(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Biodiversity Heritage Library | Children's Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "manual of mangrove plants propagation"




MS. Swaminath 
Chennai, India 



Manual on vegetative and 
micropropagation of mangroves 



P. Eganathan 

and 

C. Srinivasa Rao 




M. S. Swaminathan Research Foundation 
Chennai, India 



MSSRF/MA/01/02 



M. S. Swaminathan Research Foundation 
3 rd Cross Street, Institutional Area, 
Taramani, Chennai 600 113, India 



Telephone : +91(44) 2541 229 
+91(44) 2541 698 

Fax : +91(44) 2541319 

E-mail : msswami@mssrf.res.in 

executivedirector@mssrf .res .in 



May 2001 



© M. S. Swaminathan Research Foundation. 

All rights reserved. No part of this publication can be reproduced 

or transmitted in any form or by any means, without permission. 



ACKNO WLED GEMENT 

We express our deep sense of gratitude to Prof. M.S.Swaminathan, Chairman, 
M.S.Swaminathan Research Foundation and UNESCO- Cousteau Chair in 
Ecotechnology for guidance, encouragement and support in bringing out this 
manual. Our sincere thanks are due to Prof.P.C.Kesavan, Executive Director, 
M.S.Swaminathan Research Foundation and DAE-Homi Bliablia Chair for his 
interest and continuous encouragement. We are grateful to Prof.S.Chelliah, Project 
Director, Mangrove Conservation and Management Project for editing the 
manuscript. We are grateful to Dr.V.Selvam, State Project Coordinator, Mangrove 
Conservation and Management Project for support in the field activity and manual 
preparation. 

Our sincere thanks are due to the Department of Biotechnology, Government of 
India for providing sustained financial support for vegetative and micropropagation 
work and to India-Canada Environment Facility for its generous support in the 
preparation of the manual. 

Our heartfelt acknowledgement is due to Mr.M.M.Saravanan, Laboratory Technician, 
for assistance in tissue culture, Mrs. K.Sengani, MGR Nagar, for maintaining field 
nursery and Mr.Raja, Vadakku Pichavaram, for transport in the mangrove wetland. 

We are thankful to the Forest Departments of Tamil Nadu and Orissa for 
providing necessary permission for the field work and sharing plant materials for 
experiments. 



ABBREVIATIONS 



(NH 4 ) 2 S0 4 

10% 

IN HC1 

IN NaOH 

2ip 

Agar and Phytagel 

BA 

Ca (N0 3 ) 2 .4H 2 

CaCl 2 .2H 2 

CoCl 2 .6H 2 

CuS0 4 .5H 2 

Explant 

FeS0 4 .7H 2 

H 3 B0 3 

HgCl 2 

Hormone 

Humidifier 

IAA 

IBA 

K 2 S0 4 

KH 2 P0 4 

KI 

KN0 3 

MgS0 4 .7H 2 

MnS0 4 .4H 2 

Na 2 C0 3 

Na 2 EDTA.2H 2 

Na 2 Mo0 4 .2H 2 

Na 2 W0 4 .2H 2 

NAA 

NH 4 N0 3 

pH (-log[H+]) 



ppm 
Sphagnum moss 

Tween-20 

v/v 

w/v 

ZnS0 4 .7H 2 



Ammonium Sulphate 

10 g of Sodium carbonate (Na 2 C0 3 ) dissolved in 

80 ml distilled water and made up to 100ml 

8.77ml of 35%HC1 (Generally Available) made up to 

100ml using double distilled water. 

4g of Sodium hydroxide in 80 ml of double distilled 

water and made up to 100ml 

6-(y, y - Dimethyl allyl-Amino) purine Riboside 

Used for solidification of tissue culture media 

Benzyl Adenine 

Calcium Nitrate Tetrahydrate 

Calcium Chloride Dihydrate 

Cobalt Chloride Hexahydrate 

Copper Sulphate Pentahydrate 

Any plant part used in tissue culture 

Ferrous Sulphate Heptahydrate 

Boric Acid 

Mercuric Chloride 

Growth regulating substance in plants 

Mist forming instrument in mist chambers 

Indole Acidic Acid 

Indole Butyric Acid 

Potassium Sulphate 

Potassium Dihydrogen Phosphate 

Potassium Iodide 

Potassium Nitrate 

Magnesium Sulphate Heptahydrate 

Manganous Sulphate Tetrahydrate 

Sodium Carbonate 

Ethylene Diamine Tetra Aceticacid Disodium Salt 

Sodium Molybdate dihydrate 

Sodium Tungstate 

Naphthalene Acetic Acid 

Ammonium Nitrate 

Denotes the concentration of hydrogen ions in a 

solution 

parts per million 

A plant body creeping on the surface of a rock/soil 

in hilly areas 

Surfactant used for surface sterilization of explants 

Volume by volume 

Weight by volume 

Zinc Sulphate Heptahydrate 



FOREWORD 

The multiple benefits conferred by Mangrove wetlands in coastal estuarine areas 
are now widely recognized. For the families living along the coast, mangrove 
forests provide medicine, firewood, sustainable fish yield and protection against 
coastal storms. In the future, mangroves will assume greater ecological importance 
in view of a potential rise in sea level as a result of global warming, since they can 
tolerate varying degrees of coastal salinity. In addition, in the emerging era of 
genomics, proteomics and genetic modification, mangrove species constitute a 
source of valuable genes for a variety of useful traits including salinity tolerance. 

Unfortunately, many precious mangrove wetlands have either already been destroyed 
for alternate uses including tourism and aquaculture or are under varying degrees 
of anthropogenic pressures. Where cyclones are frequent, coastal communities are 
becoming aware the value of mangrove forests, as for example in coastal Orissa. 
There is therefore a growing interest in coastal communities in the rehabilitation of 
degraded mangrove ecosystems. What is often lacking is the availability of planting 
material of the desired species. The present publication is designed to provide 
operational procedures for undertaking the multiplication and planting of mangrove 
trees in coastal estuarine areas. Both vegetative propagation and micropropagation 
methods are described. 

In social forestry programmes, local communities are enabled to raise nurseries of 
forest tree species on the basis of a buy-back arrangement with Forest Departments. 
A similar programme is needed in the case of mangrove tree species. Local 
communities including school children can be assisted to raise mangrove nurseries. 
The planting material can be used in land belonging to forest and revenue 
departments as well as to local communities. This will help to speed up the 
restoration of degraded mangrove wastelands. 

I congratulate Mr. P. Eganathan and Dr. C. Srinivasa Rao in bringing out a practical 
manual on mangrove propagation. I also thank Prof. P. C. Kesavan, DAE-Homi 
Bhabha Chair and Dr. V. Selvam for providing overall guidance and support for this 
work. I hope this manual will help to launch a community mangrove afforestation 
programme. *j. 

M. S. Swaminathan 



CONTENTS 

FOREWORD 

ACKNOWLEDGEMENT 

ABBREVIATIONS 

PART A - VEGETATIVE PROPAGATION 

1.0 Introduction 1 

1.1 Definition 

1.2. Need for vegetative propagation 

1.3. Advantages and limitations of vegetative propagation 

1.4. Types of Vegetative Propagation 

2.0 Mangrove plants 3 

2.1. Species composition 

3.0 Plus tree 5 

3.1. Definition 

3.2. Identification of plus trees 

3.3. Plus trees of mangrove species in Pichavaram 

4.0 Vegetative propagation of species in Rhizophoraceae 7 

4.1. Propagule cutting 

4.2. Air-layering 

5.0 Vegetative propagation of other species 12 

5.1. Stem cutting 

PART B - MICROPROPAGATION 

1.0 Introduction 24 

1.1 Development of micropropagation protocols for mangrove plants 

1.2 Types of micropropagation 

1.3 Advantages and limitations in micropropagation 

2.0 Media used in micropropagation 26 

2.1. Composition of different culture media 

2.2. Preparation of stock solution 

2.3. Preparation of 20X major nutrient solution 

2.4. Preparation of 200X minor nutrient solution 

2.5. Preparation of 200X iron stock solution 

2.6. Preparation of 1000X vitamin stock solution 

2.7. Other chemicals 

2.8. Preparation of X culture medium 

2.9. Addition of growth hormones in culture media 

3.0 Methods of micropropagation 29 

3.1. Micropropagation of Excoecaria agallodia 

3.2. Micropropagation of Avicennia officinalis 

3.3. Micropropagation of Acanthus ilicifolius 

References 34 



TABLES 

Table 1 - Common mangrove species of India 3 

Table 2 - Important mangrove species in India and their utility 4 

Table 3 - Morphological and phenological characters of plus trees 

of Avicennia and Rhizophora identified at Pichavaram 6 

Table 4 - Preparation of hormone solutions of different concentrations 8 

Table 5 - Optimum concentration of root-promoting 

hormones for maximum root initiation 9 

Table 6 - Optimum concentration of root-promoting hormone used in air- 
layering in different mangrove species for maximum rooting 11 

Table 7 - Preparation of hormone treatment solutions of different 

concentrations 13 

Table 8 - Optimum concentration of hormones for maximum rooting 

in the stem cuttings of different mangrove species 14 

Table 9 - Nutrient composition of different culture media 26 

Table 10 - Composition of stock solution of 20X major nutrients 27 

Table 11 - Composition of stock solution of 200X minor nutrients 27 

Table 12 - Composition of 200X iron stock solution 28 

Table 13 - Composition of vitamin stock solution 28 

Table 14 - Combination and Concentration of growth hormones 

to be used in culture and sub-culture media 29 



PLATES 

Propagules of different species of Rhizophoraceae 15 

Root and shoot development in propagule cuttings 16 

Development and hardening of propagated plants 

in the nursery and mist chamber 17 

Air-layering in Excoecaria agallocha in the field 18 

Rooting through air layering in different mangrove species 19 

Rooting through air layering in different mangrove species 20 

Stem cuttings in Excoecaria agallocha 21 

Rooting in stem cuttings in different mangrove species 22 

Rooting in stem cuttings in different mangrove species 23 

(A) Micropropagation of Excoecaria agallocha 32 

(B) Micropropagation of Avicennia officinalis 

Plate 11 - Micropropagation of Acanthus ilicifolius 33 



1 


2 


3 


4 


5 


6 


7 


8 


9 


10 



MSSRF/MA/01/02 



M. S. Swaminathan Research Foundation 
3 rd Cross Street, Institutional Area, 
Taramani, Chennai 600 113, India 



Telephone : +91(44) 2541 229 
+91(44) 2541 698 

Fax : +91(44) 2541319 

E-mail : msswami@mssrf.res.in 

executivedirector@mssrf .res .in 



May 2001 



© M. S. Swaminathan Research Foundation. 

All rights reserved. No part of this publication can be reproduced 

or transmitted in any form or by any means, without permission. 



MANUAL ON VEGETATIVE AND 
MICROPROPAGATION OF MANGROVES 

PART A 

VEGETATIVE PROPAGATION 

1.0 INTRODUCTION 

1.1 Definition 

Vegetative propagation is a method of producing plants identical in genotype 
with the mother plant. It is a method of producing large number of plants from the 
vegetative part of a mother plant. Any part of the plant such as stem, leaf, 
propagule and root can be used to produce plants through vegetative propagation. 
It is an asexual method of propagation. It is different from producing seedlings 
from the seeds, which is through sexual method of propagation. 

The vegetative propagation forms an integral part of tree improvement 
programme. In this approach, the best planting stock with highest genetic quality 
can be obtained, which is not always possible with the sexually propagated 
progenies. Another advantage is that, by this technique, plants can be raised almost 
throughout the year and the plantable stock for some species can be obtained in 
shorter time than those raised through seeds. 

1.2. Need for vegetative propagation: 

• Vast areas of coastal wetlands potential for mangrove growth could be 
covered with mangrove vegetation for domestic and commercial utility. To 
achieve this, large scale production of planting material could be produced 
through vegetative propagation 

• In many mangrove ecosystems planting season does not coincide with the 
reproductive season of the mangrove plants and in such situations, vegetative 
propagation would be ideal to supply planting material round the year 

• Endangered species can be easily multiplied through vegetative propagation 

• Propagation of sterile hybrids is possible mainly through vegetative 
propagation 

1.3. Advantages and limitations of vegetative propagation 

Advantages : 

• Single stock can provide large number of plants 

• The clones offer the advantages of genetic uniformity 



• Seedlings produced through vegetative propagation take lesser time to 
develop, therefore, it is normally quicker and cheaper 

• Multiplication of desired hybrids is easier without loss of desirable genes 

• Helps to utilize maximum genetic gains of potential species in a 
shortest time 

• Commercialization of planting material is made attractive 

Disadvantages: 

• Only a few species are amenable for vegetative propagation 

• Standardization of methodology is time consuming and sometimes 
expensive 

• Vegetative propagation is easier with young rather than old trees 

1.4. Types of Vegetative Propagation 

1. Stem cutting 

2. Propagule cutting 

3. Air layering 

1.4.1. Stem cutting 

Production of saplings from stems and /or branches of plants is called 
vegetative propagation by stem cuttings. Stem and/or branches of plants cut into 
small pieces ranging from 12 to 20cm in length with 3 to 5 or several nodes are 
known as "stem cuttings". Stem cuttings are divided into three categories viz., i). 
Soft wood (tender branches) ii). Semi-soft wood (intermediate of soft wood and 
hard wood) and iii). Hard wood (tertiary or secondary branches). 

1.4.2. Propagule cutting 

A propagule (hypocotyl) is a seed germinated in the mother tree itself. It is 
an unique feature of mangrove plants. A propagule contains different parts such as 
pedicel, fruit, collar and radicle. In a ripened propagule, plumules can be seen 
when the fruit is removed. Viviparous propagules are produced in all the 
Rhizophoraceae species of mangroves. The mature viviparous propagule (black or 
brown or mixture of both) can be collected from the mother tree and cut into small 
pieces of 2 to 7 cm. This is called "propagule cutting", which could be used for 
vegetative propagation (Plate 1, i to vi). 

1.4.3. Air layering 

Air layering is another method of vegetative propagation popularly called as 
"Chinese Layering". In this method, roots are produced in small branches by 
applying root producing hormones and rooting media. This method can be fol- 
lowed in tertiary branches without much damage to the mother plant. 



2.0. MANGROVE PLANTS 



2.1. Species composition 

The mangrove species are a group of plants possessing specialized adaptive 
features to live in a saline, marshy estuarine environment where both physical and 
chemical properties vary widely both in space and time. There are about 30 
families, 49 genera and 80 species available in India. Table 1 gives the list of 
common mangrove species in India. 

Table 1 : Common Mangrove Species of India 



Species 



Family 



Acanthus ilicifolius Linn. 

Aegiceras corniculaturu (Linn.) Blanco 

Anioora cucullata Roxb. 

Avicennia marina (Forsk.) Vieh. 

Avicennia officinalis Linn 

Bruguiera cylindrica (Linn.) Bl. 

Bruguiera gymnorrhiza (Linn.) Lam. 

Bruguiera parviflora (Roxb.) W.& R. ex Griff 

Cerbera manghas Linn 

Cerbera odollam Gaertner 

Ceriops decandra (Griff.) Ding Hou 

Dalbergia spinosa Roxb 

Derris trifoliata Lour. 

Excoecaria agalloclia Linn. 

Heritiera fomes Buch.-Ham. 

Heritiera littoralis Dryand. 

Intsia bijuga (Colebr.) O.Kuntze 

Kandelia candel (Linn.) Druce. 

Lumnitzera racemosa Willd. 

Rliizopliora apiculata Bl 

Rhizopliora hybrid 

Rliizopliora mucronata Poir. 

Rhizopliora stylosa Griff. 

Sonneratia apetala Buch.-Ham. 

Xylocarpus granatum Koen. 

Xylocarpus mekongensis (Prain) Pierre. 

Xylocarpus ruoluccensis (Lamk.) Roem. 



Acanthaceae 

Myrsinaceae 

Meliaceae 

Avicenniaceae 

Avicenniaceae 

Rhizophoraceae 

Rhizophoraceae 

Rhizophoraceae 

Apocynaceae 

Apocynaceae 

Rhizophoraceae 

Fabaceae 

Fabaceae 

Euphorbiaceae 

Sterculiaceae 

Sterculiaceae 

Caesalpiniaceae 

Rhizophoraceae 

Combretaceae 

Rhizophoraceae 

Rhizophoraceae 

Rhizophoraceae 

Rhizophoraceae 

Sonneratiaceae 

Meliaceae 

Meliaceae 

Meliaceae 



All the above species are capable of tolerating wide range of salinity, although 
the extent of tolerance varies widely. On the basis of the salt-tolerant mechanism, 



mangrove plants are divided into salt excretory, in which excess salt is excreted 
through salt glands (e.g. Avicennia spp.), salt excluders, in which excess salt is 
excluded in the root zone itself (e.g. Rhizophora spp) and salt accumulators in 
which salt is accumulated in fleshy leaves (e.g. Suaeda spp.). In most of the species 
flowering is round the year but propagules and seeds are available only from 
September to January when the salinity is low due to heavy fresh water inflow. The 
utility of common mangrove species is presented in Table 2. 



Table 2 : Important mangrove species in India and their utility 



Species 



Family 



Time of 
flowering 



Economic value 



Avicennia marina 

Avicennia officinalis 

Brnguiera cylindrica 
Bruguiera gymnorrhiza 
Ceriops decandra 
Cerberas manghas 
Excoecaria agallocha 

Heritiera forties 

Heritiera littoralis 
lntsia bijuga 
Kandelia candel 
Rhizophora apiculata 

Rhizophora hybrid 



Rhizophora stylosa 
Sonneratia alba 



Xylocarpus granatum 
Xylocarpns mekongensis 



Avicenniaceae 

Avicenniaceae 

Rhizophoraceae 

Rhizophoraceae 

Rhizophoraceae 

Apocynaceae 

Euphorbiaceae 

Sterculiaceae 

Sterculiaceae 
Caesalpaniaceae 
Rhizophoraceae 
Rhizophoraceae 



Jun-Sep 

Jun-Sep 

Round the year 
Round the year 
Round the year 
June-Sep 
Feb-Sep 



Apr-Aug 

Jan - Aug 
June-Sep 
Round the year 
Round the year 



Rhizophoraceae Round the year 



Rhizophoraceae Round the year 
Sonnertiaceae Feb-Oct 



Meliaceae 
Meliaceae 



Jun - Sep 
Jul - Oct 



Fodder, green manure, 
glue and firewood 

Fodder, green manure, 
glue and firewood 

Firewood and fodder 

Firewood and fodder 

Firewood and fodder 

Firewood and medicinal 

Plant parts used for the 
treatment of ulcers, leprosy, 
rheumatism, paralysis 

Timber, scaffolds, boat building 
and paper industry 

Fiber, timber and firewood 

Valuable timber 

Firewood, fodder and tannin 

Firewood, tannins, leaves 
used for treating asthma. 

Timber, firewood, tannins and 
adhesive, Decoction of the bark 
is used for diarrhoea, 
dysentery and leprosy 

Firewood, tannins 

Firewood, paper pulp, 
matchwood; fermented juice is 
used to check hemorrhage 

Timber, tannins, seed oil used 
as to lit lambs and firewood 

Timber, tannins, oils extracted 
from seeds used for nerve 
disorder and antihelminthetic 



Xylocarpus moluccensis 



Meliaceae 



Jul - Oct 



Timber, tannins and firewood 



3.0 PLUS TREE 



3.1 Definition 

A Plus tree is an individual tree of a species possessing superior morphologi- 
cal and reproductive characters than other individuals of the same species. 

3.2. Identification of plus trees: 

The following characters could be used to identify plus trees. Selection of plus 
tree should be site specific since plant characters, both quantitative and qualitative 
will vary from place to place. 



Morphological characters 

Plant height 

Girth of the main stem at breast level 

Number of leaves per gram weight 

Canopy structure and Canopy area 

(Open, Closed) 

Number of pneumatophores per 

square meter 

Height of pneumatophores 

Number of stilt roots per square meter 

Length of the stilt roots 

Diameter of the stilt roots (surface 

area) 

Number of lenticels per square 

centimeter 

Characters based on physiognomy 

Pests and disease damage 

Tidal inundation (frequency and 

duration) 

Salinity levels (soil and pore water 

salinity) 

Associate species 

Reproductive characters 

Number of buds per inflorescence 



Number of flowers per inflorescence 

Number of fruits per inflorescence 

Number of propagules per 

inflorescence 

Number of propagules per tree 

Net weight of individual propagule 

Germination capacity of the 

propagules 

Germinability of viviparous 

propagules 

Viability of seeds 

Length of the propagules 

Adaptation characters 

Photosynthetic efficiency 

Biomass production 

Soil salinity 

Adaptation to different types of soils 

Pollution tolerance 

Grazing effects 

Commercial value 

Forest products, used by local people 

Timber 

Building material 

Fodder 

Fuel wood 



3.3. Plus trees of mangrove species in Pichavaram 

An example of quantitative and qualitative characters of the plus tree of 
different species of mangroves identified in the Pichavaram mangrove wetlands of 
Tamil Nadu is given Table 3. 



01 



C 

01 

« 

o 
-a 

55. 

O 

■ N 

C 

s 



s 

s 

u 
3 



IS 

ST} 



■4- 

s 

o 



« 



a. 



00 
LO <0 t> 

T-l ^ 00 



(N 



in 



0) 

TjH CO 

' o 



K "5 O CM 
^ cm ^ « 



U 



73 

CO 

o 
U 



^o 



CM g 



£j CO CO 

3 ^ v£ 



-p 



-* ^ "tf 

ON cO ^ 



-+ 



X S ^ 

00 CO" -* 



*tf 



o 



ON CO 



K S 



H-J 

ON 



00 CM 



VO 

CO LO 

CM 



MO CM 



O 
ID 



CO 



o 
o 



CO 
CM 



LO 



o 
o 



ON 



o 



00 



O 
CM 



00 
ON 



o 



-p 



an 

01 
01 
H 

s S 

o S 

SB X 

& tj 

aj »fh 

— Ch 

u l_ ' 

tt «y 

s- «; 
« 

u 



•S 
1 



CM 



VO CO 
_Z t^ \£) 

C^ <N L< 



0) ON ** 
CL, ^ CM 

o ^ 



S ^ IN LO 

^ LO LO 
LO t^ o 



T3 



u 



CO CM 






CO 
CM 



ON 



^ ON CM 
CO ^H CM 



CM 



^0 



^0 



CM 

CM t^ 

°°. 06 

O LO 



ON _h 

t— I ON 



00 



vD 



be 



so 



it 

• fH 

60 
o 

"o 
C 

01 

C 

a 

u 

• fH 

60 
O 

"o 

X 

B- 

O 



CO 

01 

[2 



- 

3 
U 



CO 

iH 

01 

«M 

u 



s 

u 



0J 

> 

0J 



co 
td 

OJ 



<« 




Cfl 


u 




.S 




1 


td 


•»H 


+j 


0J 

4-> 


60 X 

O 60 








O 


0J 


M-H 


X 


-C 


O 


Oh 


-M 


J3 



td a 



_bc 

'3 
5 



td sh 
0) 0) 

Oh 

CO 
> 

td 

0) 



T3 
OJ 
CO 

o 



U 



CO 

OJ 

S-H 

o 

Pi -5 



O- 

o 



OJ 

S-H 

u 

S-H 



3 * 



Sh C 

o G 

Oh X 

° o 
o 



-o 
c 

td 

OJ 

S-H 

o 

Oh 

O 



H , , 'h-» 



Q fa 

OJ 

C 
0- 



o 

S-H 



o a 

hH 

'-Ci 0) 



X> Oh 

6 § 

I * 

Z u 



>-. ai 

Oh pQ 

u z 



3 

0) 

Oh Mh 
hH O 
O !h 

jj ai 



■±H CO 






O 

td 

cd 

bO^ 

i-J ai 






"53 ai 



u 

C 

OJ 
OJ 

S-H 

o 



U 5h 
> 



ai y 

u C 

CO CL) 

0) cj 



^ ?hQ 



CO 

cj 0) 

Oh »-i 
CO fV 
01 CO 

° 3 



CJ 

C 

OJ 
CJ 
CO 

OJ 

Sh 

I £ 

Sh Jh 

ai a> 

Oh Oh 

CO CO 

oi ai 

3 3 

bo bo 
td cd 

Oh Oh 

o o 

Sh Sh 
Oh Oh 



td 3 



"G o o o o o 

3 Sh Sh Sh Sh Sh 

'0 0)0)0)0)01 

O pQ pD x> X> X> 

q. s s s s s 

?d d d 3 3 

tt z z z z z 



■39 

CJ 

"3 

td ,-s 

Oh ~-S 

Sh 

Oh tO 
0) 



td 

-0 
> 



3 

bo 

cd 

0- 

o 

Sh 

0- 






h- 1 Sh 
0) 0) 

2 O 



<J to 

as S 

3 ^ 

bo 

td bo 

2 1 

Oh g 

u cj 

+3 td 

k3 e5 



4.0 VEGETATIVE PROPAGATION OF SPECIES IN RHIZOPHORACEAE 

The species under the family to Rhizophoraceae are important in mangrove 
ecosystem. The genus Rliizopliora encompasses evergreen trees having strong stilt 
roots and long propagules. The stilt roots of Rliizopliora spread laterally and are 
buried deep in the mud, providing additional support to the tree. In view of this 
character, these trees are able to withstand high cyclones. Secondly, the root zones 
provide microhabitat for the juveniles of fish and prawn to grow. These species 
have become endangered in the mangrove of West Bengal. In the Pichavaram 
mangrove wetlands three Rluzopliora species viz., Rliizopliora apiculata, R. 
mucronata and Rliizopliora hybrid are present. Generally, the maternal and pater- 
nal parents of the sterile hybrids are R. mucronata and R. apiculata respectively. 

4.1. Propagule cutting 

Materials: 

Mature propagules, distilled water, solution to remove phenolics, growth 
hormones, refrigerator, knife, mist chamber, humidifier and mud filled polythene 
bags. 

Method : 

Step 1. Collection of propagules 

Collect mature propagules from the plus trees identified based on the charac- 
ters indicated in Chapter 3.0 Good propagules are fleshy, shining with red colored 
collar. 

Step 2. Cutting of propagules 

Cut the collected propagules into 2 to 5 cm pieces using a clean and sharp 
knife. The entire propagule can be used for cutting (Plate 1 A) 

Step 3. Removal of phenolic compounds 

Phenolics are a group of compounds present mostly in the bark and wood of 
almost all the mangrove species. Phenolics are essential compounds for the survival 
of mangrove plants in extreme environmental conditions, since they regulate 
growth and other physiological functions. However, in vegetative propagation they 
act as inhibitors in the formation of roots and shoots and hence, phenolics from 
propagule cuttings are to be removed before further processing. 

The following method describes the preparation of solutions and treatment 
methods to remove the phenolic compounds. Removal of phenolic compounds 
involves short-term and long-term treatments. 



Preparation of stock solution : Take 20 g of Sodium carbonate (Na 2 C0 3 ) and 
20g of Sodium tungstate (Na 2 W0 4 .2H 2 0), dissolve successively in 80 ml dis- 
tilled water and make up the final volume to 100ml. It is a 20% stock solution. 

Preparation of working solution (10% and 5%): Take 50ml from the 20% 
stock solution and add 50ml of distilled water. This gives a 10% working 
solution. Take another 25ml of stock solution and add 75ml of distilled water. 
This gives a 25% working solution. 

Short-term treatment to remove phenolic compounds : Take 10% solution in 
small cups. Keep the basal portion of the cuttings immersed in the solution 
for 5-10 minutes. Wash the treated cuttings in distilled water two to three 
times (Plate 1 B). 

Long-term treatment to remove phenolic compounds : Keep the cuttings 
treated in 10% solution, for about 20-30 minutes in 5% working solution for 
final treatment. Wash the treated cuttings in distilled water two to three times. 
Now the propagule cutting is ready for hormone treatment. 



Step 4. Hormone treatment 

Hormone treatment is necessary to induce root formation. Indole Butyric 
Acid (IBA) and Naphthalene Acetic Acid (NAA) are commonly used as root 
promoters. These promoters are available in many of the agro shops in the form of 
powder. 

• Preparation of hormone stock solution : Take lg of IBA (Himedia, Mumbai) 
and add little drops of IN NaOH until it dissolves, and make it up with 
distilled water to 100ml. The strength of this stock solution is 10,000 ppm. 

• Preparation of hormone treatment solution : The following table shows the 
method of preparing treatment solutions of different concentrations from the 
stock solution 

Table 4 : Preparation of hormone treatment solutions of different concentrations 



Quantity of 


Quantity of 


Concentration 


stock solution (ml) 


distilled water (ml) 


(ppm) 


10 


90 


1000 


15 


85 


1500 


20 


80 


2000 


25 


75 


2500 



Hormone treatment of the cuttings : Propagule cuttings treated to remove 
phenolics are again dipped in root promoting IBA hormone for 10-30 minutes. 
Since the concentration of IBA required for root promotion differs from 
species to species, propagule cuttings should be dipped in solutions of 
different concentrations as shown in Table 5. 



Table 5 : Optimum concentration of root promoting hormones for 
maximum root initiation 



Species 


Growth Hormone 


Concentration 
(ppm) 


Rooting 
<%) 


Bruguiera cylindrical 


IBA 


500 


83 


Bruguiera gymnorrliiza 


IBA 


700 


75 


Bruguiera parviflora 


IBA 


500 


90 


Bruguiera sexangula 


IBA 


500 


93 


Ceriops decandra 


IBA+NAA 


500+200 


79 


Kandelia candel 


IBA+NAA 


1000+500 


95 


Rhizophora apiculata 


IBA 


1500 


93 


Rhizophora mucronata 


IBA 


2000 


89 


Rhizophora x hybrid 


IBA 


1500 


75 



Step 5. Planting propagules in poly-bags/plastic pots : 

After hormone treatment, cuttings are planted in poly-bags containing sand 
and clay at the ratio of 2:8. Keep planted cuttings in the mist chamber at 28±2°C and 
70-80% relative humidity. 

Step 6. Hardening the cuttings in mist chamber : 

Cuttings are continuously monitored and maintained in controlled environ- 
ment in the mist chamber. Roots will form after 20 to 25 days and shoots will form 
after 30 to 35 days (Plate 2). Water is sprayed 3 to 5 times/ day inside the chambers 
to maintain the humidity and temperature. 

Step 7. Hardening the saplings in nursery : 

After 2 months, successfully established saplings should be kept under shady 
areas in nursery for 3-4 months. Before 25 days of field transfer treat the saplings 



with saline water with maximum salinity level of 20 ppt of diluted sea water, or 
mangrove water itself or water in which common salt is dissolved for desired 
concentration (Plate 3). 

Step 8. Planting in the field: 

After successful hardening, saplings are ready for planting. At the end of 
the monsoon, transfer and plant the saplings in the prepared field. 

4.2. Air-layering 

Materials : 

Growth hormones, distilled water, knife, syringe with needle, brush, sphag- 
num moss, rooting medium (clay: sand: soil), threads, and polythene. 

Methods : 

Step 1. Selection of branches: 

Select semi-hard wood and hard wood branches of plus trees. Avoid 
selecting branches that are drooping too much and twigs that are very young or 
tender. 

Step 2. Removal of bark of selected branches: 

Remove the outer bark of the selected branch at 2 to 5 cm below the node. 
The portion of the branch where the bark is removed is called, wounded portion. 
Make a bridge of bark of 2 to 4mm thickness to connect the upper end of the 
mother plant and the lower end of the daughter plant (offspring) to be 
produced. This bridge is necessary for the maintenance of some of the 
important physiological functions, since in mangrove species formation of roots is 
very slow. 

Step 3. Preparation of root promoting hormone: 

Method is explained in under 4.1 Step 4 . 

Step 4. Applying root-promoting hormones: 

Apply the hormone all around the wounded portion using a fine brush. 
Hormone should be applied twice. Different concentrations of hormone are used 
for different species as shown in Table 6. 



10 



Table 6 : Optimum concentration of root-promoting hormone used in 
air-layering in different mangrove species for maximum rooting 



Species 


Hormone 


Concentration 
(ppm) 


Rooting 

(%) 


Amoora cucullata 


IBA+NAA 


500+200 


59 


Avicennia marina 


IBA 


2500 


42 


Avicennia officinalis 


IBA 


2000 


54 


Cerbera manghas 


IBA+NAA 


1000+200 


61 


Cerbera odollam 


IBA 


1000 


55 


Excoecaria agallocha 


IBA 


2000 


48 


Heritiera fames 


IBA 


2500 


55 


Heritiera littoralis 


IBA 


2000 


51 


Intsia bijuga 


IBA 


2000 


46 


Rliizopliora apiculata 


IBA 


2000 


48 


Rhizophora mucronata 


IBA 


2500 


52 


Rliizopliora x hybrid 


IBA+NAA 


1000+500 


46 


Sonneratia apetala 


IBA 


1500 


45 


Xylocarpus granatum 


IBA 


2000 


56 


Xylocarpus mekongensis 


IBA 


1500 


64 


Xylocarpus moluccensis 


IBA 


1000 


60 



Step 5. Applying rooting medium: 

Prepare a mixture of sand and clay at 3:7 ratio and wet it with nearby 
mangrove water. This forms the first layer of the rooting medium. This first layer 
of rooting medium is followed by a layer of wet sphagnum mass (to retain 
moisture). Apply this medium of two layers around the wounded portion and 
finally cover it with a polythene cover and tie both the ends as shown in Plate 4. 
If shoot portion droops, the branch should be tied with nearby one to avoid 
drooping. Step 2 to step 5 are shown in Plate 4 A to F. 

Step 6. Monitoring the air layering: 

Periodically check the layered portion and whenever necessary inject tap 
water through a syringe. Roots will be visible after 40-60 days. 

Step 7. Hardening in growth chamber: 

After the root system is well established, separate the rooted sapling from the 
mother plant using a sharp knife. Keep rooted saplings under mist chamber in the 
field nursery at 28°C and 70% relative humidity for 2 to 3 months. The procedure 
followed in hardening the saplings produced through propagule cuttings can be 
followed for hardening the saplings produced through air layering also. 



11 



Step 8. Planting in the field : 

After 2 to 3 months, successfully established saplings should be treated with 
saline water of maximum salinity upto 20 ppt. Diluted sea water, or mangrove 
water itself or water in which common salt is dissolved for desired concentration 
could be used. 

Results obtained in different species based on our studies are presented in 
Plates 5 and 6. 

5.0. VEGETATIVE PROPAGATION OF OTHER SPECIES 

This method described hereunder is applicable to the following species: 
Acanthus ilicifolius, Amoora cucullata, Avicennia marina, Cerbera manghas, 
Excoecaria agallocha, Heritiera fames, Heritiera littoralis, Intsia bijuga, Lumnitzera 
racemosa, Sonneratia apetala and Xylocarpus granatum. 

5.1. Stem cutting 

Materials : 

Tree twigs, solution to remove phenolics, growth hormones, refrigerator, 
knife, mist chamber, humidifier and mud filled polythene bags. 

Methods : 

Step 1. Collection of branches/stems : 

Collect narrow /straight twigs from plus trees. 

Step 2. Cutting of stems : 

Select and cut stems of different types such as soft wood, semi-hard wood 
and hard wood; length of the cut stems may vary from 15 to 20 cm (Plate 7 A). 

Step 3. Removal of phenolic compounds : 

The following method describes the preparation of solutions and treatment 
methods to remove the phenolic compounds. Removal of phenolic compounds 
involves short-term and long-term treatments. 

• Preparation of stock solution : Take 20g of Sodium carbonate and 20g of 
Sodium tungstate, dissolve successively in 80 ml distilled water, and make up 
the final volume to 100ml. It is a 20% stock solution. 

• Preparation of working solution (10% and 5%) : Take 50ml from the 20% 
stock solution, and add 50ml of distilled water. This gives a 10% working 
solution. Take another 25ml of stock solution and add 75ml of distilled water. 
It is a 5% working solution. 

12 



• Short-term treatment to remove phenolic compounds : Take 10% solution 
in small cups. Keep the basal portion of the cuttings immersed in the 
solution for 5-10 minutes. Wash the treated cuttings two to three times in 
distilled water. 

• Long-term treatment to remove phenolic compounds : Keep the cuttings 
treated in 10% solution for about 20-30 minutes in 5% working solution for 
final treatment. Wash the treated cuttings two to three times in distilled water. 
Now the stem cutting is ready for hormone treatment 

Step 4. Hormone treatment 

Hormone treatment is necessary to induce root formation. Indole Acidic Acid 
(IAA), Indole Butyric Acid (IBA) and Naphthalene Acetic Acid (NAA) are com- 
monly used as root promoters. These promoters are available in most of the agro 
shops in the form of powder. 

• Preparation of hormone stock solution : Take lg of IBA (Himedia, Mumbai) 
and add little drops of IN NaOH until it dissolves, and make it up 
with distilled water to 100ml. The strength of this stock solution is 
10,000 ppm. 

• Preparation of hormone treatment solution : The following table (Table 7.) 
shows the method of preparing treatment solutions of different concentra- 
tions from the stock solution. 

Table 7 : Preparation of hormone treatment solutions of different concentrations 



Quantity of 


Quantity of 




Concentration 


stock solution (ml) 


distilled water (ml) 


(ppm) 


10 


90 




1000 


15 


85 




1500 


20 


80 




2000 


25 


75 




2500 



Hormone treatment of the cuttings : Stem cuttings treated to remove pheno- 
lics are again dipped in root promoting IBA hormone for 10-30 minutes. Since 
the concentration of IBA required for root promotion differs from species to 
species, stem cuttings should be dipped in solutions of different concentra- 
tions as shown in Plate 7 B. Table 8 shows the optimum concentration of the 
hormones for maximum rooting in the stem cuttings of different mangrove 
plants. 

13 



Table 8 : Optimum concentration of hormones for maximum rooting in the 
stem cuttings of different mangrove species 



Species 


Hormone 


Concentration 
(ppm) 


Rooting 
<%) 


Acanthus illicifolius 


IBA+NAA 


500+1000 


83 


Arnoora cucullata 


IBA 


1500 


75 


Avicennia marina 


IBA 


2000 


56 


Cerbera manghas 


IBA 


1500 


63 


Cerbera odollatn 


IBA 


1000 


69 


Excoecaria agallocha 


IBA 


2000 


68 


Heritiera fonies 


IBA 


2500 


72 


Heritiera littoralis 


IBA+NAA 


1500+500 


64 


Intsia bijuga 


IBA 


2000 


68 


Xylocarpus granatum 


IBA 


2500 


85 



Step 5. Planting stem cuttings in poly-bags/plastic pots : 

After the treatment, plant the cuttings in poly bags containing sand and clay. 
Keep planted cuttings under the mist chamber at 28±2°C and 70-80% relative humidity. 

Step 6. Hardening the cuttings in mist chamber : 

Monitor the cuttings continuously and maintain controlled environment. 
Roots can be seen after 20 to 25 days and shoots can be seen after 30 to35 days. 
Spray water inside the mist chamber 3 to 5 times/ day, using hand sprayer, to 
maintain the humidity and temperature (Plate 7 C). Plates 8 and 9 show the rooting 
from stem cuttings in different mangrove species. 

Step 7. Hardening the saplings in nursery : 

After 2 months, keep the successfully established saplings under shady areas 
in the nursery for 3-4 months. Treat the established saplings with salt water up to 
20 ppt for 25 days. 

Step 8. Planting in the field: 

After successful hardening, saplings are ready for planting. Immediately 
after the monsoon season, transfer and plant the saplings in the selected 
field. 



14 



PART B 

MICROPROPAGATION 

1.0 INTRODUCTION 

1.1 Development of micropropagation protocols for mangrove plants 

Micropropagation or plant tissue culture is a technology of growing isolated 
plant cells, tissues, organs or whole plants on semisolid or liquid synthetic nutrient 
media under aseptic and controlled environment. It is the most useful and widely 
used technology in tree improvement programmes. Mangroves are classical ex- 
amples of plants, which have adapted to the shifting, saline and muddy environ- 
ment. To fully adapt to this environment, mangroves have acquired a number of 
unique morphological, ecological and physiological characteristics. However, these 
special adaptive features make the mangrove plants recalcitrant to in vitro culture. 
There have been several inherent problems in the tissue culture of these species and 
hence, there has been only a few attempts as of now to propagate them through 
micropropagation. They are highly recalcitrant to the time tested media like 
Murashige and Skoog (1962) and Lloyd and Mc Kown (1981). The tissue browning 
occurs within few hours of inoculation, which limits the chance of survival of the 
explant tissue. There has been a high degree of contamination due to several 
microbial and fungal endophytes and the growth is very slow in the culture. The 
M.S.Swaminathan Research Foundation over came these problems and has estab- 
lished protocols for the first time for three species of mangroves viz., Excoecaria 
agalloclia, Avicennia officinalis and Acanthus ilicifolius. 

The micropropagation protocol developed for mangrove plants by the 
M.S.Swaminathan Research Foundation consists of a unique combination of macro 
nutrients while micro nutrient and vitamin composition are similar to regular MS 
media( Rao et al., 1998). The protocol is useful in propagating the plus tree of 
mangrove plants for ongoing mangrove afforestation programmes, keeping in view 
the alarming rate of mangrove forest degradation throughout the world. 

1.2 Types of Micropropagation 

There are two types of micropropagation techniques 

a. Direct organogenesis 

b. Indirect organogenesis 

In direct organogenesis, stems with internodes are grown in culture media 
to produce multiple shoots and these shoots are removed and grown in rooting 
media. Once the rooting is established, the explants, which are now called as 
saplings, will be hardened in growth chamber, field nursery and then transferred 
to the field. 

24 



Production of saplings through indirect organogenesis involves the following 
steps. In the first step, a mass of undifferentiated cells is obtained from living 
tissues of plants in a culture medium. This mass of cells is called "callus". The 
callus is removed from the medium and grown in another medium to induce green 
shoots. The next step involves the removal and isolation of individual shoots, and 
growing them in a rooting medium. Finally the rooted saplings are hardened in 
growth chamber, nursery and then transferred to the field. 

1.3 Advantages and limitations in micropropagation: 

Advantages : 

1. It is useful in rapid multiplication of plant material and can be used to 
produce both asexually (through plant parts) and sexually propagated (through 
seeds) plants. 

2. Small pieces of plants (explants) can be used to produce a large number of 
plantlets in a small space. 

3. Tissue culture provides a high degree of phenotypic uniformity. 

4. Plantlets can be stored in vitro in a small space and less labour is required for 
maintenance of stock plants. 

5. Plantlets produced through micropropgation are usually free from infection 
by bacteria, fungi and viruses. 

6. Nutrient levels, light, temperature and other factors can be precisely con- 
trolled to accelerate vegetative multiplication and regeneration. 

7. Tissue culture is independent of seasons. Tissue culture could be carried out 
round the year. 

8. Plants in vitro require minimal attention between subcultures. Therefore, 
only limited labour and materials are required. 

Disadvantages : 

1. Chemicals used in medium preparation are expensive and less readily 
available. 

2. High phenolics in mangrove plants delay the growth and thus the process 
becomes time consuming. 

3. Growth in the culture medium is slow. 

4. Endophytic fungal contamination is high in mangrove species. 



25 



2.0 MEDIA USED IN MICROPROPAGATION 

The following are the culture media used in tissue culture: i) Murashige and 
Skoog (1968) medium or MS medium, ii) Woody Plan Medium or WPM medium 
(Lloyd and McKown, 1981) and iii) Schank and Hilderbrandt medium (1972) or SH 
medium. Apart from these, M.S.Swaminathan Research Foundation has developed a 
medium, for the tissue culture of mangrove plants, which is designated in the manual 
as X medium. 

2.1 Composition of different culture media: 

The following table (Table 9) shows the nutrient composition of the different 
culture media used in the micropropagation of mangrove plants. In all the three 
species of mangroves for which protocols have been developed, cultures are 
initiated in the X medium developed by the M.S.Swaminathan Research Founda- 
tion. MS medium can be used for the subculture of Avicennia officinalis and 
Excoecaria agallocha from second subculture onwards. SH medium can be used for 
shoot elongation in Acanthus ilicifolius. Half strength MS or WPM medium can be 
used for root initiation in Avicennia officinalis and Excoecaria agallocha. 

Table 9 : Nutrient composition of different culture media 



Nutrient composition 



MS (1962) 



WPM (1981) 
(mg/1) 



SH (1972) 



X (1998) 



Major nutrients 

NH 4 NO s 

(NH 4 ) 2 S0 4 

KNO, 

Ca (NO,) 2 .4H 2 

MgS0 4 .7H 2 

CaCl^Hp 

KH 2 P0 4 



1650 


400 














300 


500 


1900 





2500 


525 





556 








370 


370 


400 


375 


440 


96 


200 


200 


170 


170 





250 





990 









Iron stock 










FeS0 4 .7H 2 


27.8 


27.8 


15 


27.8 


Na 2 EDTA.2H 2 


37.3 


37.3 


20 


37.3 



Minor Nutrients 

MnS0 4 .4H 2 

ZnS0 4 .7H 2 

H,BO, 

KI 

Na 2 Mo0 4 .2H 2 

CuS0 4 .5H 2 

CoCl 2 .6H 2 

Vitamins 
Inositol 
Glycine 
Thiamine .HC1 
Nicotinic acid 
Pyridoxine .HC1 

Sucrose (g/1) 
Agar (g/1) 



22.3 


22.3 


13.2 


22.3 


8.6 


8.6 


1 


8.6 


6.3 


6.2 


5.0 


6.3 


0.83 





1.0 


0.83 


0.25 


0.25 


0.1 


0.1 


0.025 


0.025 


0.2 


0.2 


0.025 





0.1 


0.1 


100 


100 


100 


100 


10 


2 





10 


1 


1 


5 


1 


1 


0.5 


5 


1 


1 


0.5 


5 


1 



30 



30 



30 



30 



26 



2.2 Preparation of stock solution 

In order to avoid delay in the preparation of the culture media, stock 
solutions of major and minor nutrients, iron and vitamins are prepared separately. 
These stock solutions can be stored at 4°C for about 6 months. Needed quantity of 
culture media are prepared whenever necessary by mixing these stock solutions 
and diluting them with double distilled water to get original concentration. 

2.3 Preparation of 20X major nutrient solution: 

To prepare 20X stock solution of major nutrients, dissolve all the chemicals 
except CaCl 2 .2H 2 one by one in about 500 ml of double distilled water as shown 
in Table 10. Stir the solution using a magnetic stirrer and ensure that all the 
chemicals are dissolved completely. Finally, add CaCl 2 .2H 2 and stir the solution 
till it is completely dissolved and make up the volume to 1000ml. 

Table 10 : Composition of Stock solution of 20X major nutrients 



Major nutrients 


MS (1962) 


WPM (1981) 
(g/» 


SH (1972) 


X (1998) 


NH 4 N0 3 


33 


8 








(NH 4 ) 2 S0 4 








6 


10 


KN0 3 


38 





50 


10.5 


Ca (NO,) 2 .4H 2 





11.12 








MgS0 4 .7H 2 


7.4 


7.4 


8 





CaCl 2 .2H 2 08.8 


1.920 


4 


4 




KH 2 P0 4 


3.4 


3.4 





5 


K 2 S0 4 





19.8 









2.4 Preparation of 200X minor nutrient solution: 

To prepare 200X stock solution of minor nutrients, dissolve all the chemicals 
one by one in about 500 ml of double distilled water as shown in Table 11. Stir the 
solution using a magnetic stirrer and ensure that all the chemicals are dissolved 
completely and make up the final volume to 1000ml. 

Table 11 : Composition of stock solution of 200X minor nutrients 

Major nutrients MS (1962) WPM (1981) SH (1972) X (1998) 
<g/D 

MnS0 4 .4H 2 

ZnS0 4 .7H 2 

H 3 B0 3 

KI 

Na 2 Mo0 4 .2H 2 

CuS0 4 .5H 2 

CoCl 2 .6H 2 



27 



4.46 


4.46 


2.64 


4.46 


1.72 


1.72 


0.200 


1.72 


1.26 


1.24 


1.0 


1.26 


0.166 





0.200 


0.166 


0.050 


0.050 


0.020 


0.020 


0.005 


0.005 


0.040 


0.040 


0.005 





0.020 


0.020 



2.5 Preparation of 200X iron stock solution: 

Table 12 gives the composition of the 200X iron stock solution. To prepare 500 
ml of iron stock solution, first dissolve FeS0 4 .7H 2 in about 400 ml double distilled 
hot water. After ensuring that FeS0 4 .7H 2 is completely dissolved, add 
Na 2 EDTA.2H 2 0, stir the solution and make up the volume to 500ml. It is advisable 
to prepare only low quantity of iron stock solution since it will easily get contami- 
nated. Discard the prepared solution if turbidity or precipitate is seen in the solution. 



Table 12 : 


Composition 


of 200X iron stock solution 




Iron Stock 


MS (1962) 


WPM (1981) 
(g/500 ml) 


SH (1972) 


X (1998) 


FeS0 4 .7H 2 in grams 
Na 2 EDTA~.2H 2 in grams 


2.78 

i 3.73 


2.78 

3.73 


1.5 
2.0 


2.78 
3.73 



2.6 Preparation of 1000X vitamin stock solution: 

Table 13 gives the composition of 1000X vitamin stock solution. Weigh the 
vitamins accurately and dissolve them one by one in 80 ml of double distilled 
water. Once the vitamins are completely dissolved, make the final volume to 100 
ml. Like iron stock, vitamin stock solution should also be prepared in less quantity, 
since it will also be easily contaminated. The stock solution should be used as early 
as possible. 

Table 13 : Composition of vitamin stock solution 



Vitamins 


MS (1962) 


WPM (1981) 
(mg/lOOml) 


SH (1972) 


X (1998) 


Glycine 


1000 


200 





1000 


Thiamine .HC1 


100 


100 


500 


100 


Nicotinic acid 


100 


50 


500 


100 


Pyridoxine .HC1 


100 


50 


500 


100 



2.7 Other chemicals : 

The following chemicals can be directly added to the culture medium: 

i) Inositol 100mg/l 

ii) Sucrose 30g/l 

iii) Agar 8g/l or Phytagel 2g/l 

2.8 Preparation of X culture medium : 

To prepare 1 liter of X culture medium, take 600 ml of double distilled water 
and add 50 ml of the 20X stock solution of major nutrients, 5 ml of 200X stock 

28 



solution of micro nutrients, 5 ml of 200X iron stock solution, 1 ml of 1000X vitamin 
stock solution in succession and ensure that they are completely mixed. Add lOOmg 
of Myo-inositol and 30 g of sucrose. Mix well using a magnetic stirrer until a clear 
solution is obtained. Add appropriate quantity of growth hormones such as IAA, 
IB A, NAA, Kn, BA and 2,4-D as shown in Table 14. Adjust pH to 5.75 to 5.85 
by adding IN NaOH or IN HC1. Make the final volume to 1000 ml. Add 8 g of agar 
or 2 g of Phytagel. Cook the media on a hot plate until the agar or Phytagel 
is completely dissolved. Pour 3 to 5 ml of the cooked media into sterilized 
test tubes. Close the mouth of the test tube with sterilized cotton plug. 
Autoclave the test tubes for about 15 minutes at 121°C under 15 lb pressure. After 
autoclaving, keep the test tubes in a slanting position inside a sterile chamber. 

2.9 Addition of growth hormones in culture media: 

The experiments conducted in the M.S.Swaminathan Research Foundation 
showed that for successful shoot and root formation, different mangrove species require 
different growth hormones as shown in Table 14. These combinations and 
concentrations of growth hormones should be added to the culture and sub-culture 
media. 

Table 14 : Combination and concentration of growth hormones to be used in 

culture and sub-culture media 

c . Growth hormone (ppm) 

Species rr 

BA Zeatin IBA 2ip IAA 

E .agallocha 

Shoot induction (1 st culture) 3 1 — — — 

Shoot elongation (1 st sub culture) 3 — 0.5 — — 

Rooting (2 nd subculture) — — 0.5 — — 

Avicennia officinalis 

Shoot induction (1 st culture) 1 — 0.5 — — 

Shoot elongation (1 st sub culture) — — — — — 

Rooting (2 nd subculture) — — 0.5 — — 

Acanthus ilicifolius 

Shoot induction (1 st culture) 0.5 — 0.2 1.0 

Rooting (1 st sub culture) — — 0.5 — — 

3.0 Methods of micropropagation 

Materials: 

Stock solutions of MS, WPM, SH and X media, explants, glassware, inocula- 
tion chamber, sterilized culture racks, growth chamber 

29 



Methods : 

Stepl. Collect explants, preferably shoot portion, from the field or mist chamber or 
nursery. The explant material may be leaf segments, uninodal and binodal seg- 
ments from mature trees as well as seedlings. 

Step 2. Wash the explant in running tap water for 1 hour. This is necessary 
to remove the exudates (phenolics, tannins, and mucillages) present within the 
tissue. 

Step 3. Again wash the explant with Tween 20 (2%, v/v) and rinse until traces of 
soap are removed. Take the explants to a sterile laminar flow and surface sterilize 
the explants with HgCl 2 (0.1%, w/v) followed by three washes with sterile distilled 
water. 

Step 4. Trim the explants with sterilized knife and cut them into small pieces of leaf, 
uninodal and binodal segments. Cut the lower portion of the nodal explants at an 
angle of 20 to 30 degrees to get a slanting basal portion, which facilitates in effective 
absorption of nutrition from the medium. 

Step 5. Transfer the trimmed explants to the culture media. 

Step 6. Incubate the culture at 24± 2°C and 60% relative humidity under a 16-hour/ 
day photoperiod. Provide light intensity of 50 |J,Mol nr 2 s 1 using a cool white 
fluorescent light. 

Step 7. After shoot initiation, sub-culture the explants for shoot elongation and 
multiplication. For this purpose use different combinations and concentrations of 
growth hormones as shown in Table 14. 

Step 8. After shoot elongation, remove the explants, cut the shoots in a sterile 
inoculation chamber, and transfer the shoots to rooting media for root initiation. 
Transfer the well established rooted plants into a growth chamber. 

Step 9. Harden the rooted plants in growth chamber (e.g. NK System LP-1PH) at 
80% relative humidity and 26°C for a period of three weeks. 

Step 10. Transfer the hardened plants to the mangrove nursery and after 
hardening for about 2 to 5 months, treat them with different salinities ranging from 
5 to 20 ppt. 

Step 11. Transfer the hardened plants to the site selected for plantation. 

3.1 Micropropagation of Excoecaria agallocha 

• Binodal segments respond well in X medium with a combination of BA, 
Zeatin and IBA as shown in Table 14 

30 



• The X medium has an overall low mineral content with relatively high 
concentrations of S0 4 2 ~, NH 4 + , P0 4 ~ and K + ions. Auxiliary shoot induction is 
high in this medium compared to MS and WPM media 

• Per cent shoot induction and mean shoot length is maximum (52%) when the 
X medium has BA, Zeatin and IB A. Addition of Zeatin in the culture medium 
(up to lppm) further increases shoot induction response (72%) with no 
significant effect on shoot length 

• Binodal segments give a better shoot induction over uninodal segments. 
The shoot elongation rate enhances from the second subculture onwards 
(Plate 10A) 

3.2 Micropropagation of Avicennia officinalis 

• Uninodal explants of A. officinalis responded well in the X medium with a 
combination of BA and IBA 

• There is an increase in the shoot induction response with the increase in BA 
and IBA concentrations to l.Oppm and 0.5ppm respectively 

• Rooting response is good when the regenerated shoots of 5 cm length are 
transferred to the X medium supplemented with 0.5ppm IBA 

• After 2 weeks of rooting in the growth chamber, the plantlets can be 
transferred to the potting medium consisting of 1:1 garden soil and 
sand mixture. A high humidity condition is to be maintained for another 4 
weeks (Plate 10B) 

3.3 Micropropagation of Acanthus ilicifolius 

• Uninodal explants of Acanthus ilicifolius cultured on SH medium supple- 
mented with BA (0.5 ppm), 2ip (0.2ppm) and IAA (lppm) show maximum 
shoot induction 

• Shoot elongation can be achieved when the shoots are divided and subcul- 
tured on the SH basal medium supplemented with half of the above 
concentrations of hormones 

• The individual elongated shoots subcultured on the Vi SH medium supple- 
mented with 0.5ppm IBA produce healthy roots 

• The rooted plants can be grown in pots with vermiculite in the growth 
chamber with 75% relative humidity and 26°C, to get maximum survival 
(Plate 11) 

In the experiments conducted in the M.S.Swaminathan Research Foundation, 
95% of the plantlets survived in the hardening chamber when the above procedures 
were followed. 

31 



REFERENCES 

1. Lloyd,G. and McKown,B.1981. Commercially feasible micropropagation of 
mountain laurel Kalmia latifolia by use of shoot tip cultures. Int. Plant. Soc. 
Proc. 30: 421-427. 

2. Murashige,T. and Skoog,F.1962. A revised medium for rapid growth and 
bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. 

3. Rao, C.S., PEganathan., Ajith Anand., PBalakrishna and T.PReddy.1998. Pro- 
tocol for in vitro propagation of Excoecaria agalloclia L. a medicinally impor- 
tant mangrove species. Plant Cell Reports 17:861-865. 

4. Schank, R.V. and Hilderbrandt,A.C. 1972. Medium and techniques for induc- 
tion and growth of monocotyledonous and dicotyledonous plant cell cultures. 
Can. J. Bot. 50:199-204. 



34 





Plate 2 : Root and shoot development in propagule cuttings 



A. Bruguiera cylindrica 

C. Kandelia candel 

E. Rhizophora apiculata 



B . Ceriops decandra 

D. Rhizophora mucronata 

F. Rhizophora hybrid 



16 













' 1 k 






1 



Plate 7 : Stem cuttings in Excoecaria agallocha 

A. Cutting of stem 

B. Treating the stem cuttings with hormone 

C. Development of stem cuttings into sapling 



21 




Plate 4 : Air-layering in Excoecaria agallocha in the field 

A. Removal of bark B . Application of root promoting hormone 

C. Application of rooting media D. Wrapping with polythene sheet 

E. Closing the end of the wrapping F. Air-layering 



18 




Plate 5 : Rooting through air-layering in different mangrove species 



A. Heritiera fames 

C. Xylocarpus moluccensis 

E. Sonneratia apetala 



B. Excoecaria agallocha 
D. Rhizophora hybrid 



19 




Plate 6 : Rooting through air-layering in different mangrove species 



A. Avicennia officinalis 

C. Intsia bijuga 

E. Xylocarpus granatum 



B . Avicennia marina 
D. Heritiera littoralis 



20 




Plate 3 : Development and hardening of propagated 
plants in the nursery and mist chamber 

A. General view of the field nursery with mist chamber 

B. Stem cuttings of the Acanthus ilicifolius 

C. Inside view of the mist chamber 

D. Flow of mist inside the mist chamber 

17 




& 

S- 


■+4 

i 




Plate 8 : Rooting in stem cuttings in different mangrove species 



A. Acanthus ilicifolius 
C. Excoecaria agallocha 



B.Avicennia marina 
D. Heritiera littoralis 



Plate 1 : Propagules of different species of Rhizophoraceae 

i. Rhizophora mucronata ii. Rhizophora apiculata 

iii. Rhizophora hybrid iv. Bruguiera cylindrica 

v. Ceriops decandra vi. Kandelia candel 

A. Propagule cutting - Bruguiera cylindrica B. Hormone treatment 



22 



15 




Plate 9 : Rooting in stem cuttings in different mangrove species 



A. Heritiera fames 
C. Cerbera manghas 
E. Lumnitzera racemosa 



B.Amoora cucullata 

D. Intsia bijuga 

F. Xylocarpus granatum 



23 




Plate 10 : A. Micropropagation of Excoecaria agallocha 

A. Uninodal explant B. Binodal explant 

C. Rooting of shootings D. Hardening plants 
E. Field transfer plant 

B. Micropropagation of Avicennia officinalis 

A. Uninodal explant shoot initiation B. Shoot elongation 

C. Rooting of shoots D. Field transferred plant 



32 




Plate 11 : Micropropagation of Acanthus ilicifolius 

A. Uninodal with shoot initiation B. Rooting of shoots 

C. Field transferred plant 

33 




M.S.Swaminathan Research Foundation 

3 rd Cross Street, Institutional Area, Taramani, Chennai 600 113, India 

Telephone : +91(44) 2541 229, 2541 698 Fax : +91(44) 2541319 

E-mail : msswami@mssrf.res.in executivedirector@mssrf.res.in