FACTORS AFFECTING THE GRAFT UNION OF MAMEY SAPOTE
(CALOCARPUM SAPOTA (JACQ.) MERR.)

By

MARY ANN HOLLINGSWORTH OGDEN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

Dedicated in loving Memory of

My Mother, Mary Baxley Hollingsworth (1914 to 1982);
My Father, Roger Hollingsworth (1910 to 1983);

George N. Avery (1922 to 1983), botanist, naturalist, friend.

ACKNOWLEDGEMENTS

It is a pleasure to express appreciation to all members
of my committee. I am particularly indebted to Dr. Carl
Campbell who provided friendship as well as guidance and
concern for my personal goals and professional future.
Special thanks are extended to Dr. Wayne Sherman and Dr.
Larry Jackson for encouragement, suggestions, and
opportunity for discussion. Thanks are also extended to Dr.
Walter Judd for use of the herbarium and many references,
and Dr. Tom Davenport for help in experimental design.

Other persons not on my committee also provided
invaluable assistance. Dr. Phillip Achey provided
laboratory space, equipment and supplies, and computer
instruction. Dr. Sylvia Coleman, Dr. Jasper Joiner, Dr.
Don Huber, Dr. Gloria Moore, and Dr. Sal Locascio provided
laboratory equipment and advice on techniques. Dr. Fred
Davies helped interpret my statistical analysis. Mr. Pablo
Lara assisted me with grafting, nursery work, and much
insight into the mamey sapote. Ms. Debbie Gaw and Mrs.
Anne Harper assisted me with laboratory technique. Anne
Harper assisted me in the laboratory. Mr. Steve Hiss gave
valuable advice on photography. Mr. Robert Barnum, of
Possum Trot Nursery, provided many plants for

experimentation and many hours of his time. The Rare Fruit
Council International, Inc., of Miami provided much moral
support and valuable advice on tropical fruits. Mrs. Marti
Radford and Mrs. Ann Britton assisted in proofreading the
manuscript .

Financial support in the form of a Graduate
Assistantship in Extension was provided by the Department of
Fruit Crops. Scholarships were awarded by the Dade County
Farm Bureau and the Daughters of the American Revolution.

Last, but certainly not least, I express love and
gratitude to my children, Laura Ann and Nicholas, for their
understanding and encouragement that gave me the impetus to
complete this dissertation.

IV

TABLE OF CONTENTS

Page

DEDICATION ii

ACKNOWLEDGEMENTS iii

ABSTRACT viii

CHAPTER I. INTRODUCTION 1

CHAPTER II. LITERATURE REVIEW 3

Origin and Distribution 3

Taxonomy 4

Family characters 4

Separation of POUTERIA and CALOCARPUM 6

Synonomy 6

Common names 8

Culture and Propagation 9

Cultivation in Florida 10

Propagation of tropical fruit in the Tropics 11

Propagation of mamey sapote 12

Graftage 13

Veneer graft 14

Scionwood preparation 15

Rootstocks 18

Growth regulators 18

Apical dominance 18

Seasonal and environmental effect upon graft take.. 20

Anatomy and Graft Union Formation 20

Wood anatomy relative to graft take 21

Water conduction to facilitate graft take 22

Silica content 23

Differences in juvenile and mature stem anatomy. ... 24

Anomalous secondary growth 25

Fiber band location 25

Wound healing and graft union formation 26

CHAPTER III. JUVENILITY AND SEASONAL EFFECTS 30

Introduction 30

Materials and Methods 31

Results and Discussion 32

CHAPTER IV. USE OF JUVENILE INTERSTOCKS TO FACILITATE

TOPWORKING 34

Introduction 34

Materials and Methods 34

Results and Discussion 35

v

CHAPTER V. CARBOHYDRATE ACCUMULATION RELATIVE TO GRAFT

TAKE 37

Introduction..' 37

Materials and Methods 38

Results and Discussion 39

CHAPTER VI. SUGAR PULSING 45

Introduction 45

Materials and Methods 45

Results and Discussion 46

CHAPTER VII. GROWTH REGULATORS APPLIED TO THE GRAFT

WOUND 49

Introduction 49

Materials and Methods 50

Results and Discussion 51

CHAPTER VIII. ROOTSTOCK APICAL DOMINANCE RELATIVE TO

GRAFT TAKE 53

Introduction 53

Materials and Methods 54

Results and Discussion 54

CHAPTER IX. COMPONENTS AND LOCATION OF THE VASCULAR

CAMBIUM 57

Introduction 57

Materials and Methods 58

Results and Discussion 59

CHAPTER X. GRAFT UNION FORMATION 69

Introduction 69

Materials and Methods 69

Results and Discussion 70

CHAPTER XI. FIBER BAND LOCATION WITH RESPECT TO

GENERAL ANATOMY AND THEIR EFFECT UPON

GRAFT TAKE 89

Introduction 89

Materials and Methods 89

Results and Discussion 90

CHAPTER XII. SILICA: CONTENT AND LOCATION, AND

EFFECT UPON GRAFT TAKE 93

Introduction 93

Materials and Methods 94

Results and Discussion 95

CHAPTER XIII. INTERACTIONS OF STARCH, SUGAR, AND

SILICA 107

vi

CHAPTER XIV. SUMMARY AND CONCLUSIONS Ill

LITERATURE CITED 115

BIOGRAPHICAL SKETCH 125

vii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

FACTORS AFFECTING THE GRAFT UNION OF MAMEY SAPOTE
(CALOCARPUM SAPOTA (JACQ.) MERR . )

By

Mary Ann Hollingsworth Ogden
August 1 984

Chairman: Carl W. Campbell

Major Department: Horticultural Science (Fruit Crops)

Mamey sapote is a tropical fruit from the lowlands of
Central America grown in southern Florida. Mamey is
difficult to graft, and supplies of grafted nursery stock of
superior cultivars have limited planting and production.
Seedling plants vary in quality and yield and have a long
juvenility period. If seedling trees prove to be of
inferior quality, topworking to change the cultivar is very
difficult. Research was conducted to determine the best
propagation methods, grafting period, juvenility, scion
selection, interstocks, carbohydrate accumulations, possible

viii

use of growth regulators, apical dominance, silica content,
and anatomical characteristics relative to graft union
formation .

It was found that grafting periods with warm days, cool
nights, low rainfall, and low relative humidity were the
best times to graft mamey sapote (late October to early
December or late March to early May). Juvenility affected
graft take, and scions that were reverted to a
"juvenile-like" condition were better scion materials than
mature wood. "Juvenile-like" scions with a modified veener
graft on rootstocks less than 1 meter tall were the most
succesful combination. Anatomical examinations showed an
irregular vascular cambium and large bands of fibers
surrounding the cambium that may inhibit graft formation
unless proper grafting techniques are employed.

Measurements of carbohydrates indicated that starch
accumulation enhanced graft take. Silica analysis on 3
scion maturities (seedling, "juvenile-like", and mature)
indicated that mature scions with high silica content were
the least desirable graftwood and "juvenile-like" scions
with lower silica content performed best.

Growth regulator applications were not successful and
auxins may inhibit graft take. Terminal bud removal to
break apical dominance (and eliminate a source of auxins)
enhanced graft take of mature scions during periods of
active growth during the summer (the worst time to graft
mamey) but did not improve take during the dormant periods.

IX

CHAPTER I

INTRODUCTION

Calocarpum sapota (Jacq.) Merr. is difficult to
propagate asexually (l , 68). Asexual reproduction is
desirable because the seed do not come true to type and
there is much variation in precocity fruit flavor, and yield
(1 , 10, 94). It is economically essential to asexually
propagate nursery stock because of the long juvenility
period that can last from 12-15 years (10, 94, 102).

All methods of asexual propagation are extremely
difficult. These methods include marcottage, cuttage, and
graftage (l). Once the tree has passed its juvenile period
it is extremely difficult to marcott (10, 68, 102). Rooting
of cuttings is hard at any stage, whether juvenile or adult.
Grafting is possible, although it is not easy (68, 94).

The veneer graft is considered to be the best method
for Florida conditions and in some of the Central and South
American countries. It is possible to use a chip bud or a
T-bud technique but the results are erratic (68, 94).
Approach grafts have been recommended and were at one time
the most common method of grafting in Florida nurseries
(68). This technique is tedious and cumbersome, unless the

1

2

stock and scion are perfectly matched. The stock plant
should be larger in diameter than the scion, or the graft
will fail because the rootstock dies from vascular collapse
(68, 88).

Once the tree is established and producing in the
field, it is rarely possible to top-work if a different
cultivar is desired (S. P. Lara, pers. comm.). Methods
of top-working need to be developed because many of the
trees in south Florida are seedlings and may be unacceptable
to the homeowner or for commercial production.

All fruit produced in south Florida are sold locally
with none being available for shipment to northern markets
(11). Frozen mamey pulp sold in supermarkets is generally
imported from Costa Rica and the Dominican Republic.
Increased production would make local fruit available for
processing.

Knowledge of the factors that affect the formation of
the graft union would permit the plant to be more easily
propagated and more grafted trees would be available. The
factors investigated in this study include effects of
grafting seasons, juvenility, topworking with juvenile
interstocks, stored carbohydrates (total soluble sugars and
starch), sugar pulsing, applications of growth regulators,
apical dominance, anatomy (tissue type, silica inclusions,
cambium location to determinine regularity and anomalous
secondary growth), and the formation of the graft union.

CHAPTER II

LITERATURE REVIEW
Origin and Distribution

The mamey sapote is a member of the family Sapotaceae
that includes other tropical fruits such as the sapodilla
(Manilkara zapota (L.) Royen),, green sapote (Calocarpum
yi ride Pittier), caimito (Chrysophylum cainito L. , and
canistel (Pouteria campechiana (H.B.K. ) Baehni) (5, 35).

Mamey sapote is widely distributed throughout Central

America, northern South America, the Caribbean (especially
Cuba) and south Florida (10, 33, 90, 94). Many centuries of
cultivation have made the origin of this species somewhat
obscure, but it is thought to be in the lowlands of Central
America, including southern Mexico, Honduras, Guatemala, and
Costa Rica (6, 34, 73). Popenoe (97) mentions that during
Cortez's infamous march through Central America to the
interior of Mexico, his army survived on the fruit of the
mamey that was abundant and widely distributed. The trees
can be found today growing around the ruins of ancient Mayan
cities where they are still a part of the native diet (13,
97).

3

4

Taxonomy

There are many other members of the Sapotaceae that are
indigenous to the lowland humid tropics of Central and South
America. The family is an important part of the ecosystem
in the neotropics making up 25$ of the standing timber (55).
Worldwide there are 70 genera, 800 species, with 5/4 of
these species belonging to 6 genera (84).

The Sapotaceae is a well marked and natural family, but
the familial floral characters are so uniform that generic
identification is extremely difficult. This in turn is
responsible for the extensive synonymy characterizing many
taxa within the family. (55). Identification within this
family is almost impossible without using material
consisting of leaves, flowers, fruit, and seed.
Unfortunately, species continue to be named on the basis of
flowering or fruiting material alone and this continues to
add to the already confused state of affairs (55, 56, 57,
58).

Family characters

Common family characters include well developed latex
system (that produces chicle in the genus Manilkara) ;
pubescence consisting of 2-armed hairs; mostly trilacunar
nodes; vessels with simple perforations; silica bodies;
calcium oxalate crystals; imperforate tracheary elements
with mostly simple pits; wood parenchyma generally abundant
and apotrachael, either in well defined ribbons or

5

reticulate, seldom diffuse or vasicentric. Leaves,
alternate or seldom opposite, simple, mostly entire,
occasionally with stipules. Flowers mostly small, mostly
borne singly or in cymose clusters, nearly always perfect,
sepals distinct, and imbricate or sometimes in 2 cycles;
corolla usually sympetalous; carpels 2-14, ovules 1 per
carpel, axile or axile-based placentation; fruit fleshy,
indehiscent; seed large, commonly with a relatively large,
excavated scar of attachment, seed coat shiny and usually
thick and hard; embryo large, with thin, flat cotyledons,
enclosed in a more or less well developed endosperm or with
thick cotyledons and without endosperm at maturity (23, 24,
73, 99).

The raamey sapote (Calocarpum sapota) grows to a height
of 30-35 m in the tropics and 15 m in Florida (12, 94) and
may have large buttresses. Trunk diameter can be 80-100 cm
(6, 23, 24, 99, 113). Leaves are broadly oblanceolate or
narrowly obovate mostly abruptly acuminate, about 10-30 cm
long, 4.5-15 cm wide, glabrous above with some appressed or
loose hairs along the midrib and main veins beneath;
primary lateral veins about 20-50 pair; secondary lateral
veins regular, more or less perpendicular to the primary
one, petioles subglabrous to densely pubescent or tomentose,
4 cm long or less. Flowers are clustered at defoliated
nodes, cauliflorous on upper trunk and main branches,
subsessile, or on short pedicels as much as 4 mm long;
sepals 8-12, spirally arranged,

increasing in size

6

centripetally ; corolla 9-19 mm long, white, lobes about
equal or a little exceeding the tube; filaments attached at
or a little below the sinuses, 3-5 mm long; staminodes
filiform, about 2 mm long; ovary 5-loculate. Fruit are
brown (scurfy), ellipsoid to subglobose, about 8-20 mm long;
pulp smooth to fibrous generally salmon colored to red and
sweet; seed 1-4, commonly solitary, large with broad ovate
scar (1 , 24, 35, 99).

Separation of Pouteria and Calocarpum

On a generic level there are similar characters that
link the genera Calocarpum and Pouteria and they are
difficult to distinguish on a morphological level.
Calocarpum has more numerous sepals arranged in an evident
spiral. The woods are easily distinguished anatomically.
Calocarpum has reticulate parenchyma with the corners
rounded and showing more or less distinctive intercellular
spaces or closely spaced uniseriate bands between the wood
rays, rays 1-4 seriate, radially aligned pores, and very
rarely to lacking tracheids. Pouteria has banded
parenchyma, 1-2(3) seriate rays, and pores in diffuse
arrangement, and vascular tracheids very common.

Synonomy

The scientific name of the mamey sapote has undergone
many changes since it was first described in 1703 by Plumier
(19). Sloan (19) described the mamey sapote in 1725 with a
non-Linnean name Malus persica maxima f oli is magnis .

7

Jacquin (19) placed the mamey in the genus Sideroxylum in
1760 with the first binomial description. Confusion of the
synonymy dates back to the second edition of Linnaeus'

Species plantarum that based the Linnaen name upon the type

species of Plumier's sapota (96). Since Plumier had

traveled in the West Indies he was familiar with both the
mamey and its relative the sapodilla. The confusion exists
because the drawings by Plumier combined characters of the 2
fruits. The nomenclature was based on the drawings and on a
herbarium sheet that contained the 2 different genera for
the type specimens (19, 96). A survey of the synonymy

dating from the first binomial names includes:

Sideroxylum sapota Jacq. 1760. ( 1 9 , 23, 73, 95)

Achras mammosa (Jacq.) L. 1762. (19, 23, 34, 113)

Achras zapota var. major Jacq. 1763. (19, 73)

Sapota mammosa (Jacq.) Mill. 1768. (1 9, 34, 73)

Lucuma mammosum (Jacq.) Gaertn. f. 1807. (35, 113)

Lucuma mammosa (Jacq.) DC. 1884. (19, 34, 69, 73)

Vitellaria mammosa (Jacq.) Radik. 1882. (19, 35, 34)

Calospermum mammosum (Jacq.) Pierre 1890. ( 1 9 , 35)

Calocarpum mammosum (Jacq.) Pierre 1904. ( 1 9 , 35)

Achradelpha mammosa (Jacq.) Cook 1913. (19, 35, 113)

Calocarpum sapota (Jacq.) Merr. 1923. (73, 99, 95)

Pouteria mammosa (Jacq.) Cronquist 1946. (35, 55, 73)

Pouteria sapota (Jacq.) Moore and Stearn 1967. (95)

8

Pouteria mammosa (Jacq.) Cronquist by Kukachka 1978.
(55)

Pittier (96), Merril (23), Baehni (4) ,and Lundell (63)
considered the logical name for the mamey sapote as
Calocarpum. Moore and Stearn (73) used Pouteria but said if
the genus is considered in a narrow sense that Calocarpum
could be retained. Using the anatomical characters of
Kukachka (55) the genus should be C alocarpum over Pouteria
(used by Cronquist (24) with a few morphological
characters). C_^ sapota should be used instead of C.
mammosum because Jacquin's name Sideroxylum sapota in 1760
is anterior to Miller's name Sapota mammosum in 1762.
Calocarpum sapota (Jacq.) Merr. will be used for the
remainder of this dissertation.

Common names

Common names for C_;_ sapota are just as confusing as
the lineage of the scientific names. Sapota is a derivation
from either the Aztec (l , 19) or Nahuatl (35, 96) Indian
word, tzapotl , a general name for all sweet fruit (19).
Passing into the Spanish language, the "t" was dropped and
tzapotl became zapote. When transferred to the English
language "s" is substituted for "z" and becomes sapote (19).

Confusion of the common name sapote can arise because
fruits other than C^ sapota are called sapote, because of
the usage of sapote to mean sweet fruit. Other fruits
called sapote include, white sapote (Casimiroa edulis Have)
in the Rutaceae; black sapote (Diospyros digyna Jacq.) in

9

the Ebenaceae; green sapote (C^ viride Pittier); yellow
sapote (l\_ campechiana (H.B.K. ) Baehni) in the Sapotaceae
and (Casimiroa tetrameria Millsp. ) in the Rutaceae (5, 35).

Other common names are numerous, and in the British and
French West Indies, Cuba, Colombia, Venezuela, Panama, Costa
Rica, southern Mexico (including the Yucatan peninsula)
there may be several common names per country. A listing of
the common names cited by 2 or more authors include:
atzapot iquahuitl (35, 99, 113 ); Chad haaz (35, 99);
grosse sapote (1 , 35, 99); mamee sapote (1 , 35, 99); mamey
(1 » 6, 34, 36, 69); mamey Colorado (1 , 6, 10, 12, 34, 36,
69); mamey sapote ( 1 , 1 3 , 34 , 35 , 86 , 94); mamey zapote
(1 , 35); marmalade fruit (l , 35, 99); marmalade plum (1 ,
34, 123); tenzontzapotl (2, 32, 35, 36, 69, 96); tzapotl
(23, 69, 96, 99); tsakas sabibu (35, 99); sapote (6, 10,
23, 34,); sapotier (35, 99); zapote Colorado (34, 36);
and zapote mamey (6, 34, 36, 69).

Culture and Propagation

The mamey sapote is best adapted to the lowland humid
tropics (6, 34, 73). The mamey commonly grows at 600 m in
altitude, less commonly at 900 m, and very rarely at 1200 m
(113). The green sapote (considered by some authors to be
the same species as the mamey) grows best at higher
elevations around 1200 m (5, 35). Low temperature limits
mamey distribution both in altitude and latitude (10, 90).
In Florida, -3.8 C is the lower limit for mamey (10).

10

Mamey sapote is tolerant of many soil types, including
the heavy clays of Puerto Rico, the sandy clays of
Guatemala, and the limestone and sands of southern Florida
0 > 10). The tree is not tolerant of flooding so good
drainage is essential (1 , 10).

Cultural practices vary from country to country. Most
of the trees that produce fruit for the market are dooryard
rather than plantation crops (90), because the majority of
trees are propagated by seed rather than grafting (91,94).
Lack of knowledge of vegetative propagation of the mamey
sapote has limited its culture (l ).

Seedling mamey trees have a long juvenility period
compared to grafted trees and this has fostered the myth
that all mamey trees take a long time to bear (l). Seedling
trees vary in size, fruit quality, yield and precocity (l ,
10, 94).

Cultivation in Florida

In Florida, the mamey sapote has been cultivated as a
dooryard fruit and in experimental plantings since the
mid-1800's (13). It grows well in Florida and has relatives
in the native flora (86). In 1970 Campbell (11 ) reported
few small orchards of mamey sapote. By 1982 there were at
least a total of 150 acres planted in Dade County, Florida,
with thousands of trees in dooryard plantings.

There were no vegetatively propagated trees in Florida
until the 1950s because of propagation difficulties (13).
Previously, nursery stock was limited by a lack of

11

propagating materials (both seed for stocks and trees of
superior cultivars for scionwood) and persons with
experience in grafting mamey (91). Many Florida nurseries
now regularly propagate the mamey sapote (13). There is
much interest and demand for the fruit and trees, because of
the large Latin population settled in Florida who hold the
mamey in high esteem, and who are familiar with the mamey in
their native countries (11).

Superior cultivars that are propagated in Florida
include 'Magana', 'Copan', 'Pantin' or 'Key West','
Tazumal ' , 'Chenox', 'Progresso', 'Francisco Fernandez ' , and
'Cuban no. 1'. The cultivars 'Copan', Tazumal', 'Mayapan'

(or 'No. 2'), and 'Pantin' (or 'Key West') were developed in
Florida (13).

P-ppagation of tropical fruit in the Tropics

Seasonal climatic variation in the tropics has much
less effect upon graft take than in the temperate zone (92).
Such variation is largely a matter of dry or wet conditions,
and this can be largely controlled by irrigation. Most
tropical species can be grafted most of the year. Major
factors that inhibit propagation are flowering, leaf
abscission, and growth suspension. Coordinating harvest
season and seed extraction for planting rootstocks so as to
have the rootstocks of proper maturity and grafting size is
also a limiting factor. According to Pennock (92), moderate
rainfall for field grafting is also desirable. Other
factors that affect grafting of tropical plants are phenolic

12

substances and latex released by wounding, poor stock and
scion selection, and tissue desiccation.

Some grafting techniques are recommended over others
for better and faster healing in fruit trees. In general,
Skene et al.(l09) recommends a chip bud over a T-bud for
fruit trees because the xylem develops more rapidly.

Propagation of mamey sapote

Unlike many tropical fruits, mamey is difficult to
graft and may be grafted only at certain times of the year.
Most members of the Sapotaceae are difficult to graft,
although some species are easier to graft than others (67,
68). An example of ranking in increasing order of grafting
difficulty is 1) sapodilla , 2) canistel, 3) caimito and
mamey sapote (C. W. Campbell and S. P. Lara, pers.
comm. ) .

Numerous authors report that the mamey sapote is
difficult to propagate (1 , 10, 74, 88, 91, 98). One reason
frequently cited for difficulty is that the latex exudate
released from wounding interferes with graft take (a
successful graft that has healed and sprouted (92, 98, 102).
There are other authors who feel that latex does not
interfere with graft take (68, 91 ). Lack of sufficient
callus production is another problem in the formation of the
graft union (l , 68).

Many methods have been reported for both sexual and
asexual propagation, with the most common being seedage (l ,
36, 74, 89, 91, 94, 98). Seed for either seedling trees or

13

rootstocks should be removed immediately from the fruit and
planted quickly as they remain viable for only a short
period (34, 74).

Asexual propagation methods that have been reported for
mamey sapote include cuttage, marcottage, and graftage.
Tissue culture has been unsuccessful due to a lack of callus
initiation (R. Litz, pers. comm.). Insufficient callusing
has also been reported for cuttage (l). Marcottage is
possible (74, 122,123) with generally poor success (l , 10).
The best time to marcott is in the winter (123).

Graftage

Graftage has been the most successful method of asexual
propagation for mamey. Grafting also shortens the juvenile
period of 5-15 years (10, 61, 123) by almost half (10, 94).
The types of graft reported for propagating the mamey are:
patch (1); forkert (modified chip) (l); splice (60); and
cleft, under both field conditions (59) and under mist (90).
In many countries the approach graft is utilized but it is
tedious, cumbersome, and expensive (45, 60, 68, 88, 94).
One particular problem with approach grafting is that the
scion diameter must not be equal to or larger than the
rootstock diameter, or the scion will outgrow the rootstock
(87, 88). Scion overgrowth of the rootstock causes the
scion to transpire too rapidly and this results in vascular
collapse of the rootstock and death of the scion (68, 88).
A chip bud may be used during certain times of the year, but
this method is not always dependable (10, 94).

14

Veneer graft

The veneer graft is the method of choice for grafting
mamey under Florida conditions (10, 88, 90), and other
countries as well (l , 34, 98). Veneer grafts are also used
on other species of the Sapotaceae (12, 88, 102) and in
other tropical families as well, for example mango
(Mangifera indica L_J (45, 48, 49) and guava (Psidium
gua.java) (83).

Hussain and Buhariu (45) mention that the veneer graft
is more sucessful for sapodilla because the vegetative top
of the stock plant is not lost at the time of grafting and
this supports the growth in the cambial layers of the graft
union. Height of the graft on the rootstock can make a
difference in mamey graft take when using a veneer graft
(98).

Mamey sapotes are possible topwork bub have low rate of
success (S.P. Lara, pers. comm). The new new sprouts are
veneer grafted much in the manner of topworking lychees
(Litchi chinensis Sonn) (82).

A modified veneer graft used on annonas (Annona sp. )
works well on the mamey sapote (89). This method is
different from the standard veneer graft in that a shallow
cut is made on the rootstock for the length of the scion to
be grafted. The budstick is trimmed and the vertical cut
removes only 1/8 of the diameter of the graftwood as
compared to removal of greater than 1 /2 of the budstick
diameter in the standard veneer graft. This method

15

conserves more stored carbohydrates and moisture in the
scion, which is beneficial, since the graft union formation
takes a long time, 2 months or sometimes longer, in the
mamey sapote (86, 90). The cinchona method is another type
of modified veneer graft and is used extensively in southern
Florida. This method does not use as shallow of a cut on
the rootstock (20).

Scionwood that is selected must be from terminal shoots
that are relatively short without fully expanded leaves
(10). If the wood is too hard or too gelatinous, it is not
suitable for grafting (90). Scionwood should be collected
and used the same day and not stored overnight for the best
grafting success (90).

Scionwood preparation

If the scionwood is not sufficiently mature or dormant,
it can be prepared ahead of time. This has been done on
annonas and guavas. The terminal buds are cut off on
annonas and the lateral buds are subsequently able to swell
and expand when the apical dominance is removed resulting in
the proper stage for scionwood collection (89). Guavas are
cut back severely, and the resulting sprouts when hardened
off are used for scionwood (66, 81, 83).

Maturity of the scionwood makes a difference in
grafting success for other species. Mango scionwood must be
from mature branches (49, 64, 65, 75), while the immature
scionwood is more successful in guava (81, 83).

16

Nelson (83) described the pruning back of guava from
older parent trees and the resulting flushes as juvenile
wood. This wood has larger numbers of scions with more
desirable buds. Younger parent trees produce this type of
flush without this procedure. The forcing of this
"juvenile-like" growth furnishes a larger diameter stock of
scionwood containing more scions than can normally be found
on an older parent trees.

A definition of juvenility is considered by Westwood
(121) and Zimmerman (126). Seedlings of most woody plants
pass though a juvenile stage during which the seedling
cannot be induced to flower. Actual production of flowers
is the first sign of the adult phase. The end of the
juvenile phase and the first appearance of flowers may not
coincide. This intervening period can be referred to as the
transition phase. Once the plant has reached the adult
phase it cannot revert back to the juvenile stage. There
are transitional tissues that are found between the purely
juvenile and adult sections of the stems of seedling trees.

Rejuvenation may be obtained by differentiation of
adventitious buds on the stems or the roots. This is
because the juvenile tissue remains _in situ at the base of
the seedling tree. Heavy pruning of old, weak orchard trees
is thought by some to induce juvenility. Such pruning
brings about invigoration , not juvenility. The
non-flowering phase of budded trees is referred to as the
vegetative adult phase (121).

17

By definition, the tree cannot he reverted to a
juvenile phase when pruned severely, even if the tree
exhibits morphology of the juvenile stage, such as described
in the work of Nelson and Lynch (81, 83, 66). This would be
invigorating the trees and reverting them back to the
vegetative adult transition phase as described for newly
budded trees. This tissue with the juvenile morphology,
that has been reverted from an adult phase to vegetative
adult transition phase by severe pruning, will be referred
to as 'juvenile-like' hereafter in this dissertation.

Another type of in— situ scionwood preparation is
girdling, and it is employed with several species that are
difficult to graft. Girdling is routinely done on macadamia
(Macadamia integrif olia Maiden) as preparation for cuttage
and graftage (22, 46, 51, 84). Girdled lychee branches show
a 28$ increase in carbohydrates and little nitrogen above
the girdle (51). Using the prepared scions that are high in
starch content, the graft take increased from 10$ to 75- 80$
(51 ).

Some other members of the Sapotaceae have shown a
response to girdling. Canistel had an increase in graft
take and an increase in carbohydrate accumulation (30).
Sapodilla when girdled, showed an increase in carbohydrates
but only in seasons other than spring was there an increase
in graft take (30). The green sapote showed an increase in
graft take after girdling (116). There have been tests with
the mamey sapote and girdling does increase the graft take
(117, 123) but it may be only as much as 5$ (68).

18

Rootstocks

Rootstocks must be in good condition for optimum
grafting. This is particularly true for the avocado (Persea
americana ) (41), mango (65) and sapodilla (87, 88). Stocks
for the mamey sapote should have a height of 1 m or less, or
approximately 1-1/2 years-old, under Florida conditions.
Scion diameters should be well matched with that of the
rootstock (60, 68, 88).

Other species have been tested for compatability with
mamey sapote. Canistel makes a graft union when used as a
rootstock, but not as well as mamey itself (88). Sapodilla
used as a rootstock does not form a union at all (38).

Growth regulators

Applications of growth regulators to the graft wound
have been used on mamey sapote with little success (68).
This is a general practice in some Florida nurseries (S. P.
Lara, pers. comm.) but there have been no documented
results. Kester (53) mentions that auxins, alone and in
combination with adenine, stimulated callus. He maintains
that factors such as time of year, scionwood condition, and
correct technique are of greater importance than supplying
external growth regulators.

Apical dominance

Apical dominance has been mentioned as a factor in
grafting of several species in various ways, and it is
usually mediated by auxins, although there may be growth

19

regulators involved other than auxins (93). Apical
dominance can he transmitted through experimental grafts
(25) and this may he part of the downward transport of
auxins (32). Axillary hud break is suppressed hy apical
dominance, and when the dominance is broken, lateral buds
can then expand and grow (93, 124). Axillary buds are
inhibited by auxins and this inhibition can be released by
kinetins ( 1 10).

Placing rose (Rosa sp. ) stems horizontally caused bud
break along the stem of all upward oriented buds and
inhibition of the downward oriented buds. Upper buds that
were uninhibited, were grafted on the basal part of the stem
and then inhibited; basal buds that were budded on the
upper part of the stem were still inhibited (124). Auxins
tend to collect at the basal portion of the stem (125).

Cutter and Chiu (25) mention that in grafting a
Hy grophila species, when buds were of unequal size at a
node, the larger one predominated. When the nodes were
split and grafted together again, the larger one grew out.
When a foil block was placed across the node and the node
was grafted, both buds subsequently grew out. When the
terminal bud is removed from annona ( Annona sp.) shoots, the
lateral buds swell after the dominance of the terminal bud
is broken (89). Bud dominance (dominance by buds over those
lower on a stem) resembles apical dominance and is
probably mediated by hormonal means (25).

20

Seasonal and environmental effect upon graft take

The environment affects graft take (53, 92). The mamey
sapote responds to diurnal changes and levels of relative
humidity (86). Warm days and cool nights, with relatively
low humidity, are the best conditions for grafting mamey
sapote (86). It has been found over a period of 5 years
that the best grafting times in Florida for the mamey sapote
are in the spring (March, April, and May) and in the fall
(late October, November, and December). Times may vary
according to when warm weather begins in the spring and
cooler weather begins in the fall; this is variable in
Florida (86, 88, 91). In Mexico, the mamey sapote can be
grafted best in February and April, when the trees are
defoliated (98).

The

environment also affects mango grafting (49).

In

India,

optimum

grafting

conditions

are usually

high

humidity ,

with

low night

temperatures

and medium

day

temperatures .

Anatomy and 3-raft Union Formation

There are several anatomical features that are
characteristic of the Sapotaceae. These include presence of
a white milky latex contained in laticifers, large amounts
of silica in most genera, and scleriform pitting in the
vessel members with simple perforation plates (24, 52, 77,

99). The laticifers in the Sapotaceae are articulated and
anastomosing {’ll), locations of the laticifers are in the

21

leaves, bark, and pith (24), in conjunction with phloem
fibers, and oriented at random (28, 77). The cells of the
laticifers are joined at pit connections at the center of
the transverse wall and can be identified by the absence of
a central vacuole in the protoplasm (104). In the genus
Manilkara, the copious amounts of latex are tapped for
the production of chicle (23).

Wood anatomy relative to graft take

General wood anatomy of the Sapotaceae has been
described by Kukachka in a series of 33 papers on the wood
anatomy of the neotropical Sapotaceae. In particular the
papers on the 4 economically important genera (Manilkara.
Pouteria, Calocarpum. and Chry sophy Hum) contain details for
comparison of the wood anatomy that may give insight on the
difficulty of grafting within these genera. These details
include: vessel member length, vessel diameter, fiber
length, pit diameter, ray height, presence of trachieds,
parenchyma type, and parenchyma location. The comparisons
and measurements are strictly for the secondary xylem and
are listed in Table 2-1 (55, 56, 57, 58).

The genera that are easier to graft (Manilkara. and
Pouteria) have tracheids, contain little to no silica, and
have considerably more parenchyma in the rays and the
secondary xylem. The genera that are more difficult to
graft contain a great deal of silica, rarely if ever
tracheids, very few if any bands of parenchyma in the
secondary xylem and little parenchyma in the rays (55, 56,
57, 58).

22

Guridi-Gomez (40) describes the anatomy of Calocarpum
as having simple pits in gelatinous fibers and the lumens of
these fibers as being small (these are located in the
cortex, xylem, and pith). Some of these simple pits are

only 4 in diameter. The reticulated parenchyma is

diffused and the rays are only 1-3 cells wide and 13-53
cells high (40, 99). Barajas (6) describes the gelatinous
fibers as being 2040 long, 14 diameter with a wall
thickness of 9

Pouteria and Synsepalum also have gelatinous fibers (3,
40). Vessel diameter in Pouteria is 226 with pit
diameters of 2. 5-5.0 There are distinct bands of
parenchyma in the secondary xylem. The rays are 164-2500
high and 30 cells wide (6). This could be a factor in
graft take since some parenchyma dedifferentiation to callus
comes from the medullary rays (9, 28, 112).

Water conduction to facilitate graft take.

Bin (62) describes an unnamed species of Pouteria using
factors that identify the wood as being of poor quality for
use as a timber species. The inter-vessel pit diameters are
very small with slit-like apertures and the vessel diameters
are also small. There is a high percent of fibers with
thick walls and irregular distribution of relatively small
pits, not especially adapted for liquid conduction because
flow movement between fibers is dependent primarily on
simple pits (62). Table 1 illustrates that most members of
the Sapotaceae have relatively similar sized pit openings

23

(55, 56, 57, 58). It is important in the formation of the
graft union to have water conduction, as the primary need of
the scion after budding is sufficient water supply, provided
first by the callus bridge and later by the differentiation
of the tracheid strands in the callus (71).

Silica content

Silica is one of the common characters of most genera
of the Sapotaceae (55, 56, 57, 58). There are 32 families,
90 genera, and 300 species of tropical trees known to
contain silica deposits (114).

Crystals of silica may be considered a taxonomic
diagnostic tool on the familial, generic, and specific level
(2, 16, 17, 70, 100, 106, 114, 115). Species with large
amounts of silica often yield poor timber because they are
so hard and tend to dull saws and other equipment (2, 6,
115). Timber may be soaked in water for a long period
before an attempt is made to mill the logs. This same
timber, because of its hardness, is almost impervious to
attacks of marine boring worms (2, 115). The hardness of
the wood due to silica also dulls grafting knives and in
turn the jagged cuts will not heal as well as smooth cuts.

Silica crystals are present in vertical or ray
parenchyma of many tropical families (16). In the
Lauraceae, silica crystals may be in the axile or ray
parenchyma and fibers and tyloses (100). Palms (Arecaceae)
contain silica in many species ( 1 18). Silica crystals are
found in the cells of the hypodermis and epidermis of

24

leaves, and as incrustations of lumens and lignified fibers
in certain palms (118).

Several families that contain fruit and nut bearing
trees contain silica and some of these are difficult to
graft. These families include: Lethycidaceae (Brazil nut,
Berthollet ia, and paradise nut, Lecythis) ; Proteaceae,
(Macadamia) ; Juglandaceae (Juglans) ; Sapotaceae (all
genera but Manilkara, D ipholis , and Mastichodendron) (55,
56, 57, 58, 115).

Chattaway (16) notes that the presence of crystals in
enlarged cells of idioblasts causes changes in cell walls
and the walls become sclerosed. Silica, as opposed to
calcium salts, is located not only in the cell lumen where
it fills an entire cell, but as deposits in the cell wall,
and lining the cell walls of the xylem (47). Silica may
also be found in the form of silica sand either alone, or
mixed with starch (106).

Differences in .juvenile and mature stem anatomy

There are differences in the anatomy of the juvenile
and mature stem anatomy in the Sapotaceae. Juvenile stems
of the miracle fruit (Synsepalum dulcif icum) contain 1-5
layers of fibers in the phloem and these fiber layers are
traversed by parenchyma. The pith contains taniferous
materials and many cells filled with starch grains. A few
crystals are present in the cortex and pith (3). Mature
wood in the miracle fruit has tyloses commonly present in
the xylem, and thick walled vessels with sclerotic

25

tendencies. Many gelatinous fibers are present and they are
thick walled with small lumina. Rays are exclusively
uniseriate and many cells of the rays are filled with silica
(?).

Juvenile wood is also different from mature wood in the
Sapindaceae. Juvenile lychee stems have a continuous ring
of sclerenchyma in the pericycle. In mature wood this band
of sclerenchyma is interspersed with sclereids between the
fiber bands. The mature wood is compact and of great
hardness and the plant is difficult to graft (18, 119).

Anomalous secondary growth

One of the grafting problems related to the anatomy of
the lychee is that the vascular cambium has anomalous
secondary growth (119). This cambium is actively growing at
different points along the continuous cambial ring during
different times of the year. It is hard to determine, when
selecting a twig for scionwood, where the actively growing
points are located within the vascular cylinder (119).

Fiber band location

Vascular groups in the kylem of the lychee are
interspersed with a particular type of parenchyma that has a
general appearance of tracheids in cross section. As the
stem diameter increases, the parenchymatous cells between
vascular bundles quickly differentiate into sclereids and
the rays remain only one cell wide. There is a continuous
ring of sclereids within the xylem. Scattered parenchyma

26

cells differentiate to the inside of the sclerenchymatous
ring of per icycle at the same time ( 1 19). Roth (101 )
describes the anatomy of the Mimosaceae as having numerous

fiber rings that are similar to the fiber distribution of
the Sapotaceae.

Fibers do not dedifferentiate readily (28), and cell
dedifferentiation is necessary for both differentiation of
root initials and and callus differentiation for graft
unions. Gomez (37) describes the difficulty of rooting
within the 3 races of avocados. The West Indian race has
thick, frequent fiber bands near the cambium where roots
tend to arise and this sclerenchyma ring acts as a barrier
to rooting. The Mexican race is easier to root and- has
fewer fibers. The Guatemalan race is intermediate in the
numbers of fibers and relative ease of rooting. Mamey is
also difficult to root and has many fibers in the cortex and
in the xylem (l , 6, 40).

Wound healing and graft union formation

Graft union formation and wound healing is related to
the anatomy of the plants. Differentiation is preceded by a
proliferation of parenchyma tissues (28). Collenchyraa will
dedifferentiate, and only very rarely will sclerenchyma
dedifferentiate (28, 31). Callus or wound tissue is
produced by recent cambial derivatives, parenchyma within
phloem and xylem rays (9, 104). Callus may develop from the
medullary rays in some plants. In general, it is possible
to derive callus from any meristematic tissue arising in the

27

pith, wood rays, recent secondary xylem, phloem, and cortex
(9, 11, 111).

The vascular cambium within the graft union is formed
by the mixed callus of the stock and scion. Vascular
elements within the mixed callus may develop before the
vascular cambium (28, 53).

Healing is related to tree vigor, age of the plant,
seasonal variation, and size of the wound (9, 79, 80).
Other factors in graft wound healing besides the environment
and vigor would be the influence of the stock and scion (92,
112). Auxins may play a role in differentiation of the new
xylem (120).

There are differing rates in graft union formation.
The healing stages proceed as follows: formation of an
isolation layer, an initial contact layer, a callus bridge,
vascular tissue, vascular cambium, lignif ication , and a
graft union (20, 92). Duration of these stages in the graft
union development vary according to species. Douglas fir
(Pseudotsuga) usually has initial callus formed at 10-14
days, cambium in 10-17 days, and a union around 35 days with
good conditions (21). Juniper (Juniperus) forms callus in

10-20 days, and the graft union is completed around 60 days
(29).

Citrus (Citrus sp.) appears to be faster in the initial
callus production than gymnosperms. Mendel (71 ) noted
callus initiation after 24 hours, and thick callus layers at
10 days was noted by Nanthachai (78), with the vascular

28

cambium is forming in 7-45 days (78). Xylem in citrus
differentiates before the phloem and the cambium (72, 78).
Callus may initiate from any of the outer layers of wood in
citrus but not from the inner layers (71).

Avocado grafts had callus initiation in 5 days and a
callus bridge by 11 days. This callus is derived from the
inner cortex. By the 9th day, new vascular elements appear
and a new cambium is forming. In 15 days the terminal bud
swells and the scion begins to grow (41 ).

Mango grafts have a pre-callus stage of 4 days with
obvious callus at 8 days and a callus bridge at 12 days.
The cambium develops in 38-43 days and is completely healed
in 6-8 months (112).

Little is known about the graft formation of the mamey
sapote. Healing is faster using a veneer graft (2 months)
than an approach graft (2-3 months) (88). During the summer
months the wounds of the mamey sapote heal slowly. When the
plants are placed in a cooled greenhouse, they heal more
rapidly than in a slathouse where temperature cannot be
controlled (88).

29

Table 2-1 : Anatomical Characteristics in the Sapotacece,

Manilkara. Pouteria, Calocarpum, and
Chry sophy llum {55.56 , 57 , 58)'.

z

Chare .

Manilkara

Pouteria

Calocarpum

Chrysophyllum

VML

640

396

800

700

PL

1.49 mm

1.30 mm

1.85 mm

1.64 mm

VDi

117

84.3

165

197

RHi

244

500

590

395

Pits

6-8

6

6-8

8-10

Si$

0.0$

0- . 25$

J

2.20$

.25-1 .13$

SG

1 .05

0.62

0.76

0.88

Tr

common

present

v. rare

absent

Par

diffuse

diffuse

radial

diffuse

Par-R

present

absent

present

present

AxPar

banded

banded

rare

zones free

Chare. (Anatomical characteristics)

VML (Vessel member length)

PL (Fiber length)

VDi (V essel diameter)

RHi (Ray height)

Pits (Pit diameter in vessel members)

Si$ (P ercent silica in secondary Wood)

SGr (Specific gravity)

Tr (Trachieds)

Par (Parenchyma type in secondary xylem)

Par-r (Contains reticulated parenchyma)

AxPar (Axile parenchyma, banded, rarely banded,
and parenchyma free zones)

Kukachka (55) found 2.02$ but Barajas and Echenique (6)
reported 3-5$. (analysis of the secondary xylem)

CHAPTER III

JUVENILITY AND SEASONAL EFFECTS
Introduction

Summer, Fall, Winter, and Spring are somewhat nebulous
time periods for determining grafting conditions in a
subtropical climate (88, 91, 92). Several time periods for
optimizing grafting conditions for mainey sapote have been
determined using data from local nurseries in southern
Florida. Preferred grafting periods are late October though
early December and late February through early April (both
periods have warm days and cool nights and low relative
humidity and little rainfall). May through early October
(hot days and nights, and high humidity) and late December
through early February (cool days and nights with occasional
frost) are the least desirable times. Experiments were
conducted to determine the optimum season for grafting mamey
sapote and determination of the effect of scion maturation
or juvenility upon graft take.

30

31

Materials and Methods

The general time periods described by Ogden and
Campbell (91 ) and Campbell (88) for the optimum and least
desirable time periods were followed. Precise dates were
chosen when the rootstocks were ready and the graftwood had
sufficiently matured. Dates selected for grafting in 1932
were 13 December, and for 1983, 23 February, 6 April, 12
August, and 15 November.

Scionwood was selected on the basis of tissue
maturation. The scion types were, mature (from flowering
and fruiting trees), "juvenile-like" (from grafted trees
reverted to a vegetative adult phase by pruning) , and
seedling tops (truly juvenile, collected from nursery stock
plants ) .

Modified veneer grafts were used on seedling rootstocks
as described by Ogden et al. (90), except that on the scion
the shallow cut did not extend down to the vascular cambium
and was well within the cortex, and the wound was wrapped
with clear polyethylene tape. A shallow cut was likewise
made on the rootstock that is 3/4 to 1 m high or around 1 to
1 ~ 1 /2 years old. All stocks and scions were chosen randomly
from groups of plants similar in size and age. Replication
numbers were according to the numbers of available
rootstocks that were sufficiently mature and availability of

suitable scionwood.

32

Results _ and Discussion

Grafting results are listed in Table 3-1 by scion
maturity and month grafted. Mature tissue was always lower
in graft take than either seedling or "juvenile-like"
tissue. Mature scions had a graft take ranging from 0.0-47$
with the average for the year of 20.9$. The seedling tops
and "juvenile-like" scions had a high range of 80$ graft
take .

Seedling tops had more consistent graft with a range in
graft take from 52$, 60$, 68$, 66$, and 80 $ and a the
average for the year of 65.2$. "Juvenile-like" scions had
more variation in graft take than the seedling tops or the
mature scions and the range was 36$, 80$, 72$, 26$, and 80$
with an average for the year of 53.8$. Mature scions are
the least desirable scions to use at any time of the year.
Both "juvenile-like" scions and seedling scions are good
graftwood but since the "juvenile-like" scions are clonally
propagated, they would be the graftwood of choice.

Juvenility is a factor in the graft take of maraey
scions since the scions that are morphologically juvenile
consistantly have better graft take than the mature scions.
The best grafting period would be during the spring months
of February and April and the worst times of the year would
be in the summer month of July and the followed by the cool
months when the plants are dormant in November and December.

33

TABLE 3-1 : JUVENILITY AND SEASONAL EFFECTS

Date

z

Stage

y

Gfts

$Tak

Dec .

mature

25

1 1

"juv"

25

36

seedling

25

52

Feb .

mature

15

47

"juv"

15

30

seedling

15

60

April

mature

15

24

"juv"

15

72

seedling

15

68

July

mature

15

0

"juv"

15

26

seedling

15

66

Nov.

mature

15

20

"juv"

15

46

seedling

15

80

"juv" ("juvenile-like")

Gfts (number of grafts per scion maturity)

CHAPTER IV

USE OP JUVENILE INTERSTOCKS TO FACILITATE TOPWORKING

Introduction

Top-working a mature mamey sapote is extremely difficult
once the tree has flowered and fruited. It would be
advantageous to topwork mature trees if they have fruit that
are an undesirable cultivar in quality, yield, or pest and
disease resistant. If grafted trees are frozen below the
graft union, topworking would put bearing groves back into
production .

Experimental objectives were to use seedling tops
( truely juvenile material) as interstocks and to facilitate
the use of mature scions and "juvenile-like" scions as
graf twood .

Materials and Methods

Three large trees of inferior cultivars were cut back
to a stump, a height of approximately 1 m in June, 1982.
The trunk was painted with white latex paint to prevent
sun-scald. Sprouts from the stump were permitted to grow
until they obtained a size suitable for grafting and danger
of frost had passed.

34

35

On 25 March, 1985, 45 sprouts were grafted with
seedling tops as scions, using a modified veneeer graft and
wrapped with polyethylene tape (as described in Chapter
III). The seedling tops were collected from plants grown
from seed in the nursery for use as rootstocks.

When the seedling scions had sprouted and obtained a
stem diameter of 2 to 5 cm, they were grafted with the
second graft on 8 August, 1985, using mature graftwood and
"juvenile-like" graftwood. Both types of graftwood were
from the cultivars 'No. 2' and 'Magana' in replications of
10 each, for a total of 40 grafts.

Results and Discussion

Seedling tops that were used as interstocks were
evaluated on 8 March and had a combined graft take of 92$
(Table 4-1). The mature and " juvenile-like" scions were
evaluated on 15 September , 1 985 . The ones still alive had
sprouted and were growing vigorously. The "juvenile-like"
scions had slightly different results: 100$ for 'No. 2'
and 80$ for 'Magana' , for an overall average of 90$. Both
'No. 2' and 'Magana' mature scions had 80$ graft take.
Juvenile (or seedling tops) scions are the method of choice
to be used as interstocks for topworking mature mamey trees
in the field. Both mature and "juvenile-like" scions can be
used even during August, the worst time of the year to
graft .

36

Table 4-1 : Juvenile Interstocks for Topworking Mamey Sapote

Date .

z

Stage

Cult.

y

Gf ts

% Take

March

Seedling

45

92

Aug.

" juv"

'No. 2'

10

100

" juv"

'Magana'

10

80

Mature

'No. 2'

10

80

Mature

'Magana'

10

80

"juv" ("juvenile-like")

G-fts (number of grafts per scion maturity)

CHAPTER V

CARBOHYDRATE ACCUMULATION RELATIVE TO GRAFT TAKE

Introduction

It is a general practice with several plant species to
girdle the scionwood some weeks prior to grafting (22, 51).
This is done to increase stored carbohydrate accumulations,
thereby ensuring ample food reserves for the scion while the
graft union is forming (22, 51, 84).

Mamey sapote is girdled by some propagators (117, 123),
but the wood is so brittle that the prepared shoots may snap
off i-n gusts of wind. The girdled areas may be braced with
wooden splints and wrapped with tape to prevent breakage (C.
W. Campbell, pers. comm.). There have been reports that
girdling does increase graft take (117) but it may be only
as much as 5$ (68).

Experimental objectives were to determine if graft take
is influenced by a natural rise and fall of carbohydrates,
and to determine if levels of total soluble sugars or stored
carbohydrates (starch) have effects upon graft take.

37

38

Materials and Methods

Samples of graftwood were randomly collected for the
carbohydrate analysis at the same time as those in Chapter
III for the juvenility experiments. Mature,
"juvenile-like", and seedling scions were analyzed.
Cultivars used were 'No. 2' and 'Magana'.

All samples were separated according to tissue
maturation and analyzed separately. Samples were chopped
and mixed. Three replications of 2-gm each were weighed out
from each sample. The samples were analyzed using a
phenol-sulfuric acid assay to deter total soluble
sugars (44).

Samples were ground in a tissue grinder with 10 ml of
1 00$ ethanol, centrifuged, and the supernatant filtered off.
The pellet was stored for further analysis of starch and the
liquid was analyzed for total soluble, sugars. The
supernatant was diluted 100 fold with distilled water. An
aliquot of 0.5 ml of the sample was put in a test tube with
0.5 ml of 5$ phenol and 2.5 ml of concentrated sulfuric
acid. An intense reaction took place with the tubes
becoming very hot and the sample turning a brown to red
color. After the test tubes cooled for 50 minutes, the
results were determined colorometrically on a

spectrophotometer at 490 nm using glucose standards for
reference .

39

For starch analysis, the stored pellets were incubated
in an enzyme solution for 12 hours at 34° C. The enzyme
solution consisted of: 40 ml of a 0.04 M phosphate buffer
solution pH 6.8 (8), 20 mg alpha amylase, 20 mg
amyloglucosidase , and 17.6 mg calcium chloride (44).

The starch is converted to maltose by the alpha
amylase, and the maltose is in turn converted to glucose by
the amyloglucosidase. After the incubation period, the
pellet/enzyme solution is centrifuged until a new pellet is
formed. The clear supernatant is poured off and then
diluted with distilled water 200 fold. The diluted
supernatant was then analyzed as previously described for
total soluble sugars.

Results and Discussion

Because of the similarity in concentrations of soluble
sugars and starch for the two cultivars, the results were
averaged together according to scion maturity and reported
as mg/ml glucose. On a monthly basis the results of the
total soluble sugars measurements were erratic when compared
to graft take percentages (Table 5-1).

On a seasonal basis, correlation coefficients for
soluble sugars were positive for the months of December and
November, and significant at the level.

40

For the months of February, April, and July, the results
were negatively correlated and not significant (Table 5-2).

Results were compared by scion maturation and seasons
for sugar in relation to percentage graft take. Correlation
coefficients for the overall comparisons of total soluble
sugars relative to scion maturity and grafting season were
low and not significant at the 5$ level (Table 5-5). These
comparisons show that total soluble sugars are not a strong
mediating factor in relation to graft take.

Starch accumulation was compared to graft take (Table
5-4). As with sugars, the starch content within cultivars
was similar and the results were averaged together and
reported as glucose in mg/ml.

A statistical analysis shows that on a seasonal basis,
starch content had a positive correlation coefficient. In
the months of February and April the correlation
coefficients were significant at the level, and December,
April, and November were significant at the 5^ level.
(Table 5-5).

Variablity of the graft takes was not explained upon
scion maturity alone relative to starch accumulation. In an
overall comparison of starch content with seasons and scion
maturation, the variablity of graft take and maturity of the
scionwood had a positive significant correlation coefficient
at the level (Table 5-6). This comparison indicates an
interaction between seasons and scionwood maturity in starch

accumulation .

41

As the starch content rises within a given season, the
scions with the highest starch content had the highest graft
take. Starch content was higher in the seasons prior to the
beginning of an active growth period (February and April)
for the seedling tops and the "juvenile-like" scions.
During the periods of active growth (July) and dormancy
(November), starch content was lower for mature and
"juvenile-like" scions and the graft take was lower.

Starch content, especially in mature scions within each
month, appears to have an active role in the percent graft
take. Total soluble sugar measurements do not appear to be
a reliable indication of the amounts measured relative to
percentage graft take (Tables 5-1 and 5-4).

42

Table 5-1 : Carbohydrate Accumulation — Total Soluble Sugars

Relative to Graft Take

Date

z

Stage

y

Gfts

% Take

mg/ ml

Dec .

mature

25

1 1

13.3

" j uv "

25

36

14.6

seedling

25

52

16.9

Feb .

mature

15

47

19.7

"juv"

15

80

18.1

seedling

15

60

14.1

April

mature

15

24

21 .0

"juv"

15

72

19.6

seedling

15

68

12.6

July

mature

15

0

19.9

"juv"

15

26

13.0

seedling

15

66

27.0

Nov.

mature

15

20

21 .3

"juv"

15

46

22.7

seedling

15

80

31 .0

z

"juv" ("juvenile-like/')

y

Gfts (number of grafts per scion maturity)

Table 5-2: Statistical Seasonal and Sugar Comparisons

Date

R value

z

DF

Sig

Dec .

.910

2

**

Feb .

-.164

2

NS

April

-.040

2

NS

July

-.611

2

NS

Nov.

.951

2

**

z

Significance (** 1g; NS, Non significant)

43

Table 5-3: Stastical Comparisons of Combined Scion

Maturity and Sugar Accumulation

z

R- value

DP

Sig.

0.297

14

NS

Significance (NS, non significant)

Table 5-4: Carbohydrate Accumulation — Stored Starch

relative to graft take.

Date

z

Stage

y

Gfts

Take

mg/ml

Dec .

mature

25

1 1

30.6

"juv"

25

36

33.5

seedling

25

52

51 .0

Feb .

mature

15

47

29.5

"juv"

15

80

51 .0

seedling

15

60

45.0

April

mature

15

24

24.3

"juv"

15

72

37.5

seedling

15

68

34.3

July

mature

15

00

24.3

"juv"

15

26

38.6

seedling

15

66

47.9

Nov.

mature

15

20

20.1

"juv"

15

46

38.6

seedling

15

80

43.1

"juv" ("juvenile-like")

Gfts (number of grafts per scion maturity)

44

Table 5 5: Stastical Seasonal Starch Comparisons

Date

R-value

DF

z

Sig.

Dec .

0.869

2

*

Feb .

0.932

2

**

April

0.987

2

**

July

0.883

2

*

Nov.

0.915

2

*

z

Significance

Table 5-6: Stastical Comparisons of Combined

Scion

Maturity

and Starch

Accumulation

R-value

DF

z

Sig.

0.738

14

**

z

Significance (**, \%)

CHAPTER VI

SUGAR PULSING
Introduction

Sugar pulsing is a method of increasing the shelf life
of cut flowers (26,42). Placing cut flowers in a
sugar-water solution, with a low pH (3. 0-4.0), extends
shipping and vase life for roses and carnations (42). The
effect is attributed mainly to reduction of microbial
population with lowered pH of the water, and retardation of
stem blockage which prevents a decline in flow rate of water
through rose stems and increases uptake of energy in the
form of sugar (76).

It was shown in Chapter V that mature scionwood had a
lower stored carbohydrate content (and poorer graft take)
than "juvenile-like" scions and seedling tops. The
objectives of this experiment were to determine if sugar
pulsing would increase available carbohydrates to mature
scionwood while graft unions are forming.

Materials and Methods

Two grafting periods were chosen. The first was June,
1982, when the summer heat is intense and the rainfall and

45

46

the relative humidity are high. The second period was early
February, 1983, when there are cool day and cool nights.

The pulsing solution consisted of 5$ sucrose in
distilled water with the pH adjusted to 4.0 with
hydrochloric acid.

There were 2 treatments: distilled water with a pH of
4.0 as a control, and the pulsing solution. Two cultivars
were used ('No. 2' and 'Magana') and 2 scion maturities
(mature and "juvenile-like"). The graftwood was collected
in the field and recut when placed in the 2 solutions. The
scions were left in the solutions for 24 hours at 26 0 C.

There was a total of 80 grafts: 10 of each scion
maturity for each of the solutions. The rootstocks were
grafted with a modified veneer graft and wrapped with
polyethylene tape as described in Chapter III.

In June, 1982, the weather conditions for the day of
grafting were an overcast sky and very hot. In February,
1983, the weather was cool and the sky clear. Data were
collected in July and in April, respectively.

Results and Discussion

In June, there was 0$ graft take for all treatments.
After the graftwood was soaked and before grafting, the
petiole stumps were abscissing on many of the individual
buds sticks of the "juvenile-like" cultivars. None of the
budsticks from the water treatment had abscissing petiole
stumps and neither did the mature budwood from the sucrose
treatment .

47

The 'Magana' budwood, both mature and "juvenile-like",
had a gelatinous precipitate on the wood where it had been
immersed in the water and sugar solution. There was some
precipitate on the 'No. 2' cultivars in both solutions but
not as much as on the 'Magana' cultivars.

The level of the solutions in the beakers remained the
same before and after soaking, and the scions did not appear
to take up any noticeable amount of the solutions. Both the
sucrose solution and the water treatments had a milky cast
after the budwood soaks. Presumably this was caused by the
latex exudate.

The February treatments had similar results with only
the 'Magana' "juvenile-like" scions out of all cultivars and
scion maturities having 4 takes out of a total of 80 grafts.
A statistical analysis using chi-square contingency tables
shows that sugar pulsing is not significant at any level and
does not have any appreciable effects upon graft take. Out
of the two experiments, only 4 grafts lived out of 160
plants .

It is possible that the pulsing is also deleterious,
since it has been shown that mamey does not graft well when
there is high humidity (88, 91). The budsticks were wet
when they were grafted, and the water could have caused the
latex to congeal and inhibit the graft union by sealing off
the vascular system. The plant usually breaks down the
latex when grafting conditions are good and in general there

48

is no latex effect (68) These artificial conditions could
have stimulated the latex to inhibit graft take.

CHAPTER VII

GROWTH REGULATORS APPLIED TO THE GRAFT WOUND

Introduction

Some nurseries in south Florida routinely apply growth
regulators (both auxins and cytokinins) to the graft wound
of mamey sapote and other plants (S. P. Lara, pers.
comm.). Cytex, a seaweed extract, is purported to enhance
the graft take of some tropical fruit trees. The objective
of these experiments is to determine if growth regulators
applied externally to the graft would enhance graft take.

The first test was designed to determine what effect
growth regulators have under ideal environmental conditions,
using the best rootstocks and scions. Test 2 was to
determine if growth regulators would increase graft take
under good environmental conditions, using oversized
rootstocks and mature graftwood. The third part of the test
was to determine the effects of growth regulators under
oversized environmental conditions, using poor rootstocks
and mature scions.

49

50

Materials and Methods

Growth regulators tested were a cytokinin (Benzyl
adenine — BA), auxins (Napthaleneacetic acid — NAA), and
Cytex, a commercial product purported to have cytokinin-like
activity. All were in a lanolin base. The controls were
grafts with no lanolin or growth regulators, and grafts with
lanolin alone. There were 20 replications with 2 cultivars
in the first 2 tests and 15 replications with 2 cultivars in
the next test. Replications were determined by availability
of rootstocks and graftwood. The cultivars were 'No. 2'
and 'Magana' .

The first experiment was on 22 February, 1983, and
there were 2 controls, 1.0# Cytex in lanolin, 0.1# NAA in
lanolin, and 0.1# BA in lanolin. The test was conducted in
a slathouse with 50# shade and the best rootstocks and
scions were chosen. All tests used a modified veneer graft
and polyethylene grafting tape as described in Chapter III.

Experiment 2 was on 6 April, 1983 (an ideal time to
graft mamey), and conducted at a local nursery. Rootstocks
were larger than usually used fo.r grafting and the scions
were mature graftwood. A combination of 0.01# NAA and 0.01#
NAA + 0.1# BA was added to the previous treatments. These
treatments were added to see if a lower concentration of NAA
would be more effective and to see if there was an
interaction between NAA and BA.

51

The third test was July, 1983, which was considered to
he the worst season to graft mamey. The same
controls, concentrations, cultivars, were used. There was a
total of 30 grafts per treatment, using 15 of each cultivar.

Results and Discussion

In the February test, none of the growth regulators
increased graft take over control I (Table 7-1), and only
Cytex had increased graft take over control II but was non
significant within treatment variability. Both NAA and BA
had lower graft takes than the controls. A chi-square
analysis with contingency tables showed that there was a
statistical significance of variables with the treatments at
the 5 # level.

In April, only control I (2.75#), control II (7.50#)
and the NAA/BA combination (2.75#) had any graft take. A
chi-square analysis showed no significant level of treatment

effect. In July, there was 0.0# graft take for the whole
experiment .

The data from these experiments do not indicate that
the growth regulators at the concentrations used applied to
the wound, will overcome adverse weather conditions or
stimulate a graft union when using oversized rootstocks and
mature scions. The data suggest that the growth regulators
used (NAA and BA) could have a negative effect upon graft

take .

52

Table 7-1 : Applications of Growth regulators to Graft Wounds

Date

z

Gr. Reg.

y

Reps .

$ Take

X

Sig.

Feb .

control I

40

80.0

NS

control II

40

40.0

1 .0$ Cytex

40

60.0

0.1# NAA

40

20.0

0.1$ BA

40

33.0

April

control I

40

2.75

NS

control II

40

7.50

1 .0$ Cytex

40

0.00

0.1$ NAA

40

0.00

0.01$ NAA

40

0.00

0.1$ BA

40

0.00

0.01$ NAA/0 . 1 $ BA

40

2.75

July

control I

30

0.00

NS

control II

30

0.00

1 .0$ Cytex

30

0.00

0.1$ NAA

30

0.00

0.01$ NAA

30

0.00

0.1$ BA

30

0.00

0.01$ NAA/0. 1 $ BA

30

0.00

control I (without growth regulators or lanolin)
control II (lanolin only)

y

Gfts (number of grafts per scion maturity)
Significance (NS, not significant)

CHAPTER VIII

ROOTSTOCK APICAL DOMINANCE RELATIVE TO GRAFT TAKE

Introduction

Apical dominance is strongly exhibited in the mamey
sapote under field and nursery conditions. Seedling plants,
used as nursery rootstocks, may not develop any side
branches or lateral growth until the plant is up to 2 m
high. Young mamey plants strongly exhibit apical dominance.
They have a very large terminal bud and the axillary buds
are so small that it is difficult to discern them in the
leaf axils. On branches of mature trees, axillary buds are
also small. The axillary buds on the mature trees are not
suitable for use as T-buds or chip buds because, upon visual
examination, without magnification, it is difficult to tell
when they are fully developed and mature enough to be used
for grafting. If the terminal bud is removed, the lateral
buds swell and begin to grow. At this time the buds are
suitable for grafting. This method of in situ preparation
is used to facilitate off season annona grafting (89).

Quilantan-C arreon (98) described the removal of the
terminal buds on rootstocks prior to grafting as a mechanism
to remove excess latex and enhance grafting success. Some

53

54

authors consider the latex not to be a factor in graft
inhibition (68, 88). The removal of the terminal buds on
the rootstocks may break apical dominance. The experimental
objectives were to determine if apical dominance in the
rootstocks is a factor in graft union failure.

Materials and Methods

The experiments were conducted in July, 1983 (one of
the hottest months), November, 1983, (cool days and warm
nights), and January, 1984 (one of the coolest months).
There were 2 groups of plants used in the experiments: the
first had the terminal buds removed 24 hours prior to
grafting, and the controls had the terminal buds intact.
Each group had 3 scion maturities (seedling top,
"juvenile-like" and mature) and 15 replications per scion
maturity. They were grafted using a modified veneer graft
and wrapped with polyethylene tape as described in Chapter
III.

Results and Discussion

In July, mature budwood on the topped rootstocks had a

graft take

of

26 . 0$

compared

to

0.0$

on

the

control

rootstocks

(Table 8-1 )

. "Juvenile-

■like"

on

the

topped

rootstocks

had

33.0$

compared

to

26.0$

on

the controls.

Seedling tops

used as

scions

had

0.0$

on

the

topped

rootstocks and 66.0$ on the controls (Table 8-1).

55

The experiments in November show graft take of the
mature on the topped rootstocks at 46.0$ and the controls at
26.0$. The "juvenile-like" have a graft take of 40.0$ on
the topped and 46.0$ on the controls. The seedling tops
used as scions were 80.0$ on the topped and the control
rootstocks.

Grafting results in January were the worst during the
year. Both mature and "juvenile-like" scions had 0$ graft
take with both the topped and the control treatments. The
seedling tops had 86.0$ on the controls and 46.0$ on the
topped rootstocks.

During

periods

of active growth, removal

of

the

terminal bud

on rootstocks with mature scionwood

does

increase the

graft

take percentage, and this

is

of

particular

interest

because mamey generally

cannot

be

grafted in the summer. In January, the weather is cool
enough so that the stocks are dormant and there is little to
no growth. The removal of the terminal bud does not have an
effect on graft take because the terminals are not actively
inhibiting lateral growth on a dormant plant.

56

Table 8-1 : Apical Dominance as a Factor in Graft Take

z

y

X

Date

Treatment

Stage

Gfts

% Take

July

control

mature

15

00

"juv"

15

26

seedling

15

66

topped

mature

15

26

"juv"

15

55

seedling

15

00

Nov.

control

mature

15

20

"juv"

15

46

seedling

15

80

topped

mature

15

46

"juv"

15

40

seedling

15

80

Jan.

control

mature

15

00

"juv"

15

00

seedling

15

86

topped

mature

15

00

"juv"

15

00

seedling

15

40

z

control (terminal bud intact)
topped (terminal bud removed)

"juv" ("juvenile-like")
x

Gfts (number of grafts per scion maturity)

CHAPTER IX

COMPONENTS AND LOCATION OP THE VASCULAR CAMBIUM

Introduction

Positioning of the vascular cambium relative to the
rootstock and the scion for a graft union is very important
for most species. The close developmental relationship
between the cambium in the callus and in the components of
the graft explains why proper matching of the cambium of the
stock and scion speeds up the formation of cambial
connection (28, 53). Improper matching of the cambium does
not necessarily prevent a union, but it usually delays it
(28, 53, 109).

There are some species that have an irregular cambium.
This may be due to anomalous secondary growth. There are
several types of anomalous secondary growth without extra
bundles of vascular tissue being split off from the main
cambium. One type is that described by Chalk and Chattaway
(15), referred to as the lobed stem, where the secondary
xylem is laid down unequally at different angles at
different points around the stem. The cambium cylinder
remains continuous. Venning (119) describes another type of
anomalous secondary growth in the lychee where the cambium

57

58

grows unequally during the season. Parts of the cambium are
actively dividing and growing while other sections of the
cambium are inactive at any given time. The result is a
continuous cambium with only some sections active and
capable of forming a graft union.

The purpose of these experiments was to determine if
the cambium of mamey sapote is irregular, and if any tissue
stage of scionwood or rootstocks tends to be more regular
than another. Stem morphology was also examined to see if
juvenility affected internode lengths or numbers of leaf
traces per section.

Materials and Methods

Sections for anatomical study were collected in
February, April, and July. Seedling stems and terminal
branches of the cultivars 'No. 2', and 'Magana' (both
"juvenile-like" and mature tissue types) were cut in 30 cm
lengths, and were preserved in 70$ FAA
(formalin-acetic-alcohol) (8) and dehydrated sequentially in
70$, 75$, 80$, 85$, 90$, 95$, 100$ ETOH (ethyl alcohol)
solutions under vacuum (50, 85).

An attempt was then made to cut the 30 cm specimens
into 2-3 cm sections with a single-edged razor blade. When
this failed, because the wood was so hard, a sabre saw was
used. The sections were soaked in 4$ ETD ( ethylenediamine )
to soften the wood, and 24$ HF (hydrofluoric acid) to remove
silica and crystalline materials (39, 54). This procedure

59

is used for woody materials with a specific gravity in
excess of 0.75. The chemical ETD is a swelling agent which
increases cell wall volume beyond that obtainable by simply
saturating the samples in water. The swelling of cell walls
induced by ETD is eliminated in later steps. There is no
effect by ETD on silica crystals, and other cell inclusions
(54).

The specimens were again dehydrated in ETOH and then
embedded in GMA (glycol methylacrylate) and molded in 6mm x
12 mm x 5 mm blocks for sectioning on a rotary microtome (7,
14, 53, 85, 103). Because GMA is so hard, a standard metal
blade cannot be used, and a 'Ralph' glass knife adapted to
the microtome was used instead (104, 107, 108). Sections
were cut to a thickness of 12 micrometers.

The plastic sections were floated on a clean slide on a
drop of 10$ acetone. Slide-s were then heated on a slide
dryer and stained with 0.05$ toluidine blue and differential
stain (zinc chloride, safarin, orange G, tannic acid orange
G, tannic acid, ferric ammonium sulfate) (105) for 30
seconds, rinsed, and dried again on a slide dryer (33).
When dry, a drop of permount was applied to the section.
The section was covered with a coverslip and dried for 24
hours .

Results and Discussion

The morphological examinations showed that there were
2-8 leaves per cm at the terminals. There were fewer leaves

60

per cm on the mature scions and the rootstock stems than on
the seedling subterminals and the "juvenile-like" scions.
The leaves of the mamey are alternate but are so closely
crowded toward the ends of the branches that they almost
appear whorled. There are 3 leaf traces per node (Fig. 9-1
C).

Within any one section, several leaf traces are visible
along the cambium (Fig. 9-1 A,B,C), and when the leaf trace
breaks away, the cambium cylinder is left with an undulating
and looped appearance. An enlarged leaf trace showing the
vascular connections between the trace and the cambium is
shown in Fig. 9-1 D.

"Juvenile-like" stems (Fig. 9-2 A,B) have more
irregular cambium than the mature stems, and the mature
stems (Fig. 9-3 A,B) are more irregular than the seedling
stems used as rootstocks (Fig. 9-4) (Fig. 9-2, 3, 4). The
cambium irregularities ate caused by numerous leaf traces.
Whether irregularities in the cambium are caused by
anomalous secondary growth or by numerous leaf traces, the
effect is the same. It is difficult to find the cambium on
scionwood or rootstocks when preparing the wood for grafting
if the cambium varies at different levels within the
graftwood. Since the cambium is not visible from the
outside of the branch, it is difficult to make a precise cut
that will smoothly expose the entire cambium along the
surface of the wound. Therefore it is difficult to make
complete cambial contact with the rootstock and scion and
ensure better graft take if the cambium cylinder is erratic.

Figure 9-1 : Irregularity of the vascular cambium.

A. Node with leaf trace (L Tr) and vascular
cambium (YC) located between cortex (Cx)
pith. 26x. Differential stain.

B. Fiber bands (FB) under epidermis and
outside vascular cambium, with leaf trace
(L Tr) and vascular connection (V Cn) to
node. 26x. Differential stain.

C. Node with 3 leaf traces (L Tr) and 2 leaf

gaps (LG). 26x. Toluidine blue.

D. Enlarged leaf trace (L Tr) in cortex

(Cx), with fiber bands (FB) outside
vascular cambium (VC) with vas-cular
connection (VCn) to xylem (Xy). 70x.

Toluidine blue.

62

Figure 9-2: Vascular cambium in 'juvenile-like' stems.

A. 'Magana' "juvenile-like" stem with thick
fiber bands, node with 2 leaf gaps (G)
and 3 leaf traces. Differential stain.

26x.

B. 'No. 2' "juvenile-like" stem at node with
vascular cambium (VC) and fiber bands (FB)
outside the cambium. Toluidine blue. 26x.

64

Figure 9-3: Vascular cambium in mature stems.

A. 'Magana' mature with the vascular cambium
(VC) less irregular, some leaf traces (LTr )
and leaf gaps (LG) and vascular connection
to node (VCn) from the xylem (Xy) and less
dense fiber bands (FB). Differential stain.
26X.

B. 'No. 2' mature with less undulation in the
vascular cambium due to leaf traces (LTr)
and fiber bands (FB) are less dense.
Differential stain. 26 x.

66

Figure 9-4: Vascular cambium in rootstock stem and seedling

stem sections.

A. Rootstock stem vascular cambium more regular
than mature or "juvenile-like", fiber bands
(FB) are broken interspersed with parenchyma
cells. Toluidine blue. 26 x.

B. Seedling stems with more irregular vascular
cambium and denser fiber bands (FB) than
rootstock stem. Differential stain. 26x.

68

o

CHAPTER X

GRAFT UNION FORMATION
Introduction

The experimental objectives were to determine the
sequence and duration of the graft union formation during
Fall, Winter, and Summer; and to determine if wound healing
and tissue differentiation differ, or follow the same
healing progression throughout the year.

Materials and Methods

Rootstocks were 1/2 to 3/4 m high and were grafted with
"juvenile-like" scions using a modified veneer graft and
wrapped with polyethylene tape as described in Chapter III.
On 21 October, 1982, 60 stocks were grafted, and beginning
on day 3 (day 1 being the day of grafting), 3 grafts were
collected for samples every 3-4 days. On 17 December, 1982,
30 stocks were grafted, and on 4 August, 60 stocks were
grafted. Samples were collected as in October.

Samples consisted of 30 cm sections cut from the
mid-section of the grafts. Samples were wrapped tightly
with Parafilm prior to collection to prevent graft rupture.
Samples were preserved in FAA , and dehydrated in ETOH (as
described in Chapter IX).

69

70

An attempt was made to cut 1 cm sections from the graft
union with a sabre saw, but the graft unions kept splitting
apart. The grafts unions were wrapped with another layer of
Parafilm and cut on a large band saw. The sections were
then treated with ETD, HP, sectioned on a rotary microtome
to a thickness of 12 micrometers and stained in toluidine
blue as described in Chapter IX.

Results and Discussion

The graft series with the most consistent results was
the October series compred to the December and the July
series. In October, wound response appeared on day 3 (Pig.
10-1 A), dedifferentiation on days 5 and 7 (Pigs. 10-1
B,C,D,E,F) a callus bridge on day 12 (Pigs. 10-2 A,B,C),
vessels on day 14 (Pigs. 10-2 D,E,P), vessel lignif ication
on day 17 (Pigs. 10-3 A/,B,C), cells lining up laterally on
day 21 (Pigs. 10-3 D,E,P) increased vessel lignif ication
and on days 24-26 (Pigs. 10-4 A,B,C,D), fibers on day 28
(Pigs. 10-4 E,F), cambium development on days 31-35 (Pigs.
10-4 A,B,C ,D,E,P) , increasing connection on day 38 (Pigs.
10-6 A,B), a complete connection on day 41 (Pigs. 10-6 C,D)
and an apparent union on days 44-49 (Pigs. 10-6 E,P).

In December, initial callusing was slower than in
October, with many of the sections breaking apart from lack
of connections. Only a few could be sectioned. There was
some wound response on day 3 (Pigs. 10-7 A,B), with cell
dedifferentiation on day 5 (Fig. 10-7 B). In August, many

71

of the grafts failed, hut those that took were slower in
development than those of the October series. Wound
response was on day 3-5 (Figs. 10-7 C,D), an isolation
layer on day 5 (Fig. 10-7 D), and some cell
dedifferentiation on day 9 (Figs. 1 0—7 E,F). Callus was
noted on day 12-15 (Figs. 10-8 A,B,C). Fibers in the graft
wound were merged from the edge (Fig. 10-8 D). Vessels
were noted on day 25 (Figs. 10-8 E,F), and no vascular
cambium was detected in this series. Kester (53) indicates
1-2 weeks for vessel differentiation from callus for many
species. Hartmann and Kester (43) indicate that parenchyma
can differentiate into tracheids with ease.

A graft union is considered complete when there is
enough contact from the rootstocks to transfer water to the
scion (41, 71 ), and the buds start to expand and grow (71,
72, 75). In the marney sapote the terminal buds start to
grow in up to 2 months. The primary need of the scion after
budding is a sufficient water supply (53, 71). Water supply
is first met by the callus bridge, then afterwards the
differentiation of the tracheid strands in the callus
assures the water supply of the scion. This water movement
is first based on lateral water movement from the callus,
then longitudinally from the vessels through pit
connections, then laterally from the rays (28, 71).

Lin (62) mentions that pit connections are small (5 )
in the vessel members and the fibers. There is a high
percentage of fibers in the wood, and the wood is not

72

especially adapted for liquid conduction because flow
movement between fibers is dependent primarily on simple
pits (62). Slow water movement could be a factor in the
slow healing rate of the mamey and even lack of unions.

For all time periods tested for the grafting responses
of the mamey sapote, there was a general progression of
tissue differentiation as reported; however, there were
replicates that were erratic in callusing response. The
data reported in these experiments were initial observations
of the stages, not precise delineations for each sample.
From the observations in these experiments, October is a
better month for grafting mamey than December or August,
because callusing and lignif ication were shown to be faster
in October. In mid-December the graft responses are slowed
down because the plant is no longer in active growth. In
August, the plant is in active growth, but the environmental
effects of high temperature and high humidity slow down the
graft take and subsequent healing as noted in Chapter III.

Figure 10-1: Graft union formation in October, days 3-7.

A. Day 3, wound response (WR) in graft

wound (G) between scion (Sc) and stock
(Sk) . 70x.

B. Day 5, cell dedifferentiation to form

callus (Cl) between scion (Sc) and stock
(St). 70x.

C. Day 5, same site. 200x.

D. Day 7, callus (Cl) between scion (Sc) and

stock (St). 70x.

E. Day 7, same site. 200x.

F. Day 7, same site. 500x.

A-F:

Toluidine blue stain.

74

Figure 10-2: Graft union formation in October, days 12-14.

A. Day 12, callus bridge (Cl) at graft wound
(GJ between scion (Sc) and stocks (St).
70x.

B. Same site. (Note rounded isodiametric
cells). lOOx.

C. Day 12, longitudinal secion, graft wound
(G) between xylera (Xy) of scion and stock.
70x.

D. Day 14, vessels (V ) in graft wound (G)

between scion (Sc) and stock (St). 70x.

E. Day 14, vessels (V ) with some tissue
beginning lateral lining up. 70x.

F. Same site 200x.

A-F:

Toluidine blue stain.

76

Figure 10-3: Graft union formation in October, days 17-21.

A. Day 17, healing near graft wound edge,

lignif ication of vessels between scion
(Sc) and stock (St). 70x.

B. Day 17, same site. 200x.

C. Day 17, longitudinal section. Dignified
vessels (LVj in graft wound (G) between
xylem (Xy) of scion and stock. 70x.

D. Day 19, cells forming lateral files (LF)

on the stock (St). 70x.

E. Day 21 , longitudinal section, beginning
of ray parenchyma (R-P) in graft wound
(G) between xylem (Xy) of scion and
stock.

F. Day 21, same site with rays (R). 500x.

A-F: Toluidine blue stain.

/

78

Figure 10-4: Graft union formation in October, days 24-28.

A. Day 24, large vessels (V ) in scion (Sc)
near graft wound, lateral files (LF) of
cells well developed in stock (St) .

70x.

B. Day 24, same site with silica crystal (SC)
in callus. 200x.

C. Day 26, more vessels and greater ligntfi-

cation in graft wound (G) between scion
(Sc) and stock (St). 70x.

D. Day 26, same site with lignified vessels.
200x.

E. Day 28, longitudinal section with poor
union but some fibers forming. 70x.

F. Day 28, same site with vessels (V ) and

fibers (F) . 500x.

A-F: Toluidine blue stain.

80

Figure 10-5: Graft union formation in October, days 51-55.

A. Day 51 , First indication of possible
vascular cambium (?VC) in graft wound
(G) between scion (Sc) and stock (St).
70x.

B. Day 51, same site, possible vascular

cambium (?VC) with silica crystal (Sc)
in graft wound (G). 200x

C. Day 55, poor union with numerous vessels
(V) , no cambium. 70x.

D. Day 55, same site with lignified vessels

(V). 200x .

E. Day 55, good healing with many rows of
lateral cells outside of vascular cambium
(VC) between scion (Sc) and stock (St).
70x.

F. Day 55, same site with fusiform initials

(FI). 2000x

A-F: Toluidine blue stain

82

Figure 10-6: Graft union formation in October, days 38-49.

A. Day 38, vascular cambium (VC). 70x.

B. Day 38, same site, fusiform initials
2000x.

C. Day 41, increased lateral cell formation

near cambium with good connection (Cn)
between scion (Sc) and stock (St). 70x.

D. Day 41, same site, connection (Cn) 500x.

E. Day 44, apparent water connection across

vascular cambium (VC) and large vessels
(V). 200x.

F. Day 49, completed graft union. 70x.

A-F: Toluidine blue stain

84

Figure 10-7. Graft union formations in December and August.

A. Day 3, December, wound response (WR) in

graft wound (G) between scion (Sc) and
stock (St ) . 70x.

B. Day 5, initial dedifferentiation and

wound response (WR). 500x.

C. Day 3, August, wound response (WR). 70x.

D. Day 5, isolation layer (IL). 70x.

E. Day 9, some dedifferentiation and wound
response. 70x.

F. Day 9, same site showing wound response

(WR). 200x.

A-F: Toluidine blue stain.

86

figure 10-8: Graft union formation in August, days 12-25.

A. Day 12, callus (Cl) in graft wound (G)
between sscion (Sc) and stock (St).

200x.

B. Day 12, longitudinal section with
isolation layer (IL) in graft wound (G),
vessls (V ) with sclereiform pitting in the
xylem of the stock and scion. 500x.

C. Day 15, more callus in the graft wound (G)

between scion (Sc) and stock (3t). 200x.

D. Day 18, Fibers (F) from wound edge merged

into graft wound (G). 70x.

E. Day 25, callus bridge (Cl) in graft wound
(GJ between scion (Sc) and stock (St).

70x.

F. Day 25, some non-lignif ied vessels (V ) in

callus (Cl) of graft wound (G). 200x.

A-F: Toluidine blue stain.

88

CHAPTER XI

FIBER BAND LOCATION WITH RESPECT TO GENERAL ANATOMY AND
THEIR EFFECT UPON GRAFT UNION FORMATION

Introduction

Studies in families that are difficult to root and
graft reveal bands of fibers in the cortex near the cambium.
These bands have been found in the avocado (West Indian
race) (37), the lychee (119), and in the Mimosaceae (101 ).
Gomez (37) found that the dense bands of fibers inhibited
rooting in the avocado because root initials arise from
parenchyma cells in the cortex near the vascular cambium.
Presence of fibers, that do not readily dedifferentiate,
reduced the chances of rooting. Mamey is also extremely
difficult to root from cuttings and to airlayer (l). The
experimental objectives were to determine the location and
presence of fibers, parenchyma (with or without inclusions)
and other associated cell types such as laticifers in the
proximity of the vascular cambium.

Materials and Methods

Slides prepared for Chapters IX and X were photographed
to document the locations of fiber bands around the cambia,

89

90

any bands in the cortex, and determination of any
connections or proximal locations of laticifers and fiber
bundles. Notations were made concerning the relative
abundance of parenchyma cells near the vascular cambium,
within the pith and the cortex.

The tissue maturities examined were those of the mature
scions, "juvenile-like" scions and rootstock stem sections
or material most likely to be used for grafting.

Results and Discussion

There are distinct bands of fibers on the cortical side
of the vascular cambium in all tissue types examined. In
mature scions, there are bands of fibers around the vascular
cambium, and bands within the cortex (Figs. 9-2, 9-4).
There is also a single layer of fibers under the epidermis
(Figs. 9-1, 9-2). In the "juvenile-like" scions, there are
fewer fiber bands within the cortex but more fibers around
the loops of the cambium where the leaf traces branch from
the cambium (Fig. 9-3). Stem sections have less distinct
bands and the bands are interspersed with more parenchyma
between the fibers (Fig. 9-4 A).

In the graft union series, the stock and scion may be
readily discerned by the more undulating cambium of the
scion ("juvenile-like" tissue) and fiber bands within the

cortex of the rootstock.

91

Fibers are specialized cells that rarely
dedifferentiate because of their lignified nature (28, 51).
laticifers are also specialized cells that do not
dedifferentiate. Because of the composition of the tissue
immediately surrounding the vascular cambium, it is
necessary to be precise in determining the position for
making cuts in the stock and scion preparatory to grafting.
Locations of the fiber bands and laticifers would
necessitate the cut to be precisely between the secondary
xylem and to the inside of the fiber band (the cambium is
inside this zone) (Fig. 9-2 B) . An alternative location
for the cut would be in the midsection of the cortex where
there are fewer specialized cells and more of the parenchyma
cells that dedifferentiate readily.

The fiber bands surrounding the vascular cambium in
mamey would be in the same position of the band of fibers in
the cortex-cambium region of the West Indian race of
avocados as described by Gomez (57) in his study relative to
rooting avocados. Guatemalan and Mexican races lacked these
fiber bands in the cortex-cambium region and these races
were easier to root.

In the graft union series in Chapter X, the scions that
made a stronger graft union were cut in the mid-cortex
region (Figs. 10-2 A, 10-5 A) and the ones that broke more
easily were cut in the area between the xylem and the fiber
bands (Fig. 10-5 C,D). This would support Gomez's (57)
contention that fiber bands are located where root initials

9 2

arise (next to the vascular cambium in the cortex) and that
cell dedifferentiation is difficult because of the numbers
of specialized cells in this area. The fiber bands next to
the cambium within the cortex would explain why mamey sapote
is also difficult to root and airlayer.

CHAPTER XII

SILICA: CONTENT AND LOCATION, AND EFFECT UPON GRAFT TAKE

Introduction

Silica inclusions are common in the family Sapotaceae
and are used for identification at the generic level (55,
56, 57, 58). Several tropical families (Lecythidaceae ,
Proteaceae, Sapotaceae) that contain fruit trees that are
difficult to graft, also have a high silica content.
Scurf ield (106) reports that silica lines cell walls of some
species and the lining may he found in septate fibers,
vessels, vessel tyloses, and vertical and ray parenchyma.
Silica within the cell lumen may be in the form of silica
sand, mixed with starch, and silica crystals that fill
entire cells (47, 100, 106). Silica inclusions may have a
wrinkled or uneven appearance. Some of the silica crystals
may be 70 urn in diameter and brown under normal light,
glowing under phase contrast (2, 16, 100, 106).

The experimental objectives were to determine if the
scion materials (seedling tops, "juvenile-like", and mature)
vary in silica content within tissue types and within
seasons (December, February, April, and November). Location
of silica within tissue was investigated and the tissues

93

94

examined were parenchyma of the cortex, pith, and secondary
xylem. The types of siliceous materials to be located were
silica cell wall deposits, silica crystals, and silica sand.

Materials and Methods

Materials were gathered from the field at the same time
scionwood was collected for other grafting experiments
(Chapters III, IV ) and samples were randomly chosen from the
overall collection. Two cultivars ('No. 2' and 'Magana')
were used and the tissue maturities were seedling tops,
"juvenile-like", and mature scions. Time periods were
December, 1982, February, 1983, April, 1983, July, 1983, and
November, 1983.

Tissue preparation consisted of chopping, then

air-drying the scionwood for 4 days. After the materials

/

were thoroughly dried, samples were then weighed. One gm
samples were analyzed for silica by ashing the sample in a
muffle furnace, dissolving the white ash residual in 1 N HC1
(hydrochloric acid) solution. The solutions were analyzed
at the University of Florida Soils Testing Laboratory by an
atomic absorption method. Results are reported in parts per
million and these are in turn converted to mg/ml.

Materials for microscopic examination were taken from
budstick samples collected for silica analysis. The samples
in tissue type and cultivar were the same as for the silica
analysis. Preparations for these samples consisted of
slicing into 1 cm sections on a band saw, and half of each

95

replicate was soaked in ETD and HP, dehydrated in ETCH and
embedded in GMA (as described in Chapter IX). The other
half of each sample was only dehydrated in alcohol and then
embedded in GMA. Samples were sectioned and mounted, and
stained in toluidine blue (as described in Chapter IX).

Results and Discussion

Graft taKes in relation to silica content were compared
on a tissue and monthly basis (Table 12-1). There was
little difference in cultivars and the results were averaged
together for the scion maturities.

Data were statistically analyzed using correlation
coefficients (Table 12-2). Silica did not positively affect
the graft takes and was negatively correlated in the months
of December, February, April, and July. November had a
slight positive correlation coefficient. Only April had a
significant treatment effect at the 5$ level.

Combining the effects of tissue maturation and season,
the results were negatively correlated and significant at
the 5 % level (Table 12-3).

A negative correlation would indicate that as the
measurable amount of silica increases, the graft take
decreases. A positive correlation would indicate that if
silica amounts increased, graft take would increase.

Microscopic examination of tissue sections indicates
that silica is found in the cortex in the form of a cell
wall deposit within the parenchyma (Pig. 12-1 A,B), mixed

96

with starch (Fig. 12-1 C), and as crystals. Silica
crystals appear as dark globules under normal light, but
under phase contrast they glow (Fig. 12-1 F) . Idioblasts
(containing solitary silica crystals up to 80 p in diameter)
in the cortex may be found concentrated particularly around
the phloem bundles near the vascular cambium associated with
laticifers (Figs. 12-1 D,E,F). Laticifers are found
scattered randomly in the cortex, and especially around
phloem fiber bundles. In longitudinal sections laticifers
also show silica deposits on the walls and within the latex
(Figs. 12-1 A,B).

The crystals and silica deposits may be seen, even if
the tissues are treated for silica removal with HF . When
samples of tissue not treated with HF and ETD were
sectioned, the glass knives shattered and up to 10 knives
were used to obtain one section suitable for mounting.
Tissue samples treated with ETD and HF did not shatter the
glass knives and 2-3 samples could be sectioned with
numerous slides being prepared from the use of one glass
knife .

Slides made from samples treated with ETD and HF had
very little ripping and tearing in the secondary xylem
(Figs. 12-2 A,B) compared to massive tears and cell damage
and distortion in untreated tissues (Figs. 12-2 C,D).
Phloem fibers also were torn and distorted in the untreated
tissues (Figs. 12-2 E) while the treated tissues made

smooth cuts (Fig. 12-2 F).

97

Silica may be found in the secondary xylem as deposits
on the walls of fibers and vessel members (Pigs. 12-3
A,B,C). Vessel members are easily seen in longitudinal
sections because of the scleriform pitting, contrasted to
the laticifers in the nearby pith which appear to be full of
globules (latex) (Pigs. 12-3 A,B). Under phase the same
section shows silica deposists along the vessel walls, on
the laticifer walls and within the latex (Pig. 12-3 C).

Laticifers are found scattered randomly in the pith,
(Pigs. 12-3 D,E). Under phase contrast, silica can be
found in the cortex, as well as within the secondary xylem
rays, in the forms of silica crystals, deposits on vessel
walls, within the ray parenchyma mixed with starch (Pig.
12-3 P).

The effect of silica upon graft take is possible in
several ways. Parenchyma cells become specialized when they
contain large solitary crystals (idioblasts) and specialized
cells do not readily dedifferentiate (28, 31). Vessels are
coated with silica, and this may inhibit water movement if
pit apertures become plugged (pit aperture plugging is
common in the Sapotaceae) (62). Cells hardened with silica
are subject to ripping and tearing when using a glass knife
for sectioning. A metal grafting knife used for making the
graft wounds would do a great deal of damage at the graft
site, and the tissues would require more healing than if
wounds could be cut more smoothly.

98

Silica content is lower in the species of the
Sapotaceae that are easier to graft (Manilkara (0.0$) and
Pouteria campechiana (.25$)) (6, 57, 58). The species that
are more difficult to graft contain more silica (Calocarpum
(2.2 to 5$) and Chrysophyllum (.25 to 1.13$)) (6, 55, 56).

For best grafting results, scion materials should be
selected that are lower in silica ("juvenile-like") over
materials that are higher in silica (mature).

99

'able 12-1:

Silica Conteni

; Relative

to Graft

Take

Take

z

Stage

y

Gfts

$Take

mg/ml

Dec .

Mature

25

1 1

7.2

"Juv"

25

56

5.0

Seedling

25

52

5.8

Feb .

Mature

15

47

7.2

Juv

15

80

5-9

Seedling

15

60

7.2

April

Mature

15

24

6.5

"Juv"

15

72

4.1

Seedling

15

68

4.0

July

Mature

15

00

7.6

"Juv"

15

26

4.5

Seedling

15

66

4.9

Nov.

Mature

15

20

5.2

"Juv"

15

46

5.7

Seedling

15

80

5 . 6

z

"Juv" ("

juvenile-like" )

Jf ,

Gfts (numbers of grafts

per scion

maturity)

Table 12-2: Statistical Seasonal Silica Accumulation

z

Date R-value DP Sig.

Dec .

-.700

2

NS

Peb .

-.920

2

NS

April

-.990

2

*

July

-.701

2

NS

Nov.

0.299

2

NS

z

Significance {*,5%; NS, non significant)

100

able 12-3: Statistical Comparison of Combined Tissue

Maturation and Seasonal Silica Accumulation

z

R-value

DF

Sig.

-0.535

14

*

z

Significance (*, 5$)

Figure 12-1: Silica deposits and laticifers within the

cortex, phloem, and their association with

fiber bundles and leaf traces.

A. Longitudinal section, silica crystal (SC)

in cortex adjacent to phloem (PL), and
laticifers (Lt). 500x.

B. Longitudinal section, silica found in
cortex associated with latex in the latici-
fers (Lt), silica sand (SS), deposits on
cell walls (SD) in parenchyma cells.

500x. Phase.

C. Silica sand (SS) with starch (St) in
parenchyma cell. 500x. Phase.

D. Leaf trace (LTr ) with bands of fibers (FB)
associated with numerous laticifers (Lt).
Dark bodies in cortex are silica crystals.
70x.

E. Same site. Laticifers (Lt) with epithelial
cells (EC) near leaf trace (LTr). 200x.

F. Same site. Silica deposits (SD) found in
laticifers (Lt), large silica crystals (SC)
intercellular spaces, and latex globules
visible epithelial cell (EC) of the
laticifers. 500x. Phase.

A-F: Toluidine blue stain.

102

Figure 12-2: Secondary xylem and phloem, with and without

ETD and HF treatments.

A. Secondary xylem (2oXy) with ETD and
HF treatment. 200x.

B. Same site. 500x.

C. Secondary xylem (2oXy) without ETD and
HF treatment. More ripping and tissue
distortion than treated samples. 70x.

D. Same site. 500x. Phase.

E. Fiber bundles in cortex with ETD and
HF, silica deposits in fibers. 500x.
Phase .

F. Fiber bundles in cortex with ETD and HF,
no tearing and cell distortion. 500x.

A-F: Toluidine blue stain.

Figure 12-3: Silica deposits and laticifers in the

secondary xylem and pith.

A. Longitudinal section, vascular cambium
on the left with the xylem (Xy) on the
right. 200x.

B. Longitudinal section, vessel members (VM)
with scleriform pittin in the xylem near
the laticifers (Lt) in the pith. 500x.

C. Longitudinal section, vessel members (VM)
with silica deposits (SD) and laticifers
(Lt) latex globules and silica deposits.
500x. Phase.

D. Laticifers (Lt) in the pith near the
secondary xylem and the vascular cambium
(VC) adjacent to a leaf trace (LTr). 70x.

E. Vascular cambium (VC) adjacent to second-
ary xylem (Xy) near laticifers (Lt) in
the pith. 200x.

F. Silica deposits in the secondry xylem
(Xy), located on the cell walls of the
vessel members (VM) , in deposits in fibers
(SD), and as silica crystals (SC)in the
parenchyma. 500x. Phase.

A-F: Toluidine blue stain.

104

106

CHAPTER XIII

INTERACTIONS OF STARCH, SUGAR, AND SILICA UPON GRAFT TAKE

Data from Chapters V and XII were combined in a
multiple linear regression analysis (Table 13-1). The
multiple regression model combines the 5 grafting periods, 3
scion maturities, and the 3 quantitative variables. The
interactions of starch and silica from the values in Table
13-1 indicate that during the months when starch
measurements are high relative to silica, graft take is
higher. When silica measurements are high relative relative
to starch, the graft take is lower. Mature scions have
consistently higher silica content, lower starch content,
and lower graft take percentages than the "juvenile-like"
scions.

The regression model (Table 13-2) lists the part of the
total variability, or R-square value (60.5$), as sugar
(7.4$), silica (26.1$) and starch (44.3$). Starch is the
single most important variable according to the model.
Interactions of the variables, in order of increasing
importance, are sugar and silica (32.3$), sugar and starch
(50.2), and starch and silica (55.2$).

107

108

Because only 60.5$ of the variability within the model
is explained by the regression equation, other factors in
addition to starch, silica, and sugar affect the graft take
of mamey sapote, and these have been detailed in previous
chapters .

109

Table 13-1 : Interactions of Starch, Sugar and Silica

Date

z

Stage

y

Gfts

$ Take

Analysis

mg/gm

Dec .

Mature

25

1 1

Starch

30.6

Sugar

13.3

Silica

7.2

"Juv"

25

36

Starch

33.5

Sugar

14.6

Silica

5.0

Seedling

25

52

Starch

51 .0

Sugar

16.9

Silica

5.8

Feb .

Mature

15

47

Starch

29.5

Sugar

19.7

Silica

7.2

"Juv"

15

80

Starch

51 .0

Sugar

18.1

Silica

3.9

Seedling

15

60

Starch

45.0

Sugar

14.1

Silica

7.2

April

Mature

15

24

Starch

24.3

Sugar

21 .0

Silica

6.5

"Juv"

15

72

Starch

37.5

Sugar

19.6

Silica

4.1

Seedling

15

68

Starch

34.3

Sugar

12.6

Silica

4.0

July

Mature

15

0

Starch

34.3

Sugar

19.9

Silica

7.6

"Juv"

15

26

Starch

38.6

Sugar

13.0

Silica

4.3

no

Table 13-1 - Extended

Seedling

15

66

Starch

Sugar

Silica

47.9

27.0

4.9

Nov.

Mature

15

20

Starch

Sugar

Silica

20.1
21 .3
5.2

"Juv"

15

46

Starch

Sugar

Silica

38.6

22.6

3.7

Seedling

15

80

Starch

Sugar

Silica

43.1
30.9
5 . 6

z

"Juv" ("Juvenile-like")

y

Number of grafts per test

Table 13-2: Overall Scion Multiple Linear Regression

+ :

Multiple linear regression equation:

Graf t=. 269+1 . 53 (starch)+1 .09 (sugar )-6. 21 (silica)

R-square value=60.5

P value: 1837.69/326.6=5.62 with 3,1 CP *5$ level

R-square variables in model

Model:

1 7.4$ Sugar

1 26.1$ Silica

1 44.3$ Starch

2 32.2$ Sugar and silica

2 50.2$ Starch and sugar
2 55.2$ Starch and silica

3 60.6$ Starch, silica, and sugar

CHAPTER XIV

SUMMARY AND CONCLUSIONS

Results indicate that several different factors are
involved in the formation of the graft union of mamey
sapote. These factors are: environment (mediated by
seasons that affect temperature, relative humidity, seasonal
rainfall, and diurnal effects); juvenility or tissue
maturation; seasonal carbohydrate accumulation, in
particular starch; apical dominance, perhaps mediated by
growth regulators; irregularity of the vascular cambium;
proper grafting technique; fiber bands on the cortical side
of the vascular cambium that inhibit tissue
dedifferentiation in proximity of the cambium; and silica
content .

Environmental effects (season, temperatures, rainfall,
relative humidity) allow definite grafting seasons. During
the summer months in the rainly season when the relative
humidity of very high and plant is in active growth, and
during the winter months when the plants are dormant, the
mamey sapote has poor grafting results. The spring months
of February and April, when there are warm days, cool
nights, and low relative humidity, are the best grafting
times with the highest percentage graft take.

Ill

112

Juvenile scions and scions that have been reverted to a
"juvenile-like" morphology are superior to scions collected
from mature grafted trees. "Juvenile-like" scions are the
scions of choice because they are of known genotype compared
to the seedling or juvenile scions of unknown genetic
characters .

Carbohydrate content varies according to the season of
the year depending upon whether the plant is in active
growth or dormant. Starch is a more important quantitative
variable compared to sugar relative to graft take
percentage. Mature scions have lower starch content and
lower graft take compared to the "juvenile-like" and
seedling scions.

Sugar pulsing did not enhance graft take, and it almost
inhibited graft take completely. The deleterious effect may
have been due to intolerance of the tissues to wetting or
coagulation of the latex that sealed off the vascular
system.

Applications of growth regulators to the graft wound
did not enhance graft take and some of the growth regulators
may even have inhibited graft take (auxins in particular).
Apical dominance tests indicated that removal of terminal
buds on the rootstock eliminated an inhibitor (possibly
auxins) during rapid growth periods in the summer and
partially overcame some of the grafting problems when the

season was hot.

113

Anatomical investigations revealed that the vascular
cambium is irregular and has an undulating pattern within
the stem. This undulation was found to be caused by
numerous leaf traces. Cambium irregularity makes grafting
more difficult because it is hard to line up the stock and
scion for good cambial contact and subsequent healing.

Other anatomical observations detailed fiber bands and
laticifers in the cortex in close proximity to the vascular
cambium. These cell types do not readily dedifferentiate
into callus for wound healing and differentiation into
vascular tissue necessary for a good graft union and
conduction of water and food supply.

Proper grafting technique is important because of the
irregular cambium and the position of the fiber bands. A
modified veneer graft cutting into the cortex and away form
the bands of fibers around the cambium is more successful
because there is more parenchyma in this area to
dedifferentiate into callus. This was shown by sections in
a graft union series. The grafts that were made in the
cortex were more successful than grafts that were made close
to the cambium and the fiber bands.

Silica was noted in the tissue and was found as
solitary crystals in idioblasts, lining the cell walls in
the xylem, and mixed with starch and with latex. Silica
tends to make the wood hard and this results in tissue
damage with a lot of tearing and ripping. If a smoother cut
could be made, there would be less tissue damage and faster
healing of the graft wound.

114

An analysis of the silica content was made and an
interaction of starch, sugar, and silica was indicated. A
combination of low silica content relative to a high starch
content resulted in increased graft take percentage (as
found in the "juvenile-like" and seedling scions). Mature
scions with higher silica content and lower starch content
had lower graft take percentages.

Taxonomic implications were also noted. Graft take in
4 genera was compared to data published concerning the
anatomical characters of tissue type and silica content.
Those genera (Pouter ia and Manilkara) with more abundant
parenchyma (cell types that store starch and dedifferentiate
readily to form callus) and lower silica content, are known
to graft more readily. The genera Calocarpum and
Chrysophyllum have higher silica content and less abundant
parenchyma.

The results of this study indicate that optimal
grafting conditions are the use of "juvenile-like" scions
with a modified veneer graft in late October to early
December or late March to early May (periods of warm days,
cool nights, low rainfall and low relative humidity).

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BIOGRAPHICAL SKETCH

Mary Ann Hollingsworth Ogden was born November 25,
1943, in St. Petersburg, Florida. She attended the Florida
State University (1961-63) and received the Bachelor of
Science degree in environmental studies from Florida
International University in March, 1978. She received a
Master of Science degree from the Fruit Crops Department at
the University of Florida in August, 1981 .

She has been employed as a seed analyst at the Florida
Department of Agriculture, in Tallahassee, a park ranger in
Everglades National Park, a park naturalist for Dade County
Parks and Recreation, a consultant on native plants for the
Environmental and Urban Center at the Florida International
University and had a private consulting business for the
native landscaping and identification of toxic and
allergenic plants. She is a member of the following
professional organizations: American Society for
Horticultural Science, Florida State Horticultural Society,
Tropical Region American Society for Horticultural Science,
and the Society for Economic Botany.

125

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Phi InsonViv.

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor o^ ^ - - ’

Carl W. Campbell , /Chairman
Professor of Horticultural Science

Professor of Horticultural Science

cultural Science

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy. —

cnjA

J

Tom' L. Davenport
Associate Professor of

orticultural Science

I certify that I have read this study and that in
my opinion it conforms to acceptable standards of
scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of
Doctor of Philosophy.

Walter S. Judd!

Associate Professor of Botany

This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School,
and was accepted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy.

Dean for Graduate Studies and Research