variance (for table 2) 



F calculated 

F from table at 5% level 

C.D. at 5% level 



Vitami ns 

26-765* 

2-594 

15-802 



Plant species 

10-515* 
2-152 
8-972 



* Significant at 5% level. 

Values in the parantheses represent the calculated values of f-statistic for testing the significance 

of correlation coefficients. 



chlorophyll a/b ratio is not the highest in Acalypha indica (table 1). Chlorophyll 
alb ratio is maximum (1-63) in Nerium odorum, but the vitamin content is at a 
minimum level indicating that neither chlorophyll a nor b has anything to do 
with vitamin synthesis, but it is the total chlorophyll content that is associated 
with vitamin content, both being at a lower level (tables 1 and 3). 

It is noted that there is no relationship between photosynthesis and phyllotaxy. 
Mosaic pattern of phyllotaxy is assumed to help light to fall directly on all the 
leaves without any impediment, thus making the plant photosynthetically more 
efficient. But, if a comparison is made between Acalypha and Carica (both show 
mosaic pattern) the latter is not at all efficient (table 1). Similarly Amaranthus veridfs 
tias alternate leaves, while Euphorbia hirta which is also a C 4 plant possesses 
3pposite superimposed leaf arrangement thus indicating that there is no relation 
Detween phyllotaxy and photosynthesis. In the case of opposite decussate pattern 
in Ervatamia and Euphorbia pulcherrima where overshadowing is avoided, the 
photosynthetic efficiency is somewhat better than that of Carica papaya with 
mosaic pattern. 

On the 'basis of computations it can be inferred that there is a highly positive 
correlation between total chlorophyll content and other parameters such as Hill 
activity, net photosynthesis, thiamine, riboflavin, niacin, folic acid and the total 
/itamins (table 3). The correlation coefficients of all the parameters are signi- 
icant at 5% level except with pyridoxin. Hence there is an association of chloro- 
phyll content and other parameters (except phyllotaxy). 



SCO P Gopala Rao and J Kodandaramaiah 

Even an analysis of variance (for table 2) reveals a close association among 
the parameters. F-values calculated for vitamins and plant species, are signi- 
ficant at 5% level. 



Acknowledgements 

The authors are highly thankful to Prof. V S Rama Das for his constant 
encouragement and for providing facilities and to Sri P Balasiddamuni, 
Lecturer in Statistics, SV University, Tirupati, for his help in statistical analysis. 



References 

Ainon D I 1949 Copper enzymes in isolated chloroplasts ; polyphenal oxidase in Beta viitgaris ; 

Plant Physiol. 24 1-15 

Benedict C R 1972 S. Sect. Am. Soc. Plant Physiol 7-25 
Berry J A, Downton W J S and Tregunna E B 1970 The photosynthetic carbon rn.tabalism 

o.f Zea mays and Gomphrena globosa : the location of CO a fixation and the carboxyl transfer 

reactions ; Can. J. Bot. 48 777-780 
Bonner J and Bonner H 1948 The B vitamins as plant hormones ; Vitam. Horm. (New York) 

6 225-275 
Black C S 1972 Net carbon dioxide assimilation in higher plants ; S. Sect. Am. Soc. Plant 

Physiol. 1-93 

Evans L T 1975 Crop Physiology, Cambridge University Press, pp. 336 
Gustafson F G 1948 Influence of light intersity upon the concentration of thiamine and ribo- 

flavin in plants ; Plant Physiol. 23 373-378 
James W O and Das VSR 1957 The organization of respiration in chlorophyllous cells ; New 

Phytol 56 325-343 
Jagendorf A T and Evans M 1957 The Hill reaction of red kidney bean chloroplasts ; Plant 

Physiol. 32 435-443 
Manzur-ul-Haque Hashmi 1973 Assay of vitamins m pharmaceutical preparations John Wiley and 

Sons. 
Maksyrnowych R 1973 Analysis of leaf development Cambridge University Press pp, 86 



Proc. Indian Aead. Sci. (Plant ScL), Vol. 91, Number 6, December 1982, pp. 501~5o8. 
Printed in India. 



Effect of morphactin, AMO-1618 and DPX-1840 on the endogenous 
levels of hormones and its implication on apical dominance in 
Glydne max Linn* 



I S DUA, U K KOHLI and K S CHARK 

Department of Botany, Panjab University, Chandigarh 160014, India 

MS received 4 April 1981 ; revised 24 September 1982 

Abstract. Application of morphactin, AMO-1618 and DPX-1840 to. 20 day old 
plants of soybean caused the sprouting of almost all lateral buds. However, the 
follow-up growth of newly ensued buds was manifested only with morphactin 
and not with AMO-1618 or DPX-1840. The quantitative estimations of growth 
promoters, carried out 20 days after the application of inhibitors, revealed that 
the passible mechanism through which these substances exerted their influence on 
apical dominance varied with the type of regulator. It appeared that morphactin 
lifted the apical dominance mainly through the increase in endogenous levels of 
cytakinin and partly through lowering the levels of auxins. AMO-161S checked the 
gibberellin turnover while DPX-1840 suppressed significantly the levels of auxins. 
The data show that apical dominance and not the subsequent growth of newly 
evocated buds is regulated by the ratio of cytokinins (CK) to auxins (Au) plus gibberel- 
lins (QA) and any factoi(s) which enhance(s) CK (like morphactin) or suppresses) 
GA (like AMO-1618) or Au (like DPX-1840 or morphactin) would play a key role in 
the abolition of apical dominance. 

Keywords. Apical dominance ; hormones ; growth retardants ; soybean. 



U Introduction 

Sver since Thimann and Skoog (1934) showed that an application of auxin prepa- 
ration from fungus Rhizopus onto the cut stump of decapitated Vicia faba 
3revented the growth of lateral buds, many workers have put forward a plethora 
3f chemicals which could be utilised for abolition or induction of apical dominance 
n intact plants. In spite of these successful reports by such widely diverse sub- 
itances like IAA (Thimann et al 1971), GA 3 (Bradley and Crane 1960), kinetin 
Ali and Fletcher 1970), B-995 (Brooks 1964), AMO-1618 (Ruddat and Pharis 
1966), morphactin (Tognoni et al 1967 ; Schneider 1970), TIBA (Morey and Dahl 
1975) and various ethylene releasing compounds (Morgan and Durham 1972), 
here has hardly been an attempt to divulge the underlying mechanism through 

501 



502 / S Dua, U K Kohli and K S Chark 

which these regulators might be controlling this phenomenon. Dua et at (1978) 
and Dua and Dhuria (1980) reported that morphactin-induced growth of lateral 
buds was associated with a concomitant increase in the levels of endogenous 
cytokinins. However, a comprehensive hormonal appraisal of apical dominance 
with regard to morphactia vis-a-vis a few other retardants, though unequivocally 
linked with this process, is still lacking. The present investigation attempts to 
study this aspect beside probing into the mechanism of apical dominance in 
Glycine max Linn. 



2. Materials and methods 

Glycine max Linn, cv ' Bragg ' was raised in pots of 25 cm height and 22-5 cm 
in diameter under optimum conditions and 20 day old seedlings were sprayed with 
different inhibitors with an atomiser. The treatments consisted of morphactin 
(methyl-2-chloro-9-hydtoxy-fluorene- (9)-carboxylate-chlorofluornol-methyIestei-li 
3456), DPX-1840 (3, 3a-dihydro-2-[ J p-methoxyphenyl]8H-pyiazolo-[5,l]-a isoindol- 
8-one) and AMO-1618 (ammonium (5-hydroxycaivacryl) iirimethyl chloride 
piperidine carboxylate) of 200, 1000 and 1000 ppm icspectively. The three 
regulators were selected on the basis of previous work and have been used fre- 
quently in relation to studies on apical dominance (see introduction for references). 
Twenty- days after the spray, the data for various morphological characters were 
recorded. For extractions of different endogenous plant growth substances, the 
samples from 20-day old treated or control seedlings were kept in methanol for 
48 hrs at 0C in a refrigerator. The extract was evaporated under suction to 
remove ethanol and residue (pH 3*0) was utilised for the extraction of various 
growth regulatory substances. Extraction procedures described by Nitsoh (1956) 
for auxins, by Murakami (1966) for gibberellins and by Dua and Jandaik (1979) 
for different cytokinins were adopted. Estimates for auxin activity were carried 
out on the basis of growth test of ' Kent ' oat (Avena sativa Linn.) coleoptiles as 
described by Mer et al (1962), gibberellins by the modified technique of Ogawa 
(1963) using 4 Tainan-3' rice (Oryza sativa Linn.) and cytokinin by Xanthium leaf 
disc senescence method of Osborne and McCalla (1961). Standard series of 
cytokinins (zeatin, 2iP, zeatin riboside, zeatin ribotide), gibberellins (GA 3 , GA 4 , 
GA S , GA?) and auxins (LA A, IAN) were run separately for their Rf values. The 
data were statistically analysed according to the variance method. 



3. Results 

3 1. Morphological changes 

The application of various growth retardants alone or in combinations brought 
a number of morphological changes within 20 days of treatment (table 1). The 
plant height vis-a-vis internodal length was reduced drastically. The maximum 
reduction in length (56-1% over control) was recorded when all the three regula- 
tors were present together. The most conspicuous morphological change was 
the growth of lateral buds in all treatments and 50-65% buds sprouted as compared 



Hormonal regulation of apical dominance 



503 



Table I. Effect of morphactin, AMo-1618 and DPX-1840 on the various morpho- 
logical characters in Glycine max Linn, after 20 days of treatment (average of 
5 replications). 





Plant 


Inter nodal 


Number of 


Length of the 


Treatment 


height 


length 


sprouted 


lateral buds 




(cm) 


(cm) 


lateral buds 


(cm) 


Control 


56-21 


18-25 


2-00 


2-23 


Morphactin 


42-25 


13-16 


13-00 


8-76 


AMO-1618 


34-65 


11-25 


10-00 


3-52 


DPX-1840 


38- 75 


12-30 


11-00 


3-89 


Morphactin -1- AMO-16'18 


30-25 


11-03 


16-00 


3-25 


Morphactin + opx-1840 


34-25 


11-32 


16-00 


3-25 


AMO-1618 + DPX-1840 


28-25 


10-97 


15-00 


3-30 


Moiphactin + AMO-1618 -I- DPX-1840 


26-23 


10-81 


17-00 


4-25 



Critical difference at 5 % level of 
significance 



3-63 



2-12 



1-50 



1-93 



to control where only 10% buds were growing. The effect of each chemical, in 
promoting the growth of lateral buds, was further augmented in the presence of 
the second retardant (75-80% buds growing) and maximum effect was detec- 
table when all the three regvlators were present simultaneously (85% buds ini- 
tiated). Another striking revelation was the growth of lateral buds, following 
their release from apical dominance (table 1). Though all the regulatory sub- 
stances significantly abolished apical dominance, to almost the same degree, the 
subsequent growth of the newly ensued buds was insignificant and poor in all the 
treatments except morphactin. It was only in the later treatment (morphactin) 
that a significant follow-up growth of the axillary branches occurred (approxi- 
mately 2-8 times more than control or "other treatments). Surprisingly, in 
the treatments where morphactin was combined with other regulators, the 
morphactin's promotory effect on the growth of lateral branches was also nullified. 

3.2. Behaviour of different endogenous hormones 

3.2a. Auxins'. The estimates of auxins revealed the presence of three auxins at 
Rfs 0' 1-0-2, 0-6-0'7 (IAA) and O'9-l- while IAN (Rf 0'4-0'5) was not discernible. 
Application of morphactin and DPX-1840 resulted in significantly lowering the total 
auxins (table 2 and figure 1) whereas AMO-1618 was ineffective in bringing about 
any significant change. In combined tieatments, DPX-1840 or morphactin also 
checked the levels of auxins and minimum auxin activity was noticed when both 
of them were present simultaneously. 

3.2b. Gibberellins : The data (table 2 and figure 1) showed that two gibber ellins 
at Rfs 0-4-0 '5 (GA 3 ) and 0*7-0 '8 (GA 6 ) were detectable and GA 3 contributed 
the major component (80%) of the total gibberellins. The only treatment to 
significantly check the levels of endogenous gibberellips was AMO-1618 singly or 
in combination with other retardants which themselves were ineffective in causing 
any such change. 

P.(B)-5 



504 



Duct, U K Kohti and K S Chark 



3.2c. Cytokinins : Bioassays of different extracts deciphered the presence of 
four different cytokinins, namely, zeatin riboside (Rf 0' 1-0' 2), zeatin (Rf 0-4-0-5), 
zeatin ribotide (Rf 0'8-0'9) and an unknown cytokinin (Rf 0'7-0*8) The treat- 
ment of morphactin led to an increase of total cytokinins (table 3 and figure 1). 

Table 2. Effect of morphactin, AMO-1618 and DPX-1840 on the endogenous level 
of auxins and gibberellins Og/100g fresh weight) (average of 3 replications). 



Auxins (Rf) 



Gibberellins (Rf) 



Treatments 



0-1-0-2 0-6-0-7 0-9-1-0 0'4-0'5 0-70-8 



Control 


2-53 


12-51 


1-52 


1-20 


0-30 


Morphactin 


0-85 


7-2S 


0-85 


1-15 


0-30 


AMO-1618 


2-12 


10-87 


1-35 


0-51 


Tr 


DPX-1840 


0-56 


5-33 


0-80 


1-08 


Tr 


Morphactin -f AMO-1618 


0-84 


7-35 


0-86 


0-49 


0-25 


Morphactin 4- DPX-1840 


0*51 


3-84 


0-60 


1-02 


Tr 


AMO-161S -f DPX-1840 


0-58 


5-52 


0-85 


0'44 


Tr 


Mcrphactin + AMO-1618 -f DPX-1840 


0-52 


3-91 


0-65 


0-43 


Tr 


Critical difference at 5% level of 












significance 


0-47 


1-81 


0-56 


0-39 


0-J3 



400 



2 5 



71 




Control Morph. AMO 



DPX 
IO-4O 



o 




Morph. Morph. AMO 
+ + + 

AMO DPX DPX 



Id 




Morph.-f 

AMO+ 

DPX 




Figure 1. Effect of morphactin, AMO-1618 and DPX-1840 on the endogenous 
levels of total CK and GA (small shaded blacks inside the bigger blocks) in ngm 
and auxins (in /*g) per 100 g of fiesh weight (average of 3 replications). 



Hormonal regulation of apical dominance 505 

Tables. Effect of morphaotin, AMO-1618 and DPX-! 840 on the endogenous 
levels of cytokinin //g/100g fresh weight of kiuetin equivalent (average of 
3 replications). 



Cytokinins 
Rf 

Treatment ~ 



ZR Z UC ZRT 

0-1-0-2 0-4-0-5 0*7-0-8 0-8-0-9 



Control 0-058 0-035 0-021 0-061 

Morphactin 0-091 0-083 0-062 0-096 

AMO-1618 0-053 0-056 C'023 0-053 

DPX-1840 0-057 0-059 0-028 0-041 

Morphactin ^ AMO-1618 0- 089 0- 081 0' 079 0- 073 

Morphactin H- DPX-1840 0- 093 0- 082 0- 078 0- 076 

AMO-1618 + DPX-1840 0-061 0*063 0*028 0-058 

Morphactin + AMO-1618 -h DPX-1840 0-089 0- 084 0* 080 0* 079 

Critical difference at 5% level 

of significance 0-008 O'Ol 0-01 0*009 



Both AMO-1618 and DPX-1840 were ineffective in multiplying the level of individual 
or total cytokinins. In combined treatments, the cytokinin enhancing capacity 
of morphactin was not impaired by the presence of other substances. 



4. Discussion 

The habit of growth displayed by herbaceous and woody plants or control of the 
plagiotropic position of the lateral branches are examples of correlative effects 
where the stem apex influences the growth and development of the other parts of 
the plant The way in which this phenomenon is mediated has been a matter of 
controversy and different postulations, for different species, have been envisaged. 
Some of the early investigators (Goebel 1900 ; Loeb 1918 ; Dostal 1926) and a 
few of the late reports (Mclntyre 1968 ; Wagner and Michael 1971) have empha- 
sised the importance of inorganic and organic nutrition. However, a majority 
of researches from mid-sixties were concentrated on the possibility that a corre- 
lative signal from the apex was hormonal in nature (Phillips 1969 ; Kung Woo 
?/ al 1974). Though endogenous auxins (Au) was linked with apical dominance 
quite early (Wickson and Thimann 1960) and later on confirmed by more workers 
(Phillips 1975) the evidences favouring gibberellins (GA) involvement with the same 
have been very scanty and flare. In a variety of reports, gibberellins have been 
Found to be stimulatory (Brain et al 1959 ; Marth et al 1956 ; Ruddat and 
Pharis 1966), inhibitory (Bruinsma and Patil 1963) or indifferent (Sachs and 
1964) to the growth of lateral bu4s. As compared to GA ? there has been 



506 I S Dm, U K Kohli and K S Chark 

more convincing evidence linking cytokinin (CK) to apical dominance (Guern 
and Usciati 1972 ; Thimann 1972). Unfortunately, most of the above correla- 
tions were deduced by studying the growth of lateral buds following exogenous 
application of some synthetic or natural regulators and there had been a lack of 
effort to estimate quantitatively the endogenous hormones following the evoca- 
tion of lateral buds. The present investigations show that lifting of apical 
dominance, through growth retardants, coincided with a shift in the balance of 
various hormones. The data revealed that in Glycine max this phenomenon is 
probably regulated by a ratio of CK: to Au plus GA, The growth retardants 
morphactin, opx-1840 or AMO-1618 lifted apical dominance by increasing CK or 
decreasing Au or GA respectively. The role of these retardants in successfully 
managing these changes was apparent from the studies on the changes of endo- 
genous hormones under different treatments. This also draws support from the 
work of Ruddat and Pharis (1966) and Baldev et al (1965) on AMO-1618 ; Morcy 
and Dahl (1975) and Morey (1974) on DPX-184Q ; and Bednar and Linsmaier- 
Bednar (1971), Dua et al (1978), Dua and Dhuria (1980) on morphactin. 

The present findings further point that release from the apical dominance and 
the follow-up growth of the newly sprouted buds are independent of each other 
and probably have their own specific requirements. It was seen that while 
inhibitors of auxins or gibberellins lifted the apical dominance by lowering the 
denominator in CK/Au + GA ratio, the subsequent growth was impaired owing 
to the reduction of the same. It seemed that for this latter growth, GA or An 
presence or relatively higher levels were obligatory. This inkling was further 
supported from the observation on the treatment where morphactin was in combi- 
nation with DPX-1840 or AMO-1618. In this case though the number of buds 
released from apical dominance was significantly higher (by virtue of increase in 
CK), the follow-up growth of lateral buds was insignificant (probably due to the 
non-availability of sufficient Au or GAS). 

In essence, it seems that apical dominance in soybean is regulated by a balance 
of cytokinins to auxin and gibberellin and any factor which can affect this balance 
is likely to influence the manifestation of growth of lateral buds. On tha contrary, 
the follow-up growth of newly ensued buds is independent of the above domag 
and fo* this the presence of auxin and/or gibberellins seems mandatory. 



Acknowledgements 

The authors are thankful to Pi?of. K K Nanda for his critical suggestions and 
to Late Dr H S Dhuria, Ex-Assistant Director General, ICAR, New Delhi, for 
supplying DPX-1840. 



References 

All A A and Fletcher R A 1970 Xylem differentiation in inhibited cotyledonary buds af soy- 
bean ; Can. J. Bot. 48 1139-1141 

Baldev B, Lang A and Agatep A O 1965 Gibberellin production in pea seeds by developing 
in excised pods ; effect of growth retardant AMO-1618 ; Science 147 155-157 



Hormonal regulation of apical dominance 507 

Bcdnar T W and Linsmaicr-Bednar R M 1971 Induction of cytokinin tobacoo tissues by 

substituted fluorenes ; Proc. Natl. Acad. Sci. USA 68 1178-1179 
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of cupid seed peas ; Physiologia PL 12 15-29 
Brooks H J 1964 Responses of pear seed seedlings to N-dimethylamino-succinamic acid, a 

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Moravicae 3 83-209 
I>ua I S and Dhuria H S 1980 Role of cytokinins during apical dominance release by morphactin 

in Glycine max L. ; Proc. Indian Acad. Sci. (Plant Sci.) B89 375-379 
3ua I S and Jandaik C L 1979 Cytokinins in two cultivated edible mushrooms ; Sci. Hortic. 

10 301-304 
Dua I S, Jindal K K, Srivastava L J, Dinabandhoo C L, Thakur J R and Jain R 1978 

Correlation of endogenous cytokinins with apical dominance in response to morphactin 

in soybean (Glycine max L.) ; Proc. Indian Acad. Sci. (Plant ScL) B87 319-324 
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Part L General Organography (Oxford : Clarendon Press) 
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Cung Woo, Lee Kessler B and Thimann K V 1974 The effect of hadacidin on bud develop- 
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masses of sister leaves in Bryophyllum calcyinum ; Bot. Gaz. 65 150-174 
/Earth P C, Andia W V and Mitchel J W 1956 Effects of gibberellic acid on growth and 

development of plants of various genera and species; Bot. Gaz. 118 106-111 
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the flax seedlings ; Can. J. Bot. 46 147-155 

to C L, Choudhury S N, Dattary P and Hafeez U 1962 The influence of light and tempe- 
rature on the estimation of auxin by a straight growth test ; Indian J. Plant PhysioL 5 

97-116 
forey P R 1974 Influence of 3,3-dihydro-2-(/;-methoxyphenyl)-8H-pyrazolo-(5, 1-a) isoindol-8- 

one on xylem formation in honey mesquite ; Weed Sci. 22 6-10 
Corey P R and Dahl B E 1975 Histo logical and morphological effects of auxin transport 

inhibitors on honey mesquite ; Bot. Gaz. 136 274-280 

lorgan P W and Durham J I 1972 Abscission : potentiating action of auxin transport inhi- 
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fitsch J P 1956 Methods for the investigation of natural auxins and growth inhibitors in 
The chemistry and mode of action of plant growth substances (eds) R L Wain and 
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>sborne D J and McCalla D R 1961 Rapid bioassay for kinetin and kinin using senescing 

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M B Wilkins (London : McGraw-Hill) pp. 163-202 
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achs T and Thimann K V 1964 Release of lateral buds from apical dominance ; Nature 
(London) 2 939-940 



508 / S Dua, U K Kohli and K S Chark 

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Thimann K V, Sachs T and Mathur K N 1971 The mechanism of apical dominance in Coleus ; 

Physiologia PL 24 68-72 
Thimann K V and Skoog F 1934 On the inhibition of bud development and other functions 

of growth substances in Vicia faba ; Proc. R. Soc. B114 317-339 
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and gibberellic acid on higher plants ; Plant Cell Physiol. 8 231 -239 
Wagner H and Michael G 1971 The influence of varied nitrogen supply on the production 

of cytokinins in sunflower roots ; Biocliem. Physiol. Pflanzen. 162 141-158 
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dominance ; Physiologia PL 13 539-554 



Prac. Indian A<Jad. Sci. (Plant Sci.), Vol. 01, Number 6, beceiriber Wttt PP- 
Printed in India. 



Taxonomic importance of epidermal characters in the Indian 
Thespesia Corr. (Malvaceae) 



S RAJA SHANMUKHA RAO and N RAMAYYA* 

Department of Botany, Saidar Patel College, Secunderabad 500025, India 
* Department of Botany, Osmania University, Hyderabad 500 007, India 

MS received 4 December 1981 ; revised 21 October 1982 

Abstract In Thespesia lampas and T. populnea, the foliar stomata are anisocytic, 
anomocytic and tetracytic, the first type being dominant. Further in Thespesia 
lampas and T. populnea altogether eight trichome types are recognisable mostly an 
the basis of structure. The two species can be distinguished from each other 
by the presence of multiseriate aseptate stellate hair in the former and that of 
rnultiseriate aseptate peltate hai, in the latter. T. populnea is also distinct from that 
of T. lampas due to curved to wavy epidermal walls, striated surface, absence of 
mucilaginous cells on the leaf abaxial. The present evidence also supports treat 
mcnt of T. lampas under Thespesia rather than in Hibiscus. 

Keywords. Epidermal characters ; taxonomy ; Thespesia ; Malvaceae. 



1. Introduction 

Taxonomic importance of epidermal characters in general and those of trichomes 
in particular in angiospermous plants is now widely recognised (Prat 1932 ; 
Tomlinson 1961 ; Stace 1965 ; Ghose and Davis 1973 ; Verhoeven and Schijff 
1973). However, previous studies on the epidermis are limited to few taxa and 
only to certain aspects in the Malvaceae (Solereder 1908 ; Metcalfe and Chalk 
1950 ; Inamdar and Chohan 1969; Ramayya and Shanmukha Rao 1976 ; Shan- 
mukha Rao and Ramayya 1977a, b). Hence, the present investigation was under- 
taken which deals with foliar epidermal characters along with structure and 
organographic distribution of trichomes in whole plant and their taxonomic 
importance in the two Indian Thespesia. 



2. Material and methods 

The material of Thespesia lampas Dalz. & Gibs, was collected from Caramjol, 
Goa, whereas T. populnea (L.) Sol. ex Corr. from plants growing at Sardar Patel 

509 



S Raja Shanmukha Rao and tf 

College campus, Secunderabad. Mature trfohomes were, studied either from 
epidermal peelings or those isolated by scraping the plant parts. These micro- 
preparations were stained either with anilin blue in lactophenol or safranin, 
hematoxylin and then mounted in 70% glycerine. Boiling the material with 
5-10% glacial acetic acid was useful in obtaining the peels. Microtome sections 
of shoot apices were cut at 10-14 jam thickness and stained with hematoxylin 
and basic fuchsin. The foot of the different trichomes was determined in free- 
hand and microtome sections of various parts of the species investigated. The 
walls of the different trichomes were tested with phloroglucin and 2% HC1 for 
lignin (Johansen 1940). 

The terms describing stomatal types are after Metcalfe and Chalk (1950) and 
as redefined by Shanmukha Rao and Ramayya (1977a) and for trichome types, 
after Ramayya (1972) and Shanmukha Rao and Ramayya (1977b). 



3. Observations 

3.1. Structure and distribution of epidermal and stomatal complexes on the leaf 

3 . la. Epidermal cell complex : Epidermal cells : 4-6-sided, anisodiametric ; 
contents dense, brownish in T. populnea ; sides thin, straight (leaf abaxial and 
adaxial of T. lampas and leaf adaxial of T. populnea} or curved to wavy (leaf abaxial 
of T. populnea) ; surface smooth (T. lampas) or striated (r. pojpulnea), striations 
wavy, prominent and continuous. Distribution : Confined to the interstices, 
variously oriented (figures 1-4). Costal cells : 4-sided, linear ; contents scanty 
or mostly brownish (T. populnea); sides moderately thick, straight or curved 
(leaf abaxial of T. populnea) ; surface smooth (T. lampas) or striated (T. populnea). 
Distribution : Oriented parallel to the veins, diffuse. Mucilaginous cells : 
Similar to the epidermal cells but enlarged below with opaque contents ; sides 
thin, straight or curved ; surface smooth. Distribution : Occasional either in 
leaf abaxial (T. lampas) or adaxial (T. lampas and T. populnea) (figures 4 and 10). 

3 lb. Stomatal complex : Stomata mostly anisocytic, occasionally anomocytic 
or tetracytic. Subsidiaries 3 or 4, monocyclic unequal, similar to the epidermal 
cells except that cuticular striations are absent over stomata. Distribution : 
Amphistomatic but adaxially stomata confined to margins of the midrib 
(figures 1-6). 

Data on the epidermal and stomatal complexes are given in table 1. In both 
the species, single guard cells as stomatal abnormality are occasionally observed 
in the leaf abaxial whereas they ate totally absent in the adaxial (figure 7). 

3.2. Structure and distribution of trichome complex on vegetative and floral parts 

Eight trichome types could be recognised in the two species, viz., (i) unicellular 
conical Mar, (ii) uniseriate filiform clavate hair, (iii) uniseriate filiform pyrifoum 
hair, (iv) multiseriate aseptate peltate hair, (v) multiseriate aseptate stellate hair, 
(vi) multiseriate aseptate 4-armed stellate hair, (vii) multiseriate aseptate 3-armed 
stellate hair and (viii) biseriate aseptate V-shaped hair. The details of the struc- 
ture of the trichome types are as follows : 



Taxonomic importance of epidermal characters in Thespesia 5.1 J 




Figures 1-11. Epidermis of Thespesia. T. populnea: 1 and 2. Surface views of leaf 
abaxial and adaxial respectively (cuticular stiiations not drawn). 7. Surface view 
of stomatal abnormality with single guard % ell from leaf abaxial. 10. Mucilaginous 
cell from leaf abaxial epidermis in T.S. 11. Oblipue seotion showing sunken 
unisoriate filiform clavate hair from T.S. leaf (foot cell not visible). T. lampas : 
3 and 4. Surface views of leaf abaxiial and adaxial respectively. 5. Semi- 
diagrammatic representation of stomatal distribution confined to margins of midrib 
from leaf adaxial. 6. Surface view of anomocytic stoxna from leaf adaxial. 8* 
Uniseriate filifoirm clavate hair from T.S. pedicel. 9. Side view of emergence 
from pedicel, (me = mucilaginous cell). 



P. 



512 



S Raja Shanmukha Rao and N Ramayya 







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Taxonomic importance of epidermal characters in Thespesia 513 




8 



Figures 12-28. Epidermis of Thespesia. T. populnea : 12. Multiseriate aseptate 
peltate hair from leaf abaxial. 13-15. Intermediates between multi seriate aseptate 
peltate hair and multiseriate aseptate stellate hair from petal abaxial. 16. Multi- 
seriate aseptate stellate hair from petal abaxial. 17. Isolated unicellular conical 
hair from sepal adaxial. 27. Uni seriate filiform pyriform hair of noctary from 
sepal adaxial. T. lampas : 18. Unicellular conical hair from sepal adaxial. 
19, 20. Biseriate V-shaped aseptate hair from sepal adaxial and petal abaxial 
respectively. 21, 22. Multiseriate aseptate 3-armed stellate hair from petal 
abaxial. 23. Surface view of uniseriate filiform clavate hair from pedicel. 
24, 25. Multiseriate aseptate stellate hair from leaf abaxial and pedicel respectively. 
26. Multiseriate aseptate 4-armed stellate hair from ovary. 2$. 
filiform pyriform hair of nectary from sepal adaxial. 



514 S Raja Shanmukha Rao and N Ramayya 

3.2a. Unicellular conical hair : Foot consisting of the basal end of the body 
cell, indistinct from the body, embedded in the epidermis ; neighbouring cells of 
foot annular and striated ; contents absent ; wall thick. Body representing 
extension of the foot, conical, apically pointed ; contents absent ; wall thick and 
nonlignified ; surface smooth (figures 17 and 18). 

3.2b. Uniseriate filiform clavate hair : Foot 1 -celled, square to linear or trape- 
zoidal, embedded or projected above the epidermis ; contents absent ; wall thin. 
Stalk 1-celled, rectangular to linear ; contents scanty ; wall thin ; surface smooth. 
Head clavate, 2-6-tiered, each tier 2-6-oelled ; cells square to linear ; contents 
dense; walls -thin ; surface smooth (figures 8, 11 and 23). 

3.2c. Uniseriate filiform pyriform hair : Foot 1-celled, linear, embedded or 
projected above the epidermis ; contents absent ; wail thin. Stalk 1-celled, 
rectangular to lineai ; contents scanty ; wall thin ; surface smooth. Head pyri- 
form, multiseriate, 4-10-tiered, each tier 2-6-ceUed, generally terminated by a 
pair of rounded cells ; cells rectangular to linear ; contents dense ; walls thin ; 
surface smooth (figures 27, 28). 

3.2d, Multiseriate aseptate peltate hair: Foot narrow, consisting of the juxta- 
posed basal ends of the 20-35-body cells, embedded in the epidermis, polygonal 
in outline in peels mounted upside down, occasionally subtended by an emergence ; 
contents absent ; walls thin, nonlignified. Body peltate, nearly circular or uneven, 
serrate or fcrenulate at margin, 20-35-armed, representing continuation of tho 
foot ; arms broadened near the centre of the body but tapering towards distal 
end, parallel to the epidermis ; contents brown ; walls thin, nonlignified ; surface 
smooth (figure 12). , 

3.2e. Multiseriate aseptate stellate hair : Foot as: in the above. Body stellate, !, 

5-40-armed, representing continuation of the foot ; arms tapering towards the | 

distal end, unequal in length, generally parallel to the epidermis, some obliquely 1 

raised above ; contents absent ; walls thin to moderately thick, nonlignified ; 
surface smooth (figures 9, 16, 24 and 25). 

Multiseriate aseptate 4-armed stellate hair, multiseriate aseptate 3-armed I 

stellate hair knd biseriate aseptate V-shaped hair types are similar to the multi- f 

seriate aseptate stellate hair described above except for the difference in number 
of arms of the body as indicated by the names of trichome types (figures 19-22 
and 26). The details of distribution of the various trichome types are given 
in table 2. 



4. Discussion 



Inamdar arid ChoKan (1969) recorded anisocytic and anomocytic stomata in 
Thespesia , populnea^ which is presently confirmed. Further, tetracytic stomata 
(as -defined by Shanmukha Rao and Ramayya 1977a) have also been presently 
noted, in the leaves of the two species of Thespesia studied. Among the three 
stomatal types, the anisocytic is dominant (table 1). 



Taxonomic importance of epidermal characters in Thespesia 515 

Table 2. Organographic distribution of trichome types in the Indian Thespesia. 



Plant part 



Thespesia lampas Thespesia populnea 



^eaf abaxial 


B, E 


B, D 


>af adaxial 


B, E 


B, D 


^eaf margin 


A, E 


B, D 


'etiole 


B, E 


B, D 


Stipule abaxial 


B, E 


B, D 


Stipule adaxial 


B, E 


B, D 


Stipule margin 


B, E 


D 


Stem 


B, E 


B, D 


teduncle 


B. E 


B. D 


Jracteale abaxial 


B, E 


B, D 


iracteole adaxial 


B 


B, D 


Jracteole margin 


B, E 


D 


tepal abaxi?! 




B, D 


lepal adaxial 


A, C, H 


A, C 


lepal margin 


A 


A 


>etal abaxial 


A, E-H 


B, E-H 


>etal adaxial 




... 


*etal margin 


A, E-H 


A 


Jtaminal tube 


... 


... 


Style 


... 


... 


Dvary 


A, E-H 


D 



V, Unicellular conical hair ; B, Uniscriate filiform clavate hair ; C, Uniseriate filiform pyriform 
lair ; D, Multiseriate aseptate peltate hair ; E, Multiseriate aseptate stellate hail ; F, Multiseriate 
iseptate 4-arined stellate hair ; G, Muttiseriate aseptate 3-armed stellate hair ; H, Biseriate 
iseptate V-shaped hair. 



According to Youngman and Pande (1929) the following trichome types occur 
n Thespesia : (i) single unbranched hairs, (ii) stellate hairs, (iii) peltate scales 
ind (iv) club shaped bodies. The first and third trichome types quoted above 
ire the same as the unicellular conical and multiseriate aseptate peltate hair types 
espectively, described by us. On the other hand, we consider the " stellate hair " 
ype quoted above, to be a trichome complex rather than representing a specific 
;richome type. On comparison with the trichome types delimited in the present 
investigation, the stellate hair is resolvable into the following four types : (i) multi- 
ieriate aseptate stellate hair, (ii) multiseriate aseptate 4-armed stellate hair, 
Jii) multiseriate aseptate 3-armed stellate hair and (iv) biseriate aseptate V-shaped 
hair. Similarly the club shaped body is distinguishable into two of the presently 
described trichome types : (i) uniseriate filiform clavate hair and (ii) uniseriate 
filiform pyriform hair. In the two species of Thespesia investigated thus in all 
eight trichome types are presently recognisable as described in the text. 



516 S Raja Shanmukha Rao and N Ramayya 

Though, in T. lampas, the multiseriate aseptate stellate hair on pedicel and 
sepal abaxial are 5 to many armed (figure 25), those on the leaf lamina, petiole 
and stem are 20-40-armed with thin walls (figure 24). Likewise in T. populnea 
the arms of the multiseriate aseptate peltate hair are 20-35 with thin walls on 
all the parts of its occurrence (figure 12). Many intermediate forms connecting 
the peltate and stellate types also occur on petals (figures 12-16). The characters 
which distinguish the multiseriate aseptate stellate hair of T. lampas and the 
multiseriate aseptate peltate hair of T, populnea from each other include the 
separate and tapering nature of the arms in the former and the connate condition 
and rounded ends of the arms in the latter (figures 12 and 24). As shown by 
Ramayya and Shanmukha Rao (1976) the above differences in the two trichome 
types are due to early onset of apical intrusive growth instead of symplastic growth 
in the development of arms in the multiseriate aseptate stellate hair. Thus the 
differences described are deep in origin and hence are significant in justifying 
the separation of the two trichome types. 

In the two species of Thespesia, all the vegetative parts are trichiferous whereas | 

in the floral parts, the surface of the petal adaxial, staminal tube and style are | 

non-trichiferous (table 2). Further, in each of the species, all the tiichiferous \ 

parts show at least two trichome types (viz., multiseriate aseptate peltate hair and [ 

uniseriate filiform clavate hair in T. populnea and multiseriate aseptate stellate \ 

hair and uniseriate filiform clavate hair in T. lampas) except the abaxial surface 
of the petal and the ovary which possess several trichome types each (table 2). 

Out of the eight trichome types now described, all of them occur in T. populnea 
whereas in T. lampas there are seven types, the multiseriate aseptate peltate hair I 

being absent (table 2). Thus the two species are distinguished from each other f 

due to the presence of the multiseriate aseptate peltate hair in the former and j 

that of the multiseriate aseptate stellate hair in the latter. Further, the leaves of | 

T. populnea are distinct from those of T. lampas by curved epidermal cells with \ 

striations and absence of mucilaginous cells on the abaxiai surfaces (table 1). 

In the systematic accounts, Thespesia lampas (Masters 1874 ; Borssum 1966 ; i 

Rakshit and Kundu 1970 ; Saldanha and Nicokon 1976) is considered conspecific 

with Hibiscus lampas (Schumann 1890 ; Hochreutiner 1900 ; Gamble 1957) and I 

Azanza lampas (Babu 1977). The tribe Gossypieae (includes Thespesia) differs | 

from Hibisceae (includes Hibiscus) by the gossypol glands and conduplicate | 

embryos (Fryxell 1968). The presence of gossypol glands on different parts of f 

Thespesia lampas, T. populnea and other two species (Standford and Viehoever 
1918 ; Lukefahi and Fryxell 1967) supports the separation of Thespesia from I 

Hibiscus. Further, Thespesia lampas stands out distinct in other Malvaceae (with I 

thick-walled non-lignified stellate hair) by the multiseriate aseptate thin-wailed f 

stellate hair, thus providing further evidence to treat Thespesia lampas distinct t 

from Hibiscus lampas. | 

i 

Acknowledgement ! 

\ 

One of the authors (SRR) is thankful to. the Principal, Sardar Patel College, 
Secunderabad, for providing facilities. 



Taxonomic importance of epidermal characters in Thespesia 517 

References 

Sabu C R 1977 Herbaceous flora of Dehradun (New Delhi : csm) 84 

Jorssum Waalkes J Van 1966 Malesian Malvaceae revised ; Blumea 14 1-251 

7 ryxell P A 1968 A redefinition of the tribe Gossypieae ; Bot. Gaz. 129 296-308 

Gamble T S 1957 Flora of the Presidency of Madras (Reprinted ed.) (Calcutta : BSI) Vol. 1 p. 72 

jhose M and Davis T A 1973 Stomata and trichomes in leaves of young and adult palms ; 

Phytomorphology 23 216-229 

lochreutiner B P G 1900 Revision du genera Hibiscus ; Ami. Cons. Jard. Bot. Geneve 4 23-91 
namdar J A and Chohan A J 1969 Epidermal structure and stomatal development in some 

Malvaceae and Bornbacaceae ; Ann. Bot. 33 865-878 

ahansen D A 1940 Plat't microtechnique (New York : McGraw-Hill Book Co., Inc.) 194 
^ukefahr M J and Fryxell P A 1967 Content of gossypol in plants belonging to genera i ela- 
ted to cotton ; Econ. Bot. 21 128-131 
Blasters M T 1874 Malvaceae in The flora of British India (ed) J D Hooker (London : Reeve 

and Co.) Vol. 1 184 
tfetcalfe C R and Chalk L 1950 Anatomy of the dicotyledons (Oxford : Clarendon Press) Vols. 

1 and 2 
>rat H 1932 L* Epiderme des Graminees ; Artnales des Sciences Naturelles Botanique et Biologie 

Vegetate Series 10 165-258 
Lakshit S C and Kundu B C 1970 Revision of the Indian species of Hibiscus ; Bull Bot. Surv. 

India 12 151-175 
lamayya N 1972 Classification and phylogeny of the trichomes of angiosperms in Research 

trends in plant anatomy K A Chowdhury Commemoration Volume (eds) A K M Ghouse 

and M Yunus (New Delhi : Tata-McGraw-Hill) 91-102 
lamayya N and Shanmukha Rao S R 1976 Morpholo&y phylesis and biology of the peltate 

scale stellate and tufted hair in some Malvaceae ; /. Indian Bot. Soc. 55 75-79 
laldanha C J and Nicolson D H 1976 Flora of Hassan District Karnataka India (New Delhi ; 

Amarind Publishing Company) Vol. 1 ppd 15^-156. 
Ichumann K M 1890 Malvaceae In Die natur 'lichen pflanzenfamiUen (eds) H G A Engler and 

K A E Prantl 3 
Jianmukha Rao S R and Ramayya N 1977a Storoatogonosis in the genus Hibiscus L (Malvaceae) ; 

Bot. J. Linn. Soc. 74 47-56 
Shanmukha Rao S R and Ramayya N 1977b Structure distribution and taxonomic importance 

of trichomes in the Indian species of Malvastrum ; Phytomorphology 27 40-44 
olerede' H 1908 Systematic anatomy of the dicotyledons (Oxford : Clarendon Press) Vols. I and 2 
tace C A 1965 The significance of the leaf epidermis in the taxonomy of the Combretaceae I. 

A general review of tribal, generic and specific characters ; Bot. J. Linn. Soc. 59 229-252 
tanford E E and Viehoever A 1918 Chemistry and histology of the glands of the cotton 

ylant with notes on the occurrence of similar glands in related plants ; /. Agric. Res. 13 

419-435 
'omlinson P B 1961 Anatomy of the monocotyledons. IL Palmae (Oxford : Clarendon Press) 

pp. 30-31 
r erhoeven R L and Schijff H P V D 1973 A key to the South African Combretaceae based on 

anatomical characteristics of leaf ; Phytomorphology 23 65-74 

r oungman W and Pande S S 1929 The epidermal outgrowths of the genera Thespesia and Gassy- 
plum ; Ann. Bot. 43 711-740 



toe. tndian Acad. Sci, (Plant Sci.), Vol. 91, INtumW 6, December 1982, pp. 
Printed in India. 



Embryological studies in Launaea nudicaulis Hook.* 

P S CHIKKANNAIAH and B S HIREMATH 

Department of Botany, Karnatak University, Dharwad 580 003, India 

MS received 27 February 1981 ; revised 22 September 1982 

Abstract. The ovule is anatropous, unitegmic and teruinucellate. Their funicular 
vascular strands extend almost to the base of the ovule. A large hypodermal 
archespoial cell functions directly as the megaspoje mother cell. It divides to 
form a linear tetrad. The upper three megaspores degenerate while the chalazal 
develops into an 8-nuckate embryo sac of the Polygonum type. Endothelium diffe- 
rentiates at the megaspore tetrad stage. Fertilization is porogamous. Syngamy 
and triple fusion take place almost simultaneously. The endosperm is nuclear 
but it becomes cellular subsequently. The embryo is the Senetio variation of 
Asterad type. Occurrence of polyembryony has been recorded only in one ovule. 

Keywords. Launaea nudicaulis ; embryology ; Compositae, 

1. Introduction 

The Compositae is one of the largest families of flowering plants consisting of 
more than 20,000 species, yet the embryological work in the family is scanty. 
Davis (1966) has reviewed the previous embryological literature on the family 
Compositae. Venkateswarlu and Maheswari Devi (1955a) have made embryo- 
logical studies on Launaea nudicaulis, which is widely distributed and also has 
cyto types, only a note has appeared so far (see Venkateswarlu 1939). 

The cytotypes are diploid (2n = 18) and tetraploid (2n = 36). Attempt is 
made here to present some aspects of comparative embryological studies of both 
diploid and tetraploid taxa. 

2. Material and methods 

Young heads were fixed as such but old heads were cut symmetrically into two 
halves before they were fixed in formalin-acetic alcohol. Following customary 
methods, the material was dehydrated in ethyl alcohol series and embedded in 

* Part of the Ph.D. thesis submitted by the Junior author (B S Hiremath) to the Karnatak 
University, Dharwad. 

519 



520 



P S Chikkannaiah and B S tiiremath 




8 9 

Figures 1-10. L.S. of Launaea nudicaulis ovules. 1. Showing anatropous condi- 
tion (x 150). 2. Single hypodermal archesproial cell (x 350). 3. Megaspore 
mother cell and supernumerary archesporial cells (x 350). 4, 5. Dyad and a 
tetrad of megaspares. 6-8. Uni, four and six nucleate embryo sacs (x 350). 
9, 10. Organised and mature ernbiyo sacs (x 350). ant, antipodal cells ; pel, 
periendothelial zone ; sar, supernumerary archesporial cells ; ser, secondary 
nucleus ; syn. synergid cells ; vt 9 vascular traces. 
Figures 1, 2, 4-7, 9-25 of 2n species. Figures 3, 8, 26 and 27 of 4/i species. 



Embryological studies in Launaea nudicaulis Hook. 521 

paraffin. Serial sections were cut from 6 to 15 microns and were stained in 
Heidenhains iron-alum haematoxylin using sometimes erythrocin in 90% alcohol 
as counter stain. 



3. Observations 

Detailed observations in the diploid taxon and only variations in the tetra- 
ploid species are described. The inferior, bicarpellary and unilocular ovary 
bears a single ovule at its base. The ovule is anatropous, unitegmic and tenui- 
nucellate (figure 1). The funicular vascular strands extend almost to the base 
of the ovule. There is generally a large hypodermal archesporial cell which 
functions directly as the megaspore mother cell (figure 2). In tetraploid taxon 
supernumerary archesporial cells are usually observed (figure 3). Their number 
varies from 2 to 8. An archesporial cell undergoes meiotic divisions and forms 
a linear tetrad (figures 4, 5). Further development of supernumerary archesporial 
cells in tetraploid taxon is not observed. Subsequently, the upper three mega- 
spores degenerate while the chalazal megaspore organises into an 8-nucleate 
embryo sac of the polygonum type (figures 6, 7, 9). In tetraploid taxon the 
divisions at both chalazal and micropylar ends are not synchronous. At the 
chalazal end the third mitotic division has already resulted in four nuclei while 
at the micropylar end the two nuclei are yet to divide (figure 8). 

The organised embryo sac is spindle-shaped and its chalazal end is narrow. 
The egg apparatus is at its micropylar and consists of two synergids and a centrally 
placed egg cell (figure 9). Synetrgids are hooked (figure 10). There are three 
antipodal cells which are placed in triangular or linear fashion in the narrow 
chalazal end of the embryo sac. The two polar nuclei are in the centre and are 
very near to each other (figure 9). Later the two polar nuclei, just below the 
egg apparatus, form a secondary nucleus (figure 10). 

Endothelium appears at the megaspore tetrad stage and presents its characteristic 
glandular appearance (figure 11). It arises from the inner epidermis of the integu- 
ment enclosing the nucellus (figures 7, 9 and 12). It consists of rectangular cells 
and the cells are rich in cytoplasm (figure 11). Endothelium nourishes the 
growing embryo sac. Gradually it starts losing its compactness (figures 12 to 15). 
Its peripheral layer called periendothelial zone loses its cell contents finally forming 
an empty layer (figures 13, 14). The endothelium persists as a thin layer around 
mature embryo (figure 25). 

Fertilization is porogamous. The pollen tube, after reaching the base of the 
egg, bursts open at its base discharging the two male gametes. One male gamete 
fuses with the egg and the other with the secondary nucleus (figure 12). Syngamy 
and triple fusion take place almost simultaneously (figure 12). 

The division of the endosperm nucleus precedes that of the zygote. The first 
division of the primary endosperm nucleus results in the formation of two free 
nuclei. As many as eight free nuclei have been observed around the two celled 
proembryo (figure 13). Later it becomes cellular. Hence the endosperm is of 
nuclear type. The developing embryo absorbs most of the endosperm tissue Q 



522 



P S Chikkannaiah and B S Hiremath 




ca 



Figures 11-18. L.S. L. nudicaulis nucellus. 11. Endothelium at megaspor e 
tetrad stage. 12. Syngamy and double fertilization (x 350). 13. 8-nucleat e 
endosperm (x 350). 14, 15. Stages in the development of end c sperm and endo" 
thelium (x 350). 16. Zygote (x 350). 17, 18. 2- and 4-ceUed embryos. 



Embryological studies in Launaea nuaicaulis Hook. 



523 




27 



Figures 19-27. L.S. ovules of Ir. nudicaulis. 19-24. Stages of development of 
embryo (x 350). 25. L.S. of mature seed (x 20). 26. L.S. of globular embryo 
showing long suspensor (x 350). 27. L.S. of seed showing two embryos A and B 
(x 20). cot, cotyledon ; cot tr, cotyledonary traces ; de, dermatogen initials ; el 9 
endothelium ; en, endosperm ; pe f periblem initials ; pl 9 pleurome initials ; pvt, 
stem tip ; re, root cap. 



524 P S Chikkannaiah and B S Hiremath 

that the mature seed has a thin layer of endosperm surrounding the embryo 
(figure 25). 

The zygote is pyriform (figure 16). The first division of zygote is transverse 
resulting in the formation of a terminal cell ca and a basal cell cb (figure 17). 
The next transverse division occurs in cb forming two tiers m and ci of one cell 
each (figure 18) while the cell ca divides longitudinally forming two juxtaposed 
cells resulting in a T-shaped proembryo. Further, the tier ca divides longitudi- 
nally in a plane perpendicular to the first forming a quadrant q (figures 
18-20). 

Simultaneously, the tier m divides vertically into two cells while the tier ci 
divides transversely into two tiers n and ri of one cell each (figures 19, 20). 
Generally, the transverse division in the tier ci precedes that of the longi- 
tudinal division in the tier m (figure 19). Thus, a proembryo of seven cells 
disposed in four tiers is formed (figure 19). The tier ca consists of four cells, 
m of one cell, n and ri of one cell each. The walls in the quadrant are placed 
obliquely dividing them into central cells a and peripheral cells /J (figures 21, 22). 
The peripheral cells j3 are very active and they form two cotyledons (pco) while 
the central cells a are less active initially, but contribute to stem apex (pvt) (figures 
22, 24). By this time m has undergone another vertical division to form four 
circumaxial cells (figures 21, 22). Meanwhile the tier ri has divided transversely 
forming two tiers o and p of one cell each (figures 21, 22) The cell p is highly 
elongated and vacuolated. The proembryo consists of 14 cells arranged in five 
tiers (figure 22). The terminal tier q consists of 6-cells, the tier m of four cells, 
the tier n of two cells and the tiers o and p of one cell each. Subsequently the 
periblem and pleurome initials are cut off from the tier q (figure 23). 

Each one of the four circumaxial cells in the tier m divides periclinally giving 
rise to dermatogen initials which become continuous with the dermatogen formed 
in the apical tier (figure 23). Likewise, the periblem initials which are differenti- 
ated in tier q also extend into tier m. Later, longitudinal and transverse walls 
are laid down in this tier m which finally gives rise to hypocotyl (phy) and 
radical (ice) (figure 24). 

The two juxtaposed cells in the tier n undergo periclinal division cutting off 
epidermal initials. The cells in the centre further divide both longitudinally and 
transversely to contribute to root tip (iec). The tier o divides periclinally and 
anticlinally to form a root cap (co). The tier p contributes to the suspensor (s). 
In tetraploid taxon there is a long suspensor (figure 26). It perists till the heart- 
shaped embryo stage and is totally absent in the mature embryo. The mature 
embryo is dicotyledonous and somewhat straight (figure 25). It consists of an 
elongated hypocotyl-root axis. The root apex is protected by the root-cap. The 
stem apex is centrally placed between the two cotyledons having abundant 
starch. The vascular strands run from the hypocotyl into the cotyledons. The 
following sequence represents the development of the embryo of Launaea 
nudicaulis. 

A rare instance of polyembryony has been observed in tetraploid taxon based 
on its position, the adventitious embryo seems to have been originated from one 
pf the synergids (figure 27). 



Embryoiogical studies in Launaea nudicaulis Hook, 525 



Zygote 



. ca q ' M " 

phv + irr 


U-f- 


n iAr 


n -rn 


L,r 

1 

L o $ 



I. Discussion 

ntegumentary vascular traces have been reported in some Compositae (Misra 
965 ; Chopra and Singh 1976). In Launaea nudicaulis funicular vascular strands 
ire present which extend only to the base of the chalaza. Although pluricellular 
.rchesporium has been reported earlier (Harling 195 la ; Sehgal 1966), in Launaea 
mdicaulis (2x) there is only one archesporial cell. However, in tetraploid plants 
upernumerary archesporial cells are also observed. In most of the Compositae, 
L linear tetrad of megaspores is formed in which the chalaza develops into an 
dght nucleate embryo sac of the polygonuni type (Pullaiah 1977a, b ; Sharma 
md Murthy 1978). T-shaped tetrads have also been reported in many members 
Deshpande 1964a, b ; Kaul etal 1975). Invariably in Launaea nudicaulis only 
inear tetrad of megaspores is observed. 

There are many interesting features in the embryo sac development in the 
Uompositae. Several divergences from its normal development occur (Davis 
.966). An Allium type in Ammobium spp. (Davis 1962), in Chrysanthemum 
pp. and Erigeron spp. (Harling 1951a,b) has bsen described. Three variations 
>f the tetrasporic development of the embryo sac, a Fritillaria type in Gaillardia 
ricta (Venkateswarlu and Maheswari Devi 1955b), Ratibida tagetes (Howe 1964), 
Drusa type in Chrysanthemum spp., Erigeron spp. (Harling 195 la, b) and Adoxa 
ype in Rudbeckia hirta (Palm 1934) have been reported. In Launaea nudicaulis 
he Polygonum type of embryo sac has been recorded. Large haustorial synergids, 
>ften extending into the micropyle, appears to be a common feature in the 
Hompositae (Davis 1961b). Pullaiah (1977a, b) recorded hooked synergids in 
Volidago canadensis and Achillea squarrosa. In Launaea nudicaulis, hooked 
ynergids with blunt ends are observed. There is a great variation in the number 
>f antipodal cells and in the number of nuclei in them, a feature found in many 
nembers of the Compositae. Secondly, antipodal region has been found to be 
laustorial persisting as vermiform appendage (Davis 1961a). Taigi and Taimni 
1963) have also noted the haustorial antipodals in Vernonia sinerscens. Murthy 
ind Sharma (1976) and Sharma and Murthy (1978) have observed basically three 
intipodals in all the three species, Felicia bergariana, Conyza stricta and Erigeron 
wnariensis while in Bellia perennis they have noticed an increase in the number 
>f antipodal cells up to 21. In Launaea nudicaulis there are three antipodal cells 
>laced in a triangular fashion and persist till the mature proembryo stage. But 
hey do not show any haustorial nature. 



526 P S Chikkannaiah 'and B S tfirertiath 

Endothelium is present in the Compositae (Venkateswarlu and Maheswari Devi 
1955a; Kaul et al 1975 ; Deshpande and Kothare 1976 ; Chopra and Singh 1976 ; 
Sharma and Murthy 1978 ; Sehgal 1979). It appears in the early development of 
the ovule at megaspore mother cell stage in Vernonia anthelmintica (Misra 1972) 
or at megaspore tetrad stage in Guizotia abyssinica (Chopra and Singh 1976). In 
the present study differentiation of the endothelium takes place at the megaspore 
tetrad stage. It is usually unfseriate but sometimes biseriate. Nutritive activity 
of the endothelium becomes evident during the development of the embryo sac. 
The layers of cells surrounding the endothelium show some marked changes, 
especially with respect to size, shape, form and stainability. This layer is called 
periendotheliai zone (Misra 1972). In the material under study the nutritive 
activity is at its peak at the time of fertilization. 

Both cellular and nuclear types of endosperm are known to occur in the 
Compositae, the former being more frequent (Davis 1966). Endosperm is of 
nuclear type in the taxa under study. Persistence of the endosperm as a thin layer 
around the embryo is in line with the observations of Misra (1965, 1972) , Kaul 
et al (1975) and Sharma and Murthy (1978). 

Deshpande (1961) has shown that in Eclipta prostrata obliquely oriented walls 
are laid down in the octant which is characteristic of Onagrad type. He, further, 
commented that these features constitute intermediate stages of the Onagrad type 
and the Asterad type. However, the present study does not indicate any such 
variations. 



Acknowledgement 

The authors thank Prof. M S Chennaveeraiah, Head of the Department of 
Botany, Karnatak University, Dharwad, for critically going through the 
manuscript. 



References 

Chopra S and Singh R P 1976 Effect of Gamma rays and 2,4-D en ovule, female gatnetophyte, 

seed and fruit development ; Phytomorphology 26 240-249 
Davis G L 1961a The life history of Podolepis jaceoides (Sims.) Voss. II. Megasporogcnesis, 

female gametophyte and embryogeny ; Phytomorphology 11 206-219 
Davis G L 1961b The occurrence of synergid haustoria in Cotula australis (Less) Hook. f. 

(Campositae) ; Aust. J. Sd. 24 296 
Davis G L 1962 Embryological studies in Australian Compositae. 2. Sporogenesis, gameto- 

genesis and embryogeny in Ammobium alatum ; Aust. /. Bot. 10 65-67 
Davis G L 1966 Systematic embryology of the angiosperms (New York : John Wiley) 
Deshpande P K 1961 Fertilization and development of endosperm, embryo and seed coat in 

Eclipta prostrata Linn., Bull Bot. Soc. Coll ScL Nagpur 21-8 
Deshpande P K 1964a A contribution to. tir life history of Volutarella racemosa Roxb 

/. Indian Bot. Soc. 43 141-148 
Deshpande P K 1964b A contribution to the embryology of Bidens biter ttata ; J Indian Bot 

Soc. 43 149-157 
Deshpande P K and Kathare M P 1976 Embryology of Cyathodine purpurea ; /. Indian Bot 

Soc. 55 205-212 



Embryological studies in Launaea nudicaulis Hook. 527 

Hading G 195ia Embryological studies in the Compositae. II. Anthemideac-Chrysanthcminac 

Acta Horn. Bergiani 16 1-56 
Harliflg G 19515 Embiyologtcal studies in the Compositae. in. Astcreae ; Acta Horti. 

Bergiani 16 73-120 

Howe T D 1964 Developmert of embryo sac in Ratibida tagetes; Am. /. JBot. 51 678 
Kaul V, Dathan A S R and Singh D 1975 Embryological studies on the genus Sonchus L. ; 

J. Indian Bat. Sac. 54 238-247 
Misra S 1965 Floral morphology of the family Compositae. III. Embryology of Siegesbeckia 

orientalis L. ; Aunt. J. Bot. 13 1-10 
Misra S 1972 Floral morphology of the family Compositae. IV. Vcrnonieae. Vemonia anthel- 

mintica ; Bot. Mag. (Tokyo) 85 187-199 
Murthy Y S and Sharma H P 1976 Embryol< gical studies in Compositae. II. Bellis perennis 

Lima. A casual apomict. Recent trends and contacts between Cyto genetics and Embryo- 
logy and morphology. All India UGC sponsored seminar. Nagpur pp. 251-261 
Palm B 1934 Bin nour Embryosacktypus bei Rudbeckia hirta ; Bot. Notiser 87 423-427 
Pulaiah T 1977a Embryological investigations in Solidago canader.sis Linn. (Compositae) 

Astereae ; Proc. Indian Set. Cong. 65 
Pullaiah T 1977b Embryology of Achillea squarrosa Ait (Compositae : Anthemideae) ; Proc. 

Iiidiun Sci. Cong. 65 
Sehgal C B 1966 Morphological and embryological studies on Erigeron bonariensis L. 

(E. Uniforms Willd.). ; Beitr. Biol. Pflanz. 42 161-183 
Sehgal C B 1979 Infrastructure of integumentary cells in Nicotic.ua rustica L. before and 

after fertilization ; /. Cytol. Genet. 14 192-197 
Sharma H P and Murthy Y S 1978 Embr>ok>gical studies in the Compcsitae, Astcreae II. 

Proc. Indian Acad. Set. B87 149-156 
Tiagi B and Tainnii S 1963 Floral morphology and embryology of Vemonia cinerascens Sohultz. 

and Y. cinera Less. ; Agra Univ. J. Res. (Sci.) 12 123-138 
Vcnkateswarlu J 1939 A note on the structure and development of the ovule aad the embryo 

sac in species of Launaea ; Curr. Sci. 8 556-557 
Vcnkateswarlu J and Maheswari Devi H 1955a Embryological studies in the Compositae. I. 

Launaea pintiatifida Cass. ; Proc. Indian Acad. Sci. 41 3846 
Venkateswarlu J and Maheswari Devi H 1955b Embryological studies in Compositae. If. 

Helcnicae ; Proc. Natl. Inst. S>i. India B21 149-161 



P (B)~8 



Prac. Indian Acaci. S-si. (Plant Sci.), Vol. 91, Number 6, December 1982, pp. 529-549. 
Printed in India. 



Quantitative profile structure of certain forests in the Kumaun 
Himalaya 



A K SAXENA and J S SINGH 

Department of Botany, Kumaun University, Naini Tal 263 002, India 

MS received 12 May 1982 : revised 30 September 1982 

Abstract. The structure of forests occurring within the north-western catchment 
of the river Gola in Kumaun Himalaya is quantitatively described. All the forestt 
indicated a total of four strata ; two upper strata represented by trees, the third 
stratum represented mainly by shrubs, and the fourth u-f herbs. The tree heighs 
of the A s (top most) stratum decreased with an increase in altitude. On the other 
hand, the proportion of trees devoted to the canopy in the A l and A 2 strata increased 
with an increase in altitude. In all forests, the crowns of the A- and A strata 
were more deep than wide. In general, the shrub layer in three oak forests was 
comparatively dense and the crowns of the shrubs overlapped with each other. The 
canopy index, a relative measiur of canopy coverage, of tree and shrub layers was 
maximum for Quercus flonbunda forest and minimum for Pinus roxburghii forest. 
Further, the cooler aspects developed a greater canopy index for these layers as 
compared to the warmer aspects. Oak forests exhibited a poor development of 
their herb layers. The trees in the Quercus lanuginvsa forest weere more stable, 
while in Pinus roxburghii forest they were specially susceptible to wind effect. In 
general the warmer aspects had more stable trees, while the cooler aspects showed 
a lower tree stability. The different forest types, presently studied, could be graded, 
as follows, in a decreasing order of potential for soil protection : Quercus flori- 
buncLi > Quercus leucotrichophora > Quercus lanuginosa > mixed >P/;.w roxburghii f 

Keywords. Himalayan forests ; profile structure ; canopy index. 



1. Introduction 

The Himalaya offers an array of forest types below the timber line, and is the 
cradle of major rivers of India, harbouring (thus) a net work of catchment areas. 
Growing human interference with the vegetation cover of the catchment of rivers 
has generally led to substantial reduction of forest cover which in turn has led 
to serious ecological disasters, such as, soil erosion, loss of soil fertility and cata- 
strophic floods. As the catchment efficiency depends on the type, quantity and 
stratification of vegetation, a quantitative evaluation of its vegetation is a pre-h 
requisite. However, such data are few (Ralhan et at 1982; Saxena and Singh 

529 



530 



A K Saxena and J S Singh 



1982; Saxena etal 1982; Tewari and Singh 1981). Earlier studies, mainly quali- 
tative, have been reviewed by Puri (1960) and Champion and Seth (1968). The 
present study describes the structure of the forests occurring within the north- 
western catchment of the river Gola in Kumaun Himalaya. 

Due to the presence of a large variety of growth forms, forests are generally 
highly stratified (Smith 1974). Tropical forests usually have a total of five 
strata above the soil surface, while the temperate forests have only two or three 
strata (Richards 1952). Upto some extent, light and moisture determine the 
various strata which in turn modify the environment from the canopy to the forest 
floor. The amount of light received in various strata of a community varies, 
depending upon the density of different strata, type of vegetation, opening of 
forest crown, etc. (Knight 1965). The canopy closure also plays a major role 
n the regeneration of forests by