Citation
In vitro somatic embryogenesis and plant regeneration from mango (Mangifero indica L.) nucellar callus

Material Information

Title:
In vitro somatic embryogenesis and plant regeneration from mango (Mangifero indica L.) nucellar callus
Added title page title:
Mangifera indica
Creator:
DeWald, Stephen Gregory, 1950-
Publication Date:
Language:
English
Physical Description:
xvi, 162 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Mango -- Propagation -- In vitro ( lcsh )
City of Gainesville ( local )
Callus ( jstor )
Embryos ( jstor )
Somatic embryos ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references (leaves 148-161).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Stephen Gregory DeWald.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030389576 ( ALEPH )
AER2772 ( NOTIS )
16931239 ( OCLC )

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Full Text













IN VITRO SOMATIC EMBRYOGENESIS AND PLANT REGENERATION
FROM MANGO (Mangifera indica L.) NUCELLAR CALLUS










BY

STEPHEN GREGORY DEWALD


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


1987

























This dissertation is dedicated to my cherished friend

and loving wife Maria, who has served as a constant source

of inspiration and support.


















ACKNOWLEDGEMENTS


The author expresses his sincere thanks and gratitude

to the members of his advisory committee, Drs. Gloria A.

Moore, Wayne B. Sherman, Robert J. Knight, Jr., Prem S.

Chourey, and especially to his major advisor Dr. Richard E.

Litz.

Many thanks go to his parents Stephen and Ruth Anne,

brother Joseph, and the rest of the DeWald family for their

support and encouragement.

Finally, the author extends his appreciation to the

Florida Mango Forum for their financial support and to the

graduate students, the service staff, and the faculty of the

University of Florida Fruit Crops Department, Gainesville,

and the Tropical Research and Education Center, Homestead,

for the many ways in which they have helped.















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ..........................................iii

LIST OF TABLES............................................. viii

LIST OF FIGURES...............................................xi

KEY TO ABBREVIATIONS...................................xiii

ABSTRACT......................................... .........xiv

CHAPTER

I INTRODUCTION........................... ........

II LITERATURE REVIEW..............................3

Introduction........................ .......... 3
Mango: Taxonomy, Distribution, and
Breeding Efforts............................. 3
Polyembryony .... ............................... 9
Histological Studies of Mango Embryology...... 12
Factors Affecting Polyembryony................15
The Genetics of Polyembryony...................17
The Evolutionary Significance of Apomixis.....20
Plant Tissue and Cell Culture.................21
Somatic Embryogenesis..........................24
Factors Affecting In Vitro
Somatic Embryogenesis.......................28
Tissue Culture of Mango.......................37

III SOMATIC AND ADVENTIVE NUCELLAR EMBRYONY
IN MANGO. ......................................41

Introduction.................................. 41
Materials and Methods .........................42
Results and Discussion........................43

IV CALLUS MAINTENANCE AND SOMATIC
EMBRYOGENESIS IN MANGO........................72

Introduction................................... 72
Materials and Methods .........................73










CHAPTER Page

Experiment 4-1: In Vitro Somatic
Embryogenesis from Mango Nucellar
Callus in Response to Genotype and
Plant Growth Regulators...................77
Experiment 4-2: In Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium and Major Salts
Formulations.............................78
Experiment 4-3: In Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium Formulation
and Sucrose Concentration..................79
Experiment 4-4: Callus Production from
Nonembryogenic 'James Saigon'
Nucellar Callus in Response to the
Major Salts Formulation of the
Callus Maintenance Medium................. 80
Experiment 4-5: In Vitro Somatic
Embryogenesis from 'James Saigon'
Nucellar Callus in Response to the
Solidifying Agent Used in the
Embryogenesis Medium.......................81
Experiment 4-6: In Vitro Production
of 'James Saigon' Nucellar Callus in
Response to a Liquid or a Solid
Maintenance Medium........................82
Results and Discussion........................83
Experiment 4-1: In Vitro Somatic
Embryogenesis from Mango Nucellar
Callus in Response to Genotype and
Plant Growth Regulators................... 83
Experiment 4-2: In Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium and Major Salts
Formulations ....... ............. ........ 92
Experiment 4-3: In Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium Formulation
and Sucrose Concentration..................96
Experiment 4-4: Callus Production from
Nonembryogenic 'James Saigon'
Nucellar Callus in Response to
the Major Salts Formulation of the
Callus Maintenance Medium.................98










CHAPTER Page

Experiment 4-5: In Vitro Somatic
Embryogenesis from 'James Saigon'
Nucellar Callus in Response to the
Solidifying Agent Used in the
Embryogenesis Medium......................98
Experiment 4-6: In Vitro Production
of 'James Saigon' Nucellar Callus in
Response to a Liquid or a Solid
Maintenance Medium.......................100
Conclusions .................................. 102

V SOMATIC EMBRYO MATURATION, GERMINATION,
AND PLANTLET FORMATION.......................105

Introduction..................................105
Materials and Methods ........................106
Experiment 5-1: In Vitro Somatic
Embryo Production and Maturation
from 'James Saigon' Nucellar Callus
in Response to Culture Regime............106
Experiment 5-2: In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and ABA in
a Liquid Embryo Maturation Medium......... 107
Experiment 5-3: In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and
Supplements to the Embryo Maturation
Medium...................................108
Experiment 5-4: In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Solidifying Agent and ABA in the
Embryo Maturation Medium.................111
Experiment 5-5: In Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation...........113
Experiment 5-6: In Vitro 'Parris'
Somatic Embryo Germination and Shoot

Formation in Response to the Embryo
Germination Medium Formulation...........114
Results and Discussion....................... 116
Experiment 5-1: In Vitro Somatic
Embryo Production and Maturation
from 'James Saigon' Nucellar Callus
in Response to Culture Regime.............116
Experiment 5-2: In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and ABA in
a Liquid Embryo Maturation Medium.........118










CHAPTER Page

Experiment 5-3: In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and
Supplements to the Embryo Maturation
Medium .............. .................... 120
Experiment 5-4: In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Solidifying Agent and ABA in the
Embryo Maturation Medium.................122
Experiment 5-5: In Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation........... 124
Experiment 5-6: In Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation...........126
Conclusions .................................128

VI SUMMARY AND CONCLUSIONS......................134

Histological Investigations..................134
Tissue Culture Studies.......................136
Conclusions ....................................138

APPENDIX................................... ................... 140

LITERATURE CITED..........................................148

BIOGRAPHICAL SKETCH .....................................162















LIST OF TABLES


Table Page

4-1 Basal media major salts formulations used with
in vitro culture of mango...........................74

4-2 Murashige and Skoog minor salts formulation........ 74

4-3 Basal medium components and supplements used for
in vitro culture of mango..........................76

4-4 In vitro somatic embryogenesis from
'James Saigon' nucellar callus in response to
various plant growth regulators.....................84

4-5 In vitro somatic embryogenesis from 'Parris'
nucellar callus in response to various plant
growth regulators.................................85

4-6 In vitro somatic embryogenesis from
'Tommy Atkins' nucellar callus in response to
various plant growth regulators...................86

4-7 In vitro somatic embryogenesis from 'Heart'
nucellar callus in response to various plant
growth regulators.................................87

4-8 In vitro somatic embryogenesis from mango
nucellar callus for cultivars in experiment 1......88

4-9 In vitro somatic embryogenesis in response
to various complex organic addenda................. 88

4-10 In vitro somatic embryogenesis from 'Parris'
nucellar callus in response to the callus
maintenance medium and major salts formulations....93

4-11 In vitro somatic embryogenesis from 'Parris'
nucellar callus in response to the callus
maintenance medium formulation and sucrose
concentration......................................97

4-12 Callus production from nonembryogenic
'James Saigon' nucellar callus in response to
the major salts formulation of the callus
maintenance medium.................................99


viii









Table Page


4-13 In vitro somatic embryogenesis from
'James Saigon' nucellar callus in response to
the gelling agent used in the embryogenesis
medium............................................. 99

4-14 In vitro production of 'James Saigon' nucellar
callus in response to liquid or solid callus
maintenance medium................................101

5-1 In vitro somatic embryo maturation rating
scale used in experiment 5-3......................110

5-2 In vitro somatic embryo maturation rating
scale used in experiment 5-4...................... 112

5-3 In vitro somatic embryo production
and maturation from 'James Saigon' nucellar
callus in response to the culture regime..........117

5-4 In vitro 'Parris' somatic embryo maturation
in response to sucrose concentration and ABA
in liquid embryo maturation medium................119

5-5 In vitro 'Parris' somatic embryo maturation
in response to sucrose concentration and
supplements to the embryo maturation medium........121

5-6 In vitro 'Parris' somatic embryo maturation
in response to the solidifying agent and ABA in
the embryo maturation medium.......................123

5-7 In vitro 'Parris' somatic embryo germination
and shoot formation in response to the embryo
germination medium formulation....................125

5-8 In vitro 'Parris' somatic embryo germination
and shoot formation in response to the embryo
germination medium formulation....................125

A-i Plant growth regulators (PGRs) used with
in vitro culture of mango.........................140

A-2 List of mango cultivars with successful
in vitro nucellar callus initiation...............141

A-3 In vitro somatic embryogenesis from 'Simmonds'
nucellar callus in response to various plant
growth regulators................................ 142









Table Page

A-4 In vitro somatic embryogenesis from 'Florigon'
nucellar callus in response to various plant
growth regulators................................143

A-5 In vitro somatic embryogenesis from
callus of 'Cambodiana' in response to
various plant growth regulators................... 144

A-6 In vitro somatic embryogenesis from
callus of 'Irwin' in response to various plant
growth regulators................................145

A-7 Peach embryo germination medium................... 146














LIST OF FIGURES


Figure Page

3-1 A mango fruitlet approximately 75 days-post-
bloom bisected longitudinally with intact
ovule (megasporangium) ............................49

3-2 Longitudinal section through a polyembryonic
ovule approximately 30 days-post-bloom.............50

3-3 Micropylar region of a polyembryonic ovule.........51

3-4 Longitudinal section through the micropylar
half of a polyembryonic ovule approximately
45 days-post-bloom................................52

3-5 Globular stage adventive nucellar embryos..........53

3-6 Late globular stage adventive nucellar embryo......54

3-7 The initiation of embryogenic nucellar callus......55

3-8 Close-up view of embryogenic mango nucellar
callus initiation................................. 56

3-9 Suspension culture of rapidly proliferating
mango nucellar callus....................... ...... 57

3-10 The epidermis of a late globular stage somatic
embryo prior to redifferentiation and somatic
embryogenesis.....................................58

3-11 The epidermis of a late globular stage somatic
embryo beginning redifferentiate.................... 59

3-12 Early stages of epidermal somatic embryogenesis....60

3-13 Section through the periphery of a redif-
ferentiating globular somatic embryo or callus.....61

3-14 Four early globular stage somatic embryos..........62

3-15 A globular stage somatic embryo beginning to
differentiate an epidermis and showing signs
of polarity.................................. .......63









Figure Page

3-16 A suspension culture of budding 'Parris'
somatic embryos.....................................64

3-17 Close-up of a cluster of budding 'Parris'
somatic embryos grown in suspension culture........65

3-18 Cross section, at low magnification through a
cluster of budding somatic embryos grown in
suspension culture.................................66

3-19 A section through the periphery of the
central core of a budding cluster of somatic
embryos grown in suspension culture.................67

3-20 Globular stage somatic embryo grown in
suspension culture exhibiting aberrant
development ........................................68

3-21 Globular stage somatic embryos grown in
suspension culture with a poorly formed
epidermis...........................................69

3-22 Scanning electron micrograph of a cluster
of 'Parris' somatic embryos grown on solid
medium..............................................70

3-23 Longitudinal section through the base
of a somatic embryo grown on solid medium..........71

4-1 'Parris' somatic embryogenesis in response to
major salts formulations of experiment 4-2........104

5-1 'Parris' somatic embryo maturation in
response to solidifying agents and ABA of
experiment 5-4.....................................130

5-2 A culture of germinating 'Parris' somatic
embryos from experiment 5-6.......................131

5-3 Germinated 'Parris' somatic embryos ready
to be transferred to the soil.....................132

5-4 Young 'Parris' plants regenerated via
somatic embryogenesis growing in the soil.........133















KEY TO ABBREVIATIONS


ABA: abscisic acid

BA: 6-benzylaminopurine (N6 benzyladenine)

BM: basal medium

B-5 Gamborg et al., B-5 basal medium

CH: casein hydrolysate

GA: gibberellic acid

IAA: indole-3-acetic acid

IBA: indole-3-butyric acid

LCE: liquid coconut endosperm (coconut water)

M: monoembryonic seed

MS: Murashige and Skoog basal medium formulation

NAA: 2-naphthaleneacetic acid

Na2EDTA: disodium ethylenediaminetetraacetic acid

P: polyembryonic seed

YE: yeast extract

WPM: Lloyd and McCown woody plant medium

2iP: (2-isopentenyl)adenine

2,4-D: (2,4-dichlorophenoxy)acetic acid


xiii













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



IN VITRO SOMATIC EMBRYOGENESIS AND PLANT REGENERATION
FROM MANGO (Mangifera indica L.) NUCELLAR CALLUS

BY

STEPHEN GREGORY DEWALD

May, 1987

Chairman: Richard E. Litz
Major Department: Horticultural Science (Fruit Crops)

Histological studies were conducted on polyembryonic

mango ovules and embryogenic nucellar callus to elucidate

the patterns of embryogenesis in these two systems. The

nucellar origin as well as the variable location of

adventive embryos within the micropylar portion of the

ovules was demonstrated. Adventive nucellar embryos,

characterized by densely staining cytoplasm and prominent

nuclei, were formed prior to the cellularization of the

endosperm.

Embryogenic callus was initiated from both the

micropylar and chalazal portions of excised nucelli. In

suspension culture this callus grew as compact, nonfriable

globular callus. Histological preparations of this callus

revealed that no true, undifferentiated, subculturable

tissue is produced. Proliferation occurs via secondary

budding from the epidermis of preformed somatic embryos.









Prior to somatic embryogenesis the epidermis

dedifferentiates and forms a layer, several cells thick, of

cytoplasmically dense cells with prominent nuclei. In

suspension culture, somatic embryos exhibited numerous

developmental abnormalities including poorly formed

epidermal layers, vascular system and meristems, noncompact

morphology, and secondary budding. More normal embryo

development occurred on solid medium. Somatic embryos were

shown to possess bipolar meristems with closed vascular

systems, thus unequivocally demonstrating the embryogenic

nature of plant regeneration from mango nucellar callus.

The morphogenic response of mango nucellar callus was

also investigated with emphasis on factors influencing

callus production, somatic embryogenesis, embryo maturation,

and germination. The addition of 2 mM glutamine, 5 uM 2,4-D,

5 uM kinetin, and vitamins to suspension multiplication

cultures improved the embryogenic efficiency of callus upon

transfer to a solid embryogenesis medium. Efficient somatic

embryogenesis occurred on a modified B-5 medium [lacking

(NH4)2SO4] supplemented with 5% (w/v) sucrose, 2 mM

glutamine, 11 uM NAA, 5 uM 2iP, vitamins, and amino acids.

The use of Gelrite gellan gum (0.19%, w/v) improved the

efficiency of somatic embryogenesis and maturation in

comparison to medium solidified with Difco Bacto-agar or

Sigma agar gum. Maturation and germination occurred on solid

media in which the sucrose concentration was sequentially

lowered and the synthetic plant growth regulators were









replaced with 20% (v/v) liquid coconut endosperm and 0.025%

(w/v) casine hydrolysate. Plant's were established in the

soil by watering with dilute basal salts.


















CHAPTER I
INTRODUCTION


Mango is one of the world's most important tropical

fruit crops. At the present time, production in Florida is

restricted to approximately 1000 ha of mature mango groves

(IFAS, 1983). However, despite its relatively small size in

Florida's fruit industry, mango production, both here and

abroad, should continue to expand (Bondad, 1980). One of the

major factors limiting the distribution and production of

this delicious tropical fruit crop is the lack of well

adapted, high yielding, disease resistant cultivars with

good quality fruit. Most of the industry throughout the

world is based on the use of chance seedlings maintained

clonally by means of various asexual propagation techniques.

Breeding attempts have been particularly discouraging in

comparison to many other agronomic crops.

Recent advances in biotechnology offer the plant

breeder new and exciting opportunities in cultivar

improvement and development. One of these new

biotechnologies is the field of plant cell and tissue

culture. Plant cell and tissue culture has been used

successfully to propagate large numbers of plants, eliminate

persistent pathogens, create new and economically useful










variation in existing well established genotypes, rescue

hybrid embryos from wide crosses, create homozygous breeding

lines via haploidy, produce economically important primary

and secondary plant metabolites, and elucidate complex

processes at both the cell and whole plant level. Plant

tissue culture and its associated technologies should be

particularly useful to breeders working with perennial

crops, where cultivar improvement has been slow and

difficult. Unfortunately, the woody perennials as a whole

have proved to be recalcitrant in vitro.

In vitro somatic embryogenesis from mango nucellar

cultures was first reported by Litz et al. (1982); however,

the recovery of plants from mango somatic embryos has not

been reported. The first objective of this research was to

examine histologically the processes of in vitro somatic

embryogenesis and adventive nucellar polyembryony in order

to determine more clearly the regeneration pathway in mango.

The second objective of this research was to investigate and

optimize in vitro parameters that affect somatic

embryogenesis, embryo development, and germination, thereby

developing an efficient in vitro plant regeneration system.



















CHAPTER II
LITERATURE REVIEW


Introduction


The purpose of this literature review is to introduce

the reader to the mango, its embryology, and in vitro

somatic embryogenesis. Polyembryony and somatic

embryogenesis are of particular importance to this

dissertation and will be discussed in detail. Somatic

embryogenesis in Citrus, is similar to that in mango. Citrus

In vitro culture is perhaps the best understood system for

somatic embryogenesis in woody plant species, and numerous

articles have been published since embryogenic cultures were

established by Stevenson in 1956. For these reasons the

literature pertinent to Citrus is emphasized throughout this

review.



Mango: Taxonomy, Distribution, and Breeding Efforts


Mango is one of the choicest and most popular of the

tropical fruits. With an annual world production of nearly

14 million metric tons, mango is the fifth most important

fruit crop after grape, Musa, Citrus, and apples (FAO

Production Yearbook, 1984).









The culture of mango is most intensive in India, which

accounts for 2/3 of the total world production. Singh (1960)

reports that it has been under cultivation there for almost

6,000 years. Mangos are particularly well suited to India

where they .thrive in almost every region except altitudes

above 3,000 feet (Singh, 1960). In India, the mango is

referred to as the "king of fruits" and has played an

integral part in India's folklore and religions.

Mango, Mangifera indica L., is a member of the

Anacardiaceae, a large family of mostly tropical woody

perennial plants with inconspicuous flowers often produced

in large clusters, frequently bearing attractive and edible

fruit (Purseglove, 1968). The family contains some 64 genera

and includes several economically important fruit and nut

trees, e.g., cashew (Anacardium occidentale L.), a flesh

fruit (Bouea macrophylla Griff.), pistachio (Pistacia vera

L.), several edible spondias (Spondias cythera Sonn., S.

mombin L., S. purpurea L.), and some important plant pests,

e.g., poison ivy (Rhus toxicodendron L.) and the Brazilian

pepper tree (Schinus terebinthifolius Radd.) (Popenoe, 1920;

Purseglove, 1968).

The genus Mangifera contains some 41-62 invariably

arborescent species (Mukherjee, 1972; Singh, 1969)

characterized by long, leathery leaves with a

fiberous-resinous fruit. The trees are evergreen with

several distinctive annual growth cycles or flushes. The

brownish-red to red vegetative flushes are unique in that




5




they occur in sectors rather than all over the tree during

any one cycle (Popenoe, 1920).

Mango flowers are small and generally borne on terminal

panicles. The inflorescence is polygamous producing up to

4,000 staminate and perfect flowers per panicle. The fruit

is a large, fleshy drupe consisting of an edible mesocarp

and a fiberous, stony endocarp. Mango is often grouped into

2 major categories based on embryology: monoembryonic types

contain only a single embryo per seed, while the

polyembryonic types produce multiple (usually asexual)

embryos per seed. Almost without exception the Indian mangos

are monoembryonic, while the Indochinese and Philippines

types are polyembryonic (Singh, 1976). Young and Sauls

(1979) state that many monoembryonic Indian types produce

brightly colored fruit with an attractive blush and are

often susceptible to anthracnose, [Glomerella cingulata

Stonem (S. and V.S.)]. Polyembryonic types more commonly

produce a pale green to yellow, low fibered fruit and

generally possess higher levels of resistance to

anthracnose.

Mukherjee (1950, 1972) determined that the basic

chromosome number in mango and its related species is 2n=40.

Mukherjee hypothesized that mango is a polyploid because it

has a high chromosome number with many nucleolar

chromosomes. Citing secondary meiotic associations, he

further hypothesized that a certain primitive mango type or










types probably arose through allopolyploidy and most likely

via amphidiploidy.

Most authors seem to agree that the center of origin

for mango is the Assam-Burma region (Indo-Burma), where

mangos can still be found growing wild (Singh, 1969). The

Mangifera genus probably originated in Burma, Thailand,

Indo-China, and the Malay Peninsula (Singh, 1960). A marked

center of diversity occurs in the region of

Indochina-Malaysia-Indonesia (Singh, 1976). Wild mangos

generally have fruit that is extremely fiberous-resinous and

may even be poisonous in some species of Mangifera.

Selection has been for fruits with succulence, low fiber,

high sugars, small stones, and low resin content (Singh,

1976).

Mangos were probably introduced from southern India to

Malaysia around 500 B.C., and to eastern Africa in the 10th

century by Arab merchants (Purseglove, 1968). Mukherjee

(1972) believes that mangos did not reach the Pacific

islands until the sixteenth century when the Portuguese

began opening trade routes. They are often given credit for

the extensive spread of mangos throughout the tropics

(Mukherjee, 1972; Popenoe, 1920). Their far-reaching trade

routes linked the Indian ports with the Persian gulf,

Pacific islands, and the African coast. Although the date is

uncertain, the Portuguese are reported to have brought the

Indian, monoembryonic type mangos to Rio de Janeiro around

1700 (Mukherjee, 1972). In Mexico, the earliest mango










introductions were probably polyembryonic, Indochinese

seedlings that arrived via the Spanish trade routes between

Manila and the Pacific ports of Mexico (Malo, 1977).

Henry Perrine probably first introduced mangos into the

United States from Campeche, Mexico in 1833 (Popenoe, 1920).

Rolfs (1915) divides the early history of mango cultivar

development in Florida into 2 periods. The first period is

characterized by the growing of seedling mangos by a few

dedicated and ingenious horticulturists and lasted from 1867

until the end of the 19th century (Knight, personal

communication). These mangos were polyembryonic types such

as 'Turpentine' and 'No. 11' (Young and Sauls, 1979). The

second period is characterized by the planting of grafted or

budded trees and began with the noteworthy introduction of

the monoembryonic 'Mulgoba' in 1889 (Rolfs, 1915).

'Mulgoba', an Indian cultivar brought to Florida by Van

Deman of the U.S. Department of Agriculture, was the female

parent of the open pollinated seedling 'Haden'. The

commercial potential of 'Haden' was at once realized and it

has served as the standard by which all other Florida

cultivars have been compared.

Recent surveys indicate close to 1,000 ha of commercial

mango orchards in South Florida (IFAS, 1983). In 1977 over

50% percent of the total acreage was devoted to 'Tommy

Atkins' and 'Keitt' (Malo, 1977). Other important cultivars

include 'Irwin', 'Kent', 'Van Dyke', 'Jubilee', 'Sensation',

'Palmer', and 'Haden'.










Mango breeding was initiated early in this century in

India and has been reviewed by Mukherjee et al. (1968),

Singh (1959), and Singh (1969). Unfortunately, the results

from these breeding efforts have been rather discouraging.

Singh (1959) lists some of the major problems associated

with the breeding of mango in India, e.g., long generation

times (6 years or more), one seed per fruit, high levels of

heterozygosity which makes hybrid performance unpredictable,

unique floral morphology and excessive fruit drop which

makes controlled hybridizations extremely difficult, and the

large field plots needed to grow segregating seedling

populations.

Mukherjee (1976) outlined 3 major methods for cultivar

improvement in mango. The first method is selection from

natural seedlings. This was the way that most of the early

Indian cultivars arose and it is still widely practiced in

developing nations. The second method is selection from

open-pollinated seedling progenies. This technique has been

successfully employed in Florida and Hawaii. Mukherjee

considers that this is still the most promising method for

cultivar development. The final method of improvement is

through controlled hybridization. The major problem seems to

be the overall difficulty in producing large populations of

hybrid seedlings. Hand pollinations have very low success

rates (0.3%) (Singh, 1969). These rates can be improved

somewhat if a relatively small number (40-50) of freshly

opened flowers per panicle are pollinated and the rest










removed (Mukherjee, 1976). Singh (1969) states that the most

important variable in obtaining large numbers of hybrid

seeds is the total number of panicles worked per tree and

not the total number of flowers. One method to allow

mass-pollination that has been employed in Florida with some

success is the "cage method." Parents are enclosed within a

screen tent, and pollination is accomplished by placing a

fly infested animal carcass within the enclosure (personal

communication, Knight).

Polyembryony can also restrict gene flow in controlled

hybridizations. The problem is not as pronounced in mango as

it is in Citrus, because most of the high quality Indian and

Florida cultivars are monoembryonic. Knight (1970)

recommends the increased usage of polyembryonic types and

cites favorable characteristics of fruit quality, higher

levels of disease resistance, regular high yields, and ease

of propagation. Spontaneous somatic mutations are another

source of improved cultivars and have given rise to

'Davis-Haden'.



Polyembryony


Polyembryony, the production of multiple embryos within

a single seed, is widespread but sporadically distributed

among the spermatophytes (Gustaffson, 1946). It was first

recorded by Leeuwenhoek in 1719 when he observed orange

seeds containing multiple embryos (Maheshwari, 1950).

Polyembryony is quite common in the gymnosperms but usually









involves some form of cleavage polyembryony, i.e., division

of the zygote or proembryo into 2 or more units (Maheshwari,

1950). It is less widespread in the angiosperms, but

exhibits more diversity in its expression. In the

angiosperms adventive embryos may originate from the egg

cell or synergids. This may occur within a single reduced or

unreduced embryo sac of an ovule, either following or

without fertilization. It may also occur from a fertilized

egg cell through the cleavage of the zygote or proembryo

(cleavage polyembryony) or directly from the cells of the

nucellus or inner integument of an ovule, i.e., adventitious

polyembryony (Brizicky, 1964). If the embryos are not a

result of the normal sexual process then polyembryony may be

correctly referred to as a form of apomixis, termed

agamospermy (Stebbins, 1941). Apomictic polyembryony can be

either gametophytic (diplospory or apospory, parthenogenesis

or apogamety) or sporophytic adventitiouss embryony)

(Webber, 1940; Maheshwari and Sachar, 1963).

Ernst (1918) and Schnarf (1929) (cited in Webber, 1940)

reviewed the old literature on polyembryony and

distinguished 2 main types: true and false. Their

distinction was based on whether or not the embryos arise

from or protrude into the same or different embryo sacs.

Webber (1940) considered these distinctions purely arbitrary

with no physiological or natural basis.

Polyembryony has been most widely studied in the genus

Citrus. Early reports indicated that the adventive embryos










arise in the nucellus shortly after the first division of

the zygote or sometime later (Bacchi, 1943; Rangan et al.,

1969). Substantial evidence now seems to indicate that

proembryos or proembryo initials are present in the

peripheral cells of polyembryonic nucelli prior to anthesis

(Esen and Soost, 1977; Kobayashi et al., 1979). Kobayashi et

al. (1981), studying some 74 Citrus cultivars, found

distinct primordial cells to be present at the time of

flowering in all of the polyembryonic cultivars, but did not

observe these cells in any of the monoembryonic cultivars

studied. Esen and Soost (1977) concluded that although

adventive nucellar embryogenesis seems to be independent of

pollination or fertilization, subsequent embryo development

and maturation require at least fertilization and division

of the polar nuclei endospermm). They added that previous

reports of adventive embryo development in the absence of

fertilization (Webber, 1930; Wright, 1936) were probably in

error.

Survival is a function of the relative vigor of one

embryo and its location in respect to available nutrients

within the embryo sac (Esen and Soost, 1977). Numerous

studies in Citrus have shown that, in general, the location

of the nucellar embryos provides a competitive advantage for

their survival relative to that of the zygotic embryo (Ohta

and Furusato, 1957; Frost and Soost, 1968). Similarly, it

has been found that the more nucellar embryos initiated per

ovule the less likely it is that the sexual embryo will









survive to maturity (Frost, 1926). Frost (1926) indicated

that there is evidence of heterotic vigor in zygotic embryos

from wide crosses and that selection of parents can

significantly influence the survival chances of the zygote

relative to that of its asexual neighbors.



Histological Studies of Mango Embryology


Polyembryony in mango was first recorded by Schact in

1859 (Belling, 1908). The nucellar origin of the adventive

embryos in both Citrus and mango was elucidated by

Strasburger in 1878 (cited by Belling, 1908). Juliano and

Cuevas (1932) described megasporogenesis in the mango cv.

Pico. In 'Pico', an important polyembryonic Philippine

cultivar, very few perfect flowers of the panicle contain

functional megasporangia at the time of flowering. The

megasporangium ovulee) is formed from an outgrowth of the

inner carpellary wall. It grows at approximately the same

rate as the carpel until fertilization, at which time there

is a dramatic enlargement of the carpel walls. Unilateral

growth patterns give rise to a bitegmic, crassinucellate,

anatropous ovule contained within a single unicarpous pistil

(Juliano and Cuevas, 1932). A single hypodermal cell in the

nucellus functions as the archesporial cell. This

archesporial cell does not undergo meiosis directly, but

rather divides periclinally to form an outer primary

parietal cell and an inner primary sporagenous cell

megasporee mother cell). The primary parietal cell









subsequently forms an extensive nucellus while the megaspore

mother cell is pushed deeper into the developing ovule. The

megaspore mother cell differentiates and becomes distinct

from the rest of the nucellus, with its densely staining

cytoplasm and large polygonal shape (Juliano and Cuevas,

1932).

It is at this time, apparently, that certain cells of

the nucellus, especially in the region of the micropyle

surrounding the megaspore mother cell, become

distinguishable as proembryo initials by their densely

staining cytoplasm (Belling, 1908). The megaspore mother

cell enters into meiosis that results in the formation of a

linear tetrad of daughter cells. The chalazal-most megaspore

daughter cell becomes functional (thereby pushing itself

deeper into the nucellus) and undergoes megagametogenesis to

give rise to the normal 8-celled embryo sac. The polar

nuclei, at maturity, occupy a position very close to the egg

cell apparatus.

Belling (1908) reports that at the time of

fertilization, in the polyembryonic mango 'No. 11', dense

protoplasmic nucellar cells, separated from the embryo sac

by a layer of flattened cells, can be distinguished.

Immediately following double fertilization, while still in

the flower stage, the triploid endosperm nucleus starts

dividing. These divisions take place well before any

divisions of the zygotic or adventive embryos. In fact, the

first embryonic divisions cannot be detected until the










ovules are more then 3 mm long (fruit 7 mm). As the

endosperm develops, the nucleated protoplasm becomes

appressed to the embryo sac wall opposite the places where

the adventive embryos are forming. Belling (1908) contrasts

the cellularization of the endosperm in Citrus and mango

stating that it occurs much earlier in Citrus than in mango.

Sachar and Chopra (1957) studied endosperm development

in a large number of monoembryonic and polyembryonic mangos,

concluding that the endosperm is of the nuclear type.

Numerous free nuclei are formed and distributed along the

periphery of the embryo sac. The nuclei often fuse and

exhibit large variation in size and shape. Cellularization

occurs quite late and proceeds from the micropyle to the

chalazal end of the embryo sac. They found an extremely wide

range of variation with respect to the time of adventive

embryo initiation and development in polyembryonic mangos.

It waa not uncommon to observe proembryos adjacent to

cotyledonary embryos. The location of the adventive embryos

is also quite variable, but is generally more prevalent in

the micropylar half of the ovule. Nucellar embryos are

commonly observed fused at their radical, resulting in the

production of multiple shoots on a single root.

Sachar and Chopra (1957) also described embryo

development in the monoembryonic Indian cv. Desi. The zygote

remains in a resting state until hundreds of endosperm

nuclei are produced. Two fairly rapid divisions form a

3-celled proembryo. Both the apical and the basal cells










contribute to the formation of the embryo proper with no

organized suspensor.

Several studies have been conducted in mango to

ascertain the fate of the zygotic embryo in the

polyembryonic genotypes (Belling 1908; Juliano, 1934; 1937;

Sachar and Chopra, 1957). In some cultivars, the zygotic

embryo is more or less persistent, but in most cases it

eventually aborts (Sachar and Chopra, 1957). This may be due

to its inferior location within the embryo sac.



Factors Affecting Polyembryony


Because of the economic importance of polyembryony in

Citrus breeding, numerous studies have been conducted on

ways to influence agamospermy in this genus (Traub, 1936;

Furusato et al., 1957; Furusato and Ohta, 1969; Watanabe,

1985a; 1985b). Considerable year to year variation has been

documented and a number of environmental factors have now

been shown to significantly affect the percentage of zygotic

seedlings produced by polyembryonic Citrus cultivars.

Information concerning polyembryony in general is

complicated by the fact that monoembryony does not

necessarily imply sexual reproduction (Ozan et al., 1962).

Particularly in cases where the degree of polyembryony is

low, i.e., the mean number of embryos per seed is only

slightly greater than one, a monoembryonic seed may well

contain a single asexual embryo. To avoid this problem the

more recent studies involving polyembryony in Citrus are









performed using Poncirus trifoliata L. as the pollen parent,

since this species contains a single dominant gene coding

for the easily recognizable trifoliate leaf character.

Traub (1936) was one of the first researchers to report

the artificial control of polyembryony. He indicated that by

decreasing the food supply to fruit-bearing twigs he was

able to decrease the number embryos per seed. Furusato et

al. (1957) found that the mean number of embryos per seed

was significantly higher on the north side of the tree than

on the south side, in older trees than in younger (30 vs 5

years), and in years of high yields than in off years. The

percentage of zygotic to nucellar seedlings has also

reportedly been increased by high temperature greenhouse

incubations after fertilization (Nakatani et al., 1978) and

by flower and fruit bud treatments with plant growth

regulators (PGRs) including: maleic hydrazide (Furusato and

Ohta, 1969), GA (DeLange and Vincent, 1977), and

2,3,5-triiodobenzoic acid (Yoshida, 1979). Watanabe (1985a;

1985b) was unable to significantly reduce the number of

embryos per seed by use of X-rays, NAA, coumarin, or 2,4-D

in addition to any of the previously mentioned PGRs.

Furthermore, the use of high temperatures to reduce embryo

number has not been reproduced (Moore, personal

communication). There have been several references

concerning the use of radiation to reduce polyembryony

(Spiegel-Roy et al., 1972; Ikeda, 1981; Watanbe, 1985a;

1985b). Ikeda (1981) found that a low dose of gamma rays









(1-2 kR) applied for 20 hours to small floral buds (20-30

days pre-anthesis) selectively reduced the number of

nucellar embryos per seed. The developmental period at which

the radiation was supplied was a critical factor. Watanabe

(1985b) obtained a high percentage (86-100%) of

interspecific hybrids by growing artificially pollinated

Citrus trees in a gamma field. In another study Watanabe

(1985a) demonstrated histologically that adventive embryos

were preferentially inhibited by exposure to continuous

gamma irradiation (500 R/day) and that they seldom developed

past the quartet cell stage.

Tisserat and Murashige (1977a; 1977b) isolated a

"graft-transmissible" and "diffusable embryogenic repres-

sant" from monoembryonic citrus cultivars. They found that

the chalazal half of monoembryonic C. medical L. ovules

suppressed embryogenesis in several embryogenic plant

cultures. High levels of ethanol, IAA, ABA, and GA all

suppressed embryogenic activity.

In conclusion, from these studies it is quite clear

that the expression of polyembryony can be significantly

altered by environmental conditions (Furusato et al., 1957;

Watanabe 1985a; 1985b). Generally, conditions which stress

the tree have reduced the degree of polyembryony.



The Genetics of Polyembryony


Several studies attempting to elucidate the inheritance

of polyembryony in Citrus have been conducted (Parlevliet










and Cameron, 1959; Ozan et al., 1962; Cameron and Soost

1979). Parlevliet and Cameron (1959), utilizing several

segregating seedling populations derived from hand

pollinations, sampled some 700 Citrus hybrids. The

monoembryonic cultivars produced only monoembryonic progeny,

whereas the monoembryonic X polyembryonic (MxP) crosses

produced seedling populations exhibiting a wide range of P:M

offspring (8:19-10:0). They postulated a rather simple mode

of inheritance for polyembryony, stating that 1 or possibly

2 dominant or semidominant genes along with possible

modifying genes are responsible for polyembryony. Ozan et

al. (1962) analyzed some 2,000 hybrid seedlings derived from

crosses involving three apparently obligate monoembryonic

Citrus cultivars crossed with the polyembryonic P.

trifoliata. The total percentage of P seeds ranged from

2-8%, while the range of embryos per seed was 2-6. When the

P seeds were grown out, all evidence indicated that the

seeds were not apomictic in origin but rather the result of

cleavage of the zygotic embryo. In a more recent study,

Cameron and Soost (1979) analyzed a population of 121

seedlings derived from 14 crosses involving both M and P

parents. In their discussion, Cameron and Soost, clearly

indicate that their data are in contrast to previous reports

in which generally higher proportions of P offspring are

found. To account for this, they proposed that polyembryony

in Citrus is controlled by two complementary, dominant

genes.









In summary, the mode of inheritence for polyembryony in

Citrus is more complex than was first suspected and does not

appear to be controlled by a single or even two dominant

genes. Numerous crosses have shown that the genetic

background can significantly influence the expression of

polyembryony, implicating the possible role of modifying

genes. Some crosses of P x M Citrus produce only P

offspring, while another cross between monoembryonic parents

appears to have given rise to a polyembryonic offspring

(Cameron and Soost, 1979).

The situation in mango is also unclear due to the lack

of critical genetic studies. LeRoy (1947) proposed that

polyembryony in mango is controlled by 1 or more recessive

genes. Sturrock (1968) also concluded that polyembryony in

mango is recessive and probably controlled by a single gene,

but his conclusions were based on suspected cross

pollinations rather than controlled crosses. Litz and

Schaffer (1987) found that putrescine concentration is much

higher in non-embryogenic nucellar callus of monoembryonic

mangos than in non-embryogenic or embryogenic callus of

polyembryonic cultivars. Litz (1987) speculates that

blocking of S-adenosyl-L-methionine decarboxylase (SAMDC)

activity, which has been found associated with the

accumulation of putrescine and the production of ethylene

may cause ethylene production in mango nucelli. The ethylene

production, which has been shown to inhibit somatic

embryogenesis (Tisserat and Murashige, 1977c), would then









play an important regulating role in the control of

polyembryony in mango.



The Evolutionary Significance of Apomixis


The question often arises as to the evolutionary

significance of adventive embryony and other apomictic forms

of reproduction. Several hypotheses have been proposed

regarding the adaptive significance of apomictic plants

(Marshall and Brown, 1981). One hypothesis referred to as

the "escape from sterility" hypothesis proposes that there

is no selective advantage in agamospermous reproduction over

sexual reproduction. This view stresses that the principal

advantage of apomixis is to restore fertility to sexually

sterile individuals, generally derived from polyploidy and

wide hybridization. In the past this theory was widely

accepted because it was compatible with the theory that

sexual reproduction maximizes the rate of adaptive

evolution. It also provided a ready cause for the close

association of apomixis with polyploidy and interspecific

hybridization. Clausen (1954) was one of the first to

recognize formally that apomixis, particularly facultative

apomixis, does not automatically limit an individual's

capacity for adaptive evolution. He compared the adaptive

advantage of facultative agamospermy to the mass production

of automobiles, in which the production of new genotypes is

permitted at the same time that reproductive fidelity of the

best genotypes can be faithfully maintained. The hypothesis









often referred to as the "Henry Ford" or "Model T"

hypothesis has gained wide acceptance (Marshall and Brown,

1981). Williams (1975) has cited one shortcoming associated

with this hypothesis, i.e., it ignores the cost of meiosis.

Williams explains that the cost of meiosis is incurred for

sexual reproduction at both the individual and at the group

level. At the individual level only one half of the total

genomic complement is passed on to the offspring in sexual

reproduction, while at the population or group level

valuable resources are spent on the production of male

gametes. A third hypothesis has evolved out of recent

quantitative studies which consider the cost of meiosis

(Maynard-Smith, 1978). It has been called the "automatic

advantage" hypothesis because it proposes that apomictic

plants have a twofold advantage over sexual reproduction.

The first advantage is that the apomictic offspring carry

the full genetic complement of their mother. The second

advantage is that no resources are wasted on male gametes;

hence, the cost of meiosis is taken into consideration.



Plant Tissue and Cell Culture


Plant cell and tissue culture has evolved over the past

2 centuries as a result of observations concerning the

response of excised plant parts. Several botanists in the

mid-nineteenth century observed that plants often produce

callus, in response to wounding. The initiation and growth

of this dedifferentiated tissue has proved to be of









particular importance to in vitro culture of plant tissues

and cells.

Some of the first attempts to culture excised plant

tissues were made by the German botanist, Gottlieb

Haberlandt (1854-1945). Haberlandt is credited with the

theory of totipotency, which states that every plant cell,

under the proper conditions, is capable of giving rise to a

whole plant (Haberlandt, 1902). The first successful plant

tissue cultures were derived from excised root tips

(Knudson, 1916). Using a tomato root with an intact

meristem, White (1934) was able, by subculture, to establish

an in vitro continuous culture.

Further advances in the culture of excised plant parts

awaited the discovery of a complex group of natural and

synthetic plant growth substances. Went (1926), using

detached oat coleoptiles, discovered the first plant growth

regulator, indoleacetic acid (IAA). Its subsequent isolation

and chemical characterization (Kogl et al., 1934), led Snow

(1935) to recognize the importance of this naturally

occurring auxin for cell proliferation. The incorporation of

IAA in the culture medium resulted in the first continuous

plant tissue (not organs) cultures in 1939 by White (1939),

Nobecourt (1939), and Gautheret (1939).

Using a medium enriched with liqud coconut endosperm

(LCE), Van Overbeek et al. (1941) were able to culture

immature hybrid embryos of Datura species. The isolation of

the cytokinin, kinetin by Miller et al. (1955) lead Skoog









and Miller (1957) to the recognition that the

differentiation of shoots or roots in tobacco callus was

determined by the ratio of auxin:cytokinin in the culture

medium. Although differentiation in all plant tissue and

cell cultures is apparently not controlled so simply as in

the tobacco system, its dependence on the interplay of

complex plant growth substance and nutrients does still

appear to be valid.

Steward et al. (1958a) and Reinert (1958) were able to

regenerate whole plants via somatic embryogenesis from

carrot suspension cultures. The inherent uncertainty of the

totipotency concept as observed in liquid suspensions

cultures was overcome by Vasil and Hildebrandt (1965) using

tobacco hybrid cells cultured in a suspended drop of medium

enriched with LCE and NAA. Thus, the concept of totipotency,

as envisioned by Haberlandt almost 60 years earlier had been

verified.

Two major pathways are distinguished in the de novo

regeneration of plants in vitro, organogenesis and somatic

embryogenesis. In organogenesis, plants are regenerated via

the separate formation of shoots and roots, without

exhibiting the distinct developmental stages observed in

zygotic embryos. The organs are formed via meristems with

vascular connections to the explant, and are probably not of

single cell origin. The ratio of auxin:cytokinin in the

culture medium is often very important in determining root

or shoot production (Skoog and Miller, 1957). Typically, a









shoot is formed followed by a root, which may or may not

require subculture to a different nutrient medium (Flick et

al., 1983). In contrast, regeneration via somatic

embryogenesis is characterized by the absence of vascular

connection to the maternal tissue of the explant and has

been demonstrated to have a single cell origin. Somatic

embryos pass through developmental stages similar to those

of zygotic embryos, germinate and form plantlets (Ammirato,

1983).



Somatic Embryogenesis


Somatic embryogenesis is the process of embryo

initiation from cells that are not the products of gametic

fusion, i.e., somatic cells (Tisserat et al., 1979).

Tisserat et al. (1979) suggested that adventive and somatic

embryogenesis are valid and useful synonyms for asexual

embryogenesis when referring to the general case, but

apomixis and nucellar embryony should only be used to

describe specific naturally occurring events. In this

manuscript somatic embryogenesis will be used when referring

to the in vitro situation and adventive nucellar

embryogenesis will be used to refer to the in vivo event.

An embryo is an early stage in a plant's development.

It is a bipolar structure with a closed, discrete vascular

system. The closed vascular system implies no maternal

vascular connection, while the bipolarity arises from a

shoot and root meristem at opposite ends. Ammirato (1983)









cautions against the indiscriminate use of the term in vitro

somatic embryogenesis to describe cultures where no

histology has been performed and plantlets have not been

regenerated. Although many systems superficially resemble

somatic embryogenesis, histological studies are sometimes

needed to confirm the lack of vascular connections in the

somatic embryos.

Haberlandt's theory of totipotency (1902) was verified

by Reinert (1958) and Stewart et al. (1958a) who

independently reported somatic embryogenesis in tissue

cultures of carrot (Daucus carota). Since that time in vitro

somatic embryogenesis has been reported in a number of

monocots, dicots, and gymnosperms (Tisserat et al., 1979).

Several theories have been advanced to explain the

phenomenon of in vitro somatic embryogenesis based upon

observations of embryogenic carrot cultures, the model

system for somatic embryogenesis. Sharp et al. (1980) have

arbitarily distinguished 6 theories.

One of the most widely accepted theories postulates

that cells must undergo dedifferentiation before they can

attain embryological competency (Halperin, 1970). Indeed

this seems to be the case in carrot, where a recognizable

callus stage is a prerequisite to somatic embryogenesis.

However, not all embryogenic systems have a distinguishable

dedifferentiated stage, e.g., Citrus (Button et al., 1974)

and Ranunculus (Konar et al., 1972).









Another theory postulates that a cell must be

physiologically isolated from repressive factors of its

neighboring cells. This theory evolved mainly from

observations with the meiocytes of higher plants and has

been corroborated by observation with Citrus (Button et al.,

1974).

A third theory has been developed primarily to account

for observations which are not readily explained by the

theory of dedifferentiation. Street (1978) proposed that

dedifferentiation, i.e., callus formation, itself does not

guarantee embryological competence, but rather the

physiological status of the individual explant cells at the

time of culturing together with the subsequent culture

environment are the primary determinants. This theory is

strongly supported by the embryogenic qualities of nucelli

and other reproductive tissue in general.

A fourth theory emphasizes the importance of

intercellular communication and subsequent cytodifferentia-

tion for somatic embryogenesis (Halperin, 1967). It evolved

from observations on the importance of callus formation

prior to somatic embryogenesis, and has been supported by

observations with cultured protoplasts in which a cell mass

is formed prior to somatic embryogenesis.

The fifth theory hypothesizes that the physiological

status of the explant itself is the primary determinant of

embryological competence (Tisserat et al., 1979). The









culture environment only enhances or represses embryogenic

determination.

Finally, Sharp et al. (1980) have developed a working

hypothesis which attempts to integrate many aspects of the

previously discussed theories. They propose that in vitro

somatic embryogenesis follows 2 major developmental

patterns. In the first pattern somatic embryogenesis occurs

directly from cells in the explant that have been

predetermined to form somatic embryos. The culture

environment can be envisioned as permissively allowing these

predetermined cells to undergo the process of embryogenesis.

Examples of this pattern would be Citrus and mango nucellar

cultures. In contrast, the second pattern of somatic

embryogenesis hypothesizes that the explant must

dedifferentiate before it can be induced in a directed

manner to undergo embryogenesis. An example of indirect

somatic embryogenesis would be the carrot pith cultures. In

both cases the cultured cells are induced to undergo

embryogenesis, but in the first case it is a permissive

induction, while in the latter it is a directive induction.

Sharp's hypothesis appears to delineate a possible role

for plant growth regulators as agents that contribute to the

determination of the cells but that do not directly alter

their embryogenic competence. It also provides some

explanation for the disparities observed in the effects of

auxins on somatic embryogenesis in various plant culture

systems. In the direct pattern of embryogenesis, auxins are









viewed as merely cloning agents which produce multiple

copies of the predetermined cells while in indirect

embryogenesis auxins are envisioned as mitogenic substances

which redetermine the callus cells to an embryogenic state.



Factors Affecting In Vitro Somatic Embryogenesis


Somatic embryogenesis is a dynamic phenomenon

involving many genes and gene complexes, all controlled in a

highly coordinated fashion (Sung et al., 1984). Several

factors have a direct or indirect effect on somatic

embryogenesis, and a number of reviews have addressed this

(Raghavan, 1976a and 1976b; Reinert et al., 1977; Kohlenbach

1977; 1978; Tisserat et al., 1979; Ammirato, 1983; 1984;

Bhojwani and Razdan, 1983; Ozias-Akins and Vasil, 1985).

Plant genotype. Certain taxa have been more amenable to

in vitro somatic embryogenesis, e.g., Umbelliferae, but

within a given species or taxonomic group, individual

genotypes often show pronounced differences in their

embryogenic potential. In alfalfa (Kao and Michaluk, 1980),

clover (Keys et al., 1980), corn (Lu et al., 1982), carrot

(Steward et al., 1975), and indeed almost every culture

system where somatic embryogenesis has been investigated,

significant differences have been found among individual

cultivars (Ammirato, 1983).

Explant source. The explant source and its

physiological qualities are perhaps the most significant

factors in determining whether an embryogenic culture can be









initiated. Other in vitro influences merely enhance or

repress the embryogenic response (Tisserat et al., 1979).

For some plant species, e.g., D. carota, almost any explant

taken at any stage of development can be used to establish

embryogenic cultures. However, for other, more recalcitrant

plant species, choice of explant at an appropriate

developmental stage may be critical, e.g., sweet potato (Liu

and Cantliffe, 1984). Floral tissues, in general, have been

very useful for initiating embryogenic cultures in a number

of plant species (Ammirato, 1983). Ovules of several fruit

species have been successfully cultured, e.g., Citrus

(Kochba and Button, 1974), grape (Srinivasan and Mullins,

1980), apple (Eichholtz, 1979), mango (Litz et al., 1982),

papaya (Litz and Conover, 1982), jaboticaba (Litz, 1984c),

and Eugenia spp. (Litz, 1984a). Zygotic embryos have also

proved to be very useful for initiating embryogenic

cultures, e.g., cotton (Joshi and Johri, 1972), Ranunculus

sceleratus (Sachar and Guha, 1962), and barley (Norstog,

1970). Anthers and microspores are also frequently used,

e.g., Triticale (Sun et al., 1973), Ranunculus sceleratus

(Nataraja and Konar, 1970), Prunus avium (Zenkteler et al.,

1975), Coffea arabica (Sharp et al., 1973), and Datura (Guha

and Maheswari, 1964) The use of nonmaternal tissues, e.g.,

microspores and zygotic embryos, has limited the application

of somatic embryogenesis for propagation and for improvement

of clonally propagated crops. Meristems, e.g., coffee (Sharp

et al., 1973), Ranunculus (Nataraja and Konar, 1970), and









immature vegetative tissue, e.g., millets (Vasil and Vasil,

1981), sorghum (Brettell et al., 1980) are other important

sources of explant material.

The physiological condition of the explant at time of

culturing also significantly affects its embryogenic

potential. In many recalcitrant plant species there appears

to be a rather discrete developmental window, during which

the cells of the explant are receptive to outside influences

and somatic embryogenesis can be induced. With Citrus

Gmitter (1985) found that ovules of C. sinesis Osb. cv.

Hamlin became more responsive with increased fruit maturity.

The size and shape of the explant can also be important.

Explants below a certain minimum size may not have enough

viable cells in contact with the medium (Flick et al.,

1983). The polarity of the explant can also influence its

responsiveness. In Pennisetum, Vasil and Vasil (1981) found

that excised zygotic embryos were most responsive when both

the root and the shoot were in direct contact with the

culture medium. The ploidy level of the explant does not

seem to be a limiting factor, and somatic embryogenesis has

occurred from haploid, diploid, and polyploid tissues.

Plant growth regulators. Perhaps the most widely

studied variable affecting in vitro somatic embryogenesis

has been the kind and quantity of PGRs used in the culture

medium. Of the PGRs, auxin, particularly 2,4-D, has been the

most often cited as being useful for in vitro somatic

embryogenesis. In carrot, callus is maintained on









2,4-D-supplemented media. The callus is transferred to an

auxin-free medium in order to induce somatic embryogenesis

(Halperin and Wetherell, 1964; Halperin, 1966). In general,

the presence of auxin or of an auxin-like substance is

required for the initiation of embryogenic cultures, while

the quantitative lowering or qualitative changing of the

auxin source generally allows somatic embryo growth and

maturation to proceed (Ammirato, 1983). When in vitro

somatic embryogenesis has occurred without auxin in the

culture medium, it has involved tissues that were embryonic,

e.g., cotyledons of Ilex (Hu and Sussex, 1971) or

reproductive, e.g., ovules of Citrus (Kochba and

Spiegel-Roy, 1973). In Citrus and mango (Litz and Schaffer,

1987) it appears that somatic embryogenesis can occur

without the addition of PGRs, but the addition of exogenous

auxins does increase callus proliferation. The addition of

2,4-D to the culture medium is more effective for callus

proliferation in Citrus than either NAA or IAA (Murashige

and Tucker, 1969); however, Moore (1985) and Kochba and

Spiegel-Roy (1977) found that the addition of various auxins

significantly reduces somatic embryogenesis in several

Citrus tissue culture systems even at relatively low

concentrations (0.5 uM). Raghavan (1984a) points out that

many of the confusing results concerning the role of auxins

in somatic embryogenesis are related to the use of diverse

tissues (freshly isolated vs. habituated callus), the size

of the inoculum, and the concentration and type of auxin.









Sharp et al. (1980) make a strong distinction between the

auxin requirement for cultures that undergo indirect somatic

embryogenesis and those that undergo direct embryogenesis,

e.g., Citrus. They concluded that there is an absolute

requirement for exogeneous application of auxin in those

cultures undergoing indirect embryogenesis, and its role is

hypothesized to be one of a directive inducer, while in

systems undergoing direct embryogenesis, there does not seem

to be a strict auxin requirement. The role of auxin in these

systems is thought to be as a cloning agent for

pre-embryogenic determined cells.

Cytokinins are another class of PGRs routinely used in

embryogenic culture systems. Their quantitative interaction

with auxins and their role in developmental control are well

documented in organogenesis (Skoog and Miller, 1957). The

type and concentration of cytokinin often differ in media

used for callus multiplication, embryo induction, and embryo

maturation media. Evans et al. (1981) reported that

cytokinins have been employed in the initiation medium of

over 65% of all crop plants that have been cultured.

Abscisic acid (ABA) has been used successfully in a

large number of embryogenic plant culture systems. It is an

inhibitor of embryo development, whereby it allows embryo

maturation to proceed in a more normal fashion. This

normalizing effect has been reported in caraway (Ammirato,

1974), carrot (Kamada and Harada, 1981), Pennisetum (Vasil

and Vasil, 1981), and Citrus (Kochba et al., 1978).










According to Ammirato (1983) ABA also suppresses secondary

embryo proliferation and precocious germination.

Gibberellins have been used successfully in a few

instances to induce a normal pattern of embryo development

and stimulate germination and shoot elongation, e.g., Citrus

(Kochba et al., 1974), Panicum (Lu and Vasil, 1981), and

corn (Lu et al., 1982). Ethylene and other volatile gases

may exert a significant effect on closed tissue culture

systems but little research has been done to establish these

effects (Ozias-Akins and Vasil, 1985). Ethephon, which

liberates ethylene, was reported to stimulate embryogenesis

in Citrus cultures (Kochba et al., 1978).

Nitrogen. Elemental nitrogen occurs in the highest

concentration of all the macroelements included in most

culture media. The total nitrogen concentration of the

various nutrient media ranges from 3.3 mM in White's to 60.0

mM in MS (sulfur ranges from 1-5 mM while sodium ranges from

0.2-8.0 mM). Of all the mineral nutrients, the form in which

nitrogen (oxidized or reduced, organic or inorganic) is

supplied, probably has the most dramatic effect on both

growth and differentiation of cultured cells (Ozias-Akins

and Vasil, 1985). The importance of total nitrogen and the

form in which it is supplied to in vitro somatic

embryogenesis has been well established (Halperin and

Wetherell, 1965). A direct requirement for nitrogen either

as ammonium or in another of its reduced forms is necessary.










The time at which nitrogen is applied also appears to

be important. Halperin and Wetherell (1965) found that

cultures initiated on a medium supplied with nitrogen in the

nitrate form were less embryogenic than cultures initiated

on media containing the same total nitrogen, but supplied in

the ammonium form. Gamborg et al. (1968) found that when the

total nitrogen requirement of a suspension culture medium is

supplied in the form of the ammonium ion, the pH has a

tendency to drop and may ultimately restrict the

availability of nitrogen. Although the importance of

nitrogen and the form in which it is supplied has been well

established, there seems to be no general formula which

optimizes embryogenesis in all plant tissue culture systems.

Cultured cells can differ in their ability to utilize the

various forms of nitrogen. Carrot cells in vitro

characteristically have a very well developed glutamine

synthetase system (Caldas, 1971). Thus, the reduced forms of

nitrogen may not be as important as in other plant cultures.

The concentration of 2,4-D in the medium can also

significantly alter the culture's ammonium requirement

(Halperin and Wetherell, 1965; Halperin, 1966).

Many plant culture systems respond well to the addition

of reduced forms of nitrogen, i.e., amino acids, to the

culture medium. In general, glutamine and to a lesser

extent, glycine and aspartic acid have been the most useful

amino acids for increasing the efficiency of somatic

embryogenesis (Ammirato, 1983). Complex organic addenda such










as endosperm extracts, casein hydrolysate (CH), and malt and

yeast extracts serve as important sources of amino acids and

other reduced forms of nitrogen (Ozias-Atkins and Vasil,

1985).

Carbohydrates. Carbohydrates serve as a carbon source

for plant cultures, and because they are often present at

such high concentrations (1-12%) they are effective as an

osmoticum. The disaccharide sucrose is usually the most

effective sugar (Verma and Dougall, 1977), but other sugars

are also used. Maltose increases the germination of Citrus

aurantifolia somatic embryos (Miller, 1986). Lactose and

galactose increase somatic embryogenesis in various Citrus

spp. (Kochba et al., 1982), and sorbitol is effective in

Malus cultures (Chong and Taper, 1974). Inositol

(myo-inositol) has only rarely been shown to be essential in

plant tissue culture, e.g., in tobacco (Linsmaier and Skoog,

1965), but it does serve an important role in carbohydrate

metabolism (Loewus and Loewus, 1983) and is routinely

included in the culture medium.

Complex organic addenda. Complex, undefined organic

substances not only are important source of nitrogen, but

also are sources of PGRs, carbohydrates, and vitamins (Dix

and van Staden, 1982). They have also been useful for

somatic embryogenesis. In the early studies with carrot,

Steward et al. (1958a) thought that the addition of LCE to

the culture medium was essential for somatic embryogenesis;

however, efficient somatic embryogenesis has been achieved









on completely defined media (Steward et al., 1964). Many

early plant tissue culture systems using low salt media

relied on the addition of complex, undefined naturally

occurring substances to supplement the simple inorganic salt

formulations being used (Ozias-Atkins and Vasil, 1985).

Various combinations of PGRs, particularly auxins and

cytokinins, can often replace the requirement for complex

organic addenda. Casein hydrolysate was shown to increase

somatic embryogenesis and development in one of the earliest

report of Citrus tissue culture (Maheswari and Rangaswamy,

1958). Malt extract, which has been shown to contain

cytokinins, giberellins, and auxins (Dix and van Staden,

1982), is commonly added to embryogenic cultures of Citrus

(Kochba and Spiegel-Roy, 1973; Moore, 1985).

Other factors. Several undefined gelling agents have

been used to solidify culture media, the most common of

which is agar. The impurities present in agar often are an

important source of trace elements (Heller, 1953). Many

different commercial preparations of agar are now available

which vary significantly in quality. Gelrite gellan gum is a

self-gelling hydrocolloid that forms a transparent gel in

the presence of soluble solids (Pasqualetto et al., 1986).

A number of vitamins have also been used in plant

cultures. Thiamine is the one vitamin which has been

demonstrated repeatedly to significantly affect plant

regeneration (White, 1951; Murashige and Skoog, 1962;

Linsmaier and Skoog, 1965; Gamborg et al., 1968). Other









vitamins included in culture media are pyridoxine, nicotinic

acid, and glycine (Ozias-Akins and Vasil, 1985).

The osmolarity of the culture medium can also affect

somatic embryogenesis and embryo development. Litz (1986)

working with Carica and Wetherell (1984) with carrot found

that pretreatment exposures of embryogenic suspension

cultures to media with high osmolarities (0.3-1.0 M)

followed by subculture to media with lower osmolarities,

resulted in increased levels of somatic embryogenesis. These

results are thought to mimic the changes in osmolarity found

in the endosperm of developing ovules, in which high

concentrations are associated with the earliest stages of

embryogenesis and development (Raghavan, 1976b). High

osmotic concentrations are also thought to suppress organ

differentiation and prevent precocious germination in young

somatic embryos (Ammirato, 1983).

In Citrus, various culture stresses can increase

somatic embryogenesis. 'Shamouti' cultures become more

embryogenic when relatively long culture intervals are used

and when callus is briefly subcultured onto a medium devoid

of sucrose (Kochba and Button, 1974).



Tissue Culture of Mango


The in vitro potential of mango was clearly recognized

by Maheshwari and Rangaswamy (1958); however, no cultures

were actually initiated. The first successful reports of

tissue culture in mango were by Rao et al. (1982) and Litz









et al. (1982). Rao et al. (1982) reported callus initiation

from mango cotyledons on MS medium supplemented with 15%

LCE, 4% sucrose, 27 uM NAA, and 11.5-23 uM kinetin. Cultures

consisted of a dark compact callus that differentiated

prolific roots. Root proliferation continued with subsequent

subcultures, but no shoots or plantlet formation was

observed.

Litz et al. (1982) obtained callus initiation and

somatic embryogenesis from cultured ovules of polyembryonic

mangos collected approximately 2 months post-pollination.

They initiated cultures on a modified solid MS medium with

1/2 strength MS major salts, 6% sucrose, 0.8% Difco-Bacto

agar, 2.73 mM glutamine, 0.57 mM ascorbic acid with or

without 20% filter-sterilized LCE or 4.4-8.8 uM BA. The

nucelli were excised from the cultured ovules after 1-3

weeks and subcultured to fresh media. Embryogenic calli were

transferred to either solid or liquid media containing 20%

LCE. Cultures were incubated in a growth chamber at 25 C

with a 16 hr photoperiod (1000 lux). Liquid cultures were

maintained on a rotary shaker at 100 r.p.m. The most

efficient media for somatic embryogenesis was reported to be

liquid media with 20% LCE. The embryogenic response was

cultivar-dependent with 5 of the 9 polyembryonic cultivars

producing embryogenic callus. No root, shoot, or plantlet

production was reported from somatic embryos transferred to

LCE-free media.









There have been additional reports on in vitro somatic

embryogenesis of mango (Litz, 1984b; Litz et al., 1984; Litz

and Schafer, 1987; Litz, 1987). Nucelli and somatic embryos

were excised from cultured mango ovules 1-2 weeks after

culturing and transferred to a liquid medium containing 9 uM

2,4-D instead of LCE. Cultures were maintained in liquid for

3 months with monthly subcultures, and then transferred to

liquid MS medium without PGRs and with or without 0.5%

activated charcoal. Somatic embryos were transferred to a

solid germination medium supplemented with 0-22 uM BA

together with 0.01% CH, 0.01% ME, or 10% LCE. Activated

charcoal was reported to be deleterious to embryo

maturation. Limited root formation was observed on solid

media supplemented with 4.4-8.8 uM BA (Litz et al., 1984).

Excised nucelli from monoembryonic mango ovules when

cultured on a modified solid MS media (described previously)

supplemented with either 4.5-13.5 uM 2,4-D or to a lesser

extent 5.4-27.0 uM NAA, gave rise to a "loose and friable"

slowly growing callus (Litz, 1984b). Somatic embryos were

reported to have arisen indirectly from the nucelli via

callus formation. This observation contrasted with reports

of in vitro somatic embryogenesis in monoembryonic Citrus

where embryos were reported to have arisen directly from the

nucellus without intermediate callus formation (Rangan et

al., 1968). The cytokinins BA and 2iP were both reported by

Litz (1984b) to be ineffective in initiating callus. The

addition of 20% filter-sterilized LCE to the culture media









somewhat delayed a gradual necrosis that was observed in all

the embryos, while casein hydrolysate (CH), malt extract

(ME), and reducing agents were found to be ineffective.

Alterations in physical culture conditions such as light vs.

dark and solid vs. liquid were also reported to be

ineffective in the alleviation of the gradual necrosis.

Limited germination (root formation) was observed in liquid

media without PGRs. Litz and Schaffer (1987) reported that

although 2,4-D enhances callus formation from mango nucelli,

it does not significantly affect somatic embryogenesis.

There have been no histological studies to verify that

regeneration from mango nucellar callus is via somatic

embryogenesis, although the evidence is persuasive. Somatic

embryogenesis is most efficient in liquid cultures

supplemented with 20% LCE or 2,4-D. Polyembryonic and to a

lesser extent monoembryonic mangos both are capable of

somatic embryogenesis. The response is genotype-dependent.

Limited root growth has been reported but no plantlet or

shoot formation. Embryos exhibited a gradual and progressive

terminal necrosis.
















CHAPTER III
SOMATIC AND ADVENTIVE NUCELLAR EMBRYONY IN MANGO


Introduction


The term somatic embryogenesis has been used throughout

this dissertation to refer to the initiation of embryos from

somatic tissues under in vitro conditions. The terms

adventive embryogenesis and adventive nucellar embryogenesis

have been used only to describe the natural in vivo

production of adventive embryos from cells of the nucellus.

Somatic embryos are generally thought to pass through all

the developmental stages exhibited by zygotic and adventive

embryos in vivo (Tisserat et al., 1979; Ammirato, 1983),

although minor differences in the first series of cell

divisions have been reported with carrot somatic embryos in

comparison to zygotic embryo development (McWilliams et al.,

1974). The most notable difference between somatic embryo

and zygotic embryo development in most plant cultures is the

lack of suspensors in the somatic embryos, e.g., Citrus

(Esan 1973), Daucus (Halperin and Jensen, 1967), and

Ranunculus (Konar et al., 1972). Cotyledon abnormalities

have often been associated with the development of somatic

embryos and include polycotyledony (Halperin, 1964), fused









cotyledons (Rashid and Street, 1973), unequally sized pairs

(Rao 1965), and fasciation (Rao 1965).



Materials and Methods


Flowers and fruitlets were gathered during the 1984 and

1985 season from the mango germplasm collection of the

University of Florida, Tropical Research and Education

Center in Homestead. Ovules over 1 cm in length were

dissected from the fruit and placed directly into the

fixative. The material was fixed and stored in FAA [90:5:5

of (95%) ethanol, formalin, glacial acetic acid].

Embryogenic calli were initiated from excised nucelli

of the polyembryonic 'Parris' on solid, modified MS medium

(callus initiation medium, Table 4-3). Embryogenic nucellar

callus was multiplied in suspension culture on a modified

B-5 medium supplemented with kinetin and 2,4-D (callus

maintenance medium C, Table 4-3). Embryo production,

maturation and germination occurred on a modified B-5 media

solidified with 0.19% Gelrite gellan gum and supplemented

with NAA and 2iP (for embryo production) or 20% liquid

coconut endosperm and 0.025% casein hydrolysate (for

maturation and germination). Callus and somatic embryos used

for histology were killed, fixed, and stored for 48 hours in

FAA (prepared with 70% ethanol).

All material used for light microscopy was removed from

FAA fixative, dehydrated with tertiary butyl alcohol, and

embedded in paraffin (Paraplast) according to Johansen









(1940). Sections were cut at 8-9 uM using an American

Optical rotary microtome and stained in safranin and fast

green.

Clusters of cotyledonary somatic embryos produced on

solid media were used for scanning electron microscopy.

Embryos were harvested and fixed for 1 hour in a solution of

4.25% gluteraldehyde with 0.05 M sodium cacodylate buffer.

The material was then rinsed 3 times for 20 minutes in the

cacodylate buffer. Material was postfixed for 1 hour in a 2%

OsO4 solution with 0.05 M cacodylate buffer. Specimen

fixation was followed by three 20 minute rinses in distilled

H20 and dehydration was accomplished using a 6 step series

of ethanol solutions (25, 50, 75, 95, 100, and 100%). Any

remaining moisture was removed by critical point drying.

Material was gold coated under vacuum and observed with a

Philips scanning electron microscope.



Results and Discussion


Adventive Nucellar Embryony. The mango fruit is a drupe

which contains a large fibrous, hard endocarp enclosing the

seed. Figure 3-1 shows a young mango fruit approximately 75

days post-bloom. At this stage the tough, fiberous endocarp

has not yet developed around the ovule (megasporangium). The

fruit is easily cut open to expose the ovule which is borne

on a funiculus or stalk. The micropyle is just below the

point where the funiculus joins the ovule proper. The

chalazal region is at the opposite end of the ovule.









Mangos possess crassinucellate ovules which are

characterized by having well developed and persistent

nucelli. Figure 3-2 is representive of a polyembryonic mango

ovule approximately 30 days post-bloom. In this 'James

Saigon' ovule (3 mm length) the nucellus has already

undergone considerable degeneration. The nucellus appears as

a layer, several cells thick, consisting of mostly flattened

cells surrounding the entire embryo sac cavity. In this

ovule the nucellus is more developed and persistent near the

micropyle. The endosperm has not become cellularized at this

stage of development and is still in the free-nuclear

condition. It is characteristically a thin line of densely

staining nuclei and cytoplasm which is appressed against the

nucellus by the central vacuole. This free-nuclear endosperm

appears to be more concentrated in the micropylar region of

the ovule. The adventive nucellar embryos are a small

cluster of densely stained cells at the extreme micropylar

end of the embryo sac cavity (Figures 3-2; 3-3). In these

micrographs the micropyle is clearly defined. The epidermal

layer of the the integument lines the micropyle and

delineates the cells of the nucellus. In Figure 3-3 six

adventive embryos can be clearly distinguished. They range

from a two-celled proembryo to a twenty-four-celled early

globular stage adventive embryo. In the earliest stages of

adventive nucellar embryony, the proembryos contain cells

with rich densely staining cytoplasm, thin cell walls,

little vacuolation, and prominent nuclei. At approximately









45 days post bloom the adventive nucellar embryos have

enlarged and protrude into the embryo sac cavity as observed

in the 7 mm length 'Parris' ovule (Figure 3-4). At this

stage the endosperm has become fully cellularized. Adventive

nucellar embryos at the early to late globular stage are

demonstrated in Figures 3-5 and 3-6. The 0.5 mm length

adventive embryo in Figure 3-6 possesses a well developed

epidermis; however, no embryonic organs or vascularization

are distinguishable.

In Vitro Somatic Embryony. Embryogenic nucellar callus

can be initiated from both the micropylar and the chalazal

portions of excised nucelli cultured on solid callus

initiation medium (Table 4-3, see Figure 3-7). Whatever

factors limit adventive embryogenesis in the chalazal

portion of the nucellus in vivo are apparently not present

or are inhibited in vitro. This is in contrast with the

initiation of Citrus nucellar cultures as reported by

Kobayashi et al. (1979), in which the morphologically

distinct primordiumm cells" of the adventive embryos

proliferate to form embryogenic ovular callus in

polyembryonic Citrus cultures.

Embryogenic mango nucellar callus is compact,

nonfriable and globular in form (Figure 3-8). It consists of

numerous light-dark brown globules (0.5-2.0 mm diameter)

with cream-white somatic embryos 2-5 mm in length arising

from the periphery. When this callus is transferred to









liquid medium (callus maintenance medium, Table 4-3), it

proliferates rapidly as globular callus (Figure 3-9).

Somatic embryogenesis from globular callus grown in

suspension is restricted primarily to the epidermal layer of

the globular callus or somatic embryos. The epidermis of

globular callus or somatic embryos prior to the initiation

of embryogenesis is a smooth and continuous layer of cells

completely surrounding the somatic embryo. The cells are

highly vacuolated without densely staining cytoplasm (Figure

3-10). Eventually the epidermis begins to redifferentiate

and is no longer a continuous single cell layer (Figure

3-11). Numerous embryonic initials, i.e., cells with densely

staining cytoplasm are visible. As dedifferentiation

proceeds the layer of densely staining cells with prominent

nuclei becomes thicker (Figure 3-12), causing the somatic

embryos to have a rough and bumpy appearance at low

magnification. The process of dedifferentiation of somatic

embryos from the epidermal region of a late globular stage

somatic embryos is shown in Figure 3-13. Numerous distinct

somatic embryos from the single cell to the octet stage of

embryo development can be observed. Subsequently, more

advanced stages of somatic embryo development become evident

(Figures 3-14 and 3-15). In Figure 3-15 a globular stage

somatic embryo with a distinct epidermis is shown.

The embryogenic 'Parris' suspension cultures used in

the regeneration experiments of Chapters IV and V have a

rather distinctive growth morphology. Proliferating globular









'Parris' callus in suspension appears very differentiated.

Cultures consist of clusters of somatic embryos 0.5-1.5 cm

in diameter which have formed via the budding of secondary

embryos (Figures 3-16 and 3-17). These clusters of budding

somatic embryos contain a distinct central core (Figure

3-18), which consists of a late globular to early torpedo

stage somatic embryo. Numerous somatic embryos at various

stages of development radiate from the outer portions of the

epidermis-derived cell layer. The somatic embryos which form

the central core are still composed of living tissue.

However, the nonembryogenic epidermis-derived regions of the

somatic embryos become necrotic (Figure 3-18). The necrotic

portions of the epidermis along with some of the underlying

cell layers are sloughed off (Figure 3-19).

Somatic embryos produced in suspension culture exhibit

a number of developmental abnormalities, some of which are

evident at very early stages (Figures 3-20 and 3-21).

Globular somatic embryos without a distinct epidermis are

prevalent. Moreover, their overall morphology appears to be

less compact than typical adventive embryos (Figures 3-5 and

3-6).

Fewer developmental abnormalities occur when somatic

embryos are produced on solid medium even though

polycotyledony and unequal sized cotyledons were present

(Figure 3-22). After the differentiation of cotyledons on

embryogenesis medium (Table 4-3) the bipolar nature of the

regenerated somatic embryos together with their closed





48



vascular system was apparent (Figure 3-23). Thus, the nature

of regeneration via somatic embryogenesis was unequivocally

demonstrated.










































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CHAPTER IV
CALLUS MAINTENANCE AND SOMATIC EMBRYOGENESIS IN MANGO


Introduction


The efficient regeneration of plants via somatic

embryogenesis is partially dependent on the production of

large quantities of uniform, embryogenically competent

callus. This callus must then be induced by means of the

culture environment to undergo efficient and preferably

synchronized embryogenesis. Control of somatic embryo

maturation and germination is a final prerequisite for

normal plantlet formation.

In chapters IV and V a series of experiments are

described that addresses somatic embryogenesis and plant

recovery in mango. In Chapter IV, callus production and

somatic embryogenesis are considered. In Chapter V

parameters that affect the maturation and germination of the

somatic embryos are considered.

Callus production parameters have been quantified by

both biomass production and by measuring the embryogenic

competence of the callus produced. Somatic embryogenesis

parameters have been quantified by total cotylodonary embryo

production. Ratings have also been used to evaluate the









somatic embryos based on absence of necrotic or vitrified

regions, large relative size (5-8 mm in length), single

cotyledons without fasciations, and no secondary embryo

proliferation.



Materials and Methods


Immature mango fruits of both monoembryonic and

polyembryonic cultivars were harvested 2-3 months post-

pollination from the mango germplasm collections of the

University of Florida Tropical Research and Education Center

in Homestead and from the U.S.D.A. Subtropical Horticultural

Research Unit in Miami, Florida. Surface-sterilization of

the fruitlets was accomplished using the following protocol:

10 minutes in 70% ethanol, 30 minutes in a 1% sodium

hypochlorite solution with 2-3 drops of Tween 20 or Tween

80, followed by 2 rinses in sterile distilled water. Ovules

were dissected from the fruitlets using sterile forceps and

scapels. Embryogenic callus was initiated from whole

immature ovules, immature ovule halves without embryo(s),

zygotic embryos, nucellar embryos, and excised nucelli. All

callus was initiated on a modified solid Murashige and Skoog

(MS) medium consisting of 1/2 concentration major salts

(Table 4-1), MS minor salts (Table 4-2), 6% sucrose, 0.8%

Difco Bacto-agar, 2.74 mM (400 mg 1-1) glutamine, 0.55 mM

(100 mg 1-1) inositol, 0.57 mM (100 mg 1-1) ascorbic acid,

1.2 uM (0.4 mg 1-1) thiamine HC1, and 4.5 uM (1 mg 1-1)

2,4-D (callus initiation medium, Table 4-3). All media were










Table 4-1. Basal media major salts formulations
used with in vitro culture of mango.


Formulation mM(mg 1-1)

Major Salts MSz 1/2 MS WPMy B-5x

NH4NO3 20.6(1650) 10.3(825) 5.0(400)
KNO3 18.8(1900) 9.4(950) 25.0(2500)
Ca(N03)2 4H20 2.4(556)
K2SO4 -- (990)
MgSO4 7H20 1.5(370) 1.8(185) 1.5(370) 1.0(250)
CaC12 2H20 3.0(440) 1.5(220) 0.7(96)
KH2PO4 1.3(170) 0.6(85) 1.3(170)
NaH2PO4 H20 1.1(150)

ZMurashige and Skoog, 1962.
YWoody Plant Media, Lloyd and McCown, 1982.
Gamborg B-5, Gamborg, et al., 1968, without
(NH4)2SO4.






Table 4-2. Murashige and Skoog minor salts formulation.


Minor saltsz uM(mg 1-1) Minor salts uM(mg 1-1)

Na2EDTA 100(37.3) KI 5.0(0.830)
FeSO4 7H20 100(27.8) Na2MoO4 2H20 1.0(0.250)
MnSO4 4H20 100(22.3) CoSO4 6H20 0.1(0.025)
ZnSO4 7H20 30(8.6) CuS04 5H20 0.1(0.025)
H3B03 100(6.2)

ZMurashige and Skoog, 1962.










adjusted to pH 5.75 using HC1, KOH, or NaOH prior to
-2
autoclaving at 121 C and 1.1 kg cm for 18 minutes. Plant

growth regulators (PGRs) were dissolved in 1N HC1, KOH, NaOH

or 95% ethanol according to Table A-i.

Explants were cultured on sterile medium in 60 X 15 mm

plastic petri dishes, stored in clear plastic boxes and

incubated either in growth chambers at a constant
-i
temperature of 25 C, with a 16 hr photoperiod (40 umol s
-2
m ) provided by cool-white fluorescent lamps or at ambient

room temperature without supplemental light. Embryogenic

callus was established from both monoembryonic and

polyembryonic mango cultivars (Table A-2).

Original explants were subcultured onto fresh medium

several days after the initial culturing to remove the

tissues from oxidation products that had accumulated in the

media. They were subcultured again 1 week later and

subsequently at 3-4 week intervals.

One hundred to 500 mg of excised proliferating callus,

visible 1-3 months after culturing, was transferred to

fresh, solid callus initiation medium (Table 4-3) or

directly into liquid medium for multiplication and

maintenance. Liquid cultures consisted of 50 ml of basal

medium (BM) in 125 ml Erlenmeyer flasks maintained at 100

r.p.m. on rotary shakers. Liquid callus cultures were

subcultured at 2 week intervals until the fresh weight of

the callus was approximately 10 g per flask. Cultures were





















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o *
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(0



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- I


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-I










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then subdivided into 2 flasks of 5 g callus per flask with

fresh medium.



Experiment 4-1: In Vitro Somatic Embryogenesis from Mango
Nucellar Callus in Response to Genotype and Plant Growth
Regulators

Calli from 10 mango cultivars (8 polyembryonic, 2

monoembryonic), initiated 3 months previously on solid media

(Table 4-3), were multiplied in liquid medium of the

composition. Treatments consisted of 200 mg callus

inoculated onto 12 ml of sterile BM in 60 X 15 mm plastic

petri dishes supplemented with 1 or more combinations of the

following PGRs, 2,4-D, NAA, IBA, or IAA at 0, 5, 10, or 20

uM and kinetin, 2iP, or BA at 0, 5, 20, or 80 uM. In

addition to the various synthetic PGRs, 3 complex organic

growth addenda were used, 20% (v/v) liquid coconut endosperm

(LCE), 0.015% (w/v) casein hydrolysate (CH), and 0.05% (w/v)

yeast extract (YE). All PGRs were prepared according to

Table A-i.

A modified 10 X 10 X 13 unreplicated factorial

experimental design was used, with 10 cultivars and 130 PGR

treatment combinations plus 6 treatment combinations with

complex organic addenda. Petri dishes were stacked in clear

plastic boxes and incubated in a growth chamber at a

constant temperature of 25 C, with a 16 hr photoperiod

provided by low intensity illumination (40 umol s- m )

from cool-white fluorescent lamps. The total number of









somatic cotyledonary embryos per dish was determined 2

months after inoculation.



Experiment 4-2: In Vitro Somatic Embryogenesis from 'Parris'
Nucellar Callus in Response to the Callus Maintenance
Medium and Major Salts Formulations

One-year-old embryogenic 'Parris' callus, initiated and

maintained on a solid modified MS medium (callus initiation

medium, Table 4-3) was transferred to liquid medium for

callus multiplication. Based on the results from Experiment

4-1 2 liquid callus maintenance media formulations were

tested for their effects on somatic embryogenesis. Callus

maintenance medium A (Table 4-3) was supplemented with 4.5

uM (1 mg 1-1) 2,4-D and 18.6 uM (4 mg 1-1) kinetin. Callus

maintenance medium B (Table 4-3) was supplemented with 4.5
-i -1
uM (1 mg 1-1) 2,4-D, 4.6 uM (1 mg 1-1) kinetin, and 2.05 mM

(300 mg 1-) glutamine. Both callus maintenance media

formulations contained the same BM consisting of: 1/2 MS

major salts, MS minor salts, 5% sucrose, 0.55 mM (100 mg

1-1) inositol, and 1.2 uM (0.4 mg 1-1) thiamine HC1.

Four BM major salts formulations MS, 1/2 MS, Lloyd

McCown Woody Plant Medium (WPM), and a modified Gamborg B-5

(B-5) were tested for their effect on somatic embryogenesis

(Table 4-1). All embryogenesis media contained: MS minor

salts, 5% sucrose, 0.75% Difco Bacto-agar, 2.05 mM (300 mg

1-1) glutamine, 0.55 mM (100 mg 1-1) inositol, 53.3 uM (4 mg

1-1) glycine, 3.0 uM (1 mg 1-1) thiamine HCI, 4.1 uM (0.5 mg

1-1) nicotinic acid, 2.4 uM (0.5 mg 11) pyridoxine HC1,










10.8 uM (2 mg 1-1) NAA, and 4.9 uM (1 mg 1-1) 2iP

(embryogenesis medium, Table 4-3).

Callus (300 mg/inoculum) was inoculated onto 12 ml of

solid sterile embryogenesis medium in 60 X 15 mm plastic

petri dishes. Cultures were incubated at room temperature

(24-26 C) in a clear plastic box with no supplemental light.

The experimental design was a 2 X 4 factorial with 6

replicates. Treatments were scored 45 days after inoculation

by counting the total number of somatic cotyledonary embryos

produced.



Experiment 4-3: In Vitro Somatic Embryogenesis from 'Parris'
Nucellar Callus in Response to the Callus Maintenance
Medium Formulation and Sucrose Concentration

One-year-old embryogenic 'Parris' callus, initiated and

maintained on a solid modified MS medium (callus initiation

medium, Table 4-3), was transferred to liquid medium for

callus multiplication. Two callus maintenance media

formulations, A and B (Table 4-3) were tested for their

effects on somatic embryogenesis.

Six sucrose concentrations: 0, 2, 3, 4, 5, and 6% were

also tested for their effect on somatic embryogenesis. Only

sucrose concentrations and callus maintenance medium

formulations were altered between treatments. All

embryogenesis media contained the same BM components

(embryogenesis medium, Table 4-3) and were solidified with

0.75% (w/v) Difco Bacto-agar.









Callus (300 mg/inoculum) was inoculated onto 12 ml of

solid sterile embryogenesis medium in 60 X 15 mm plastic

petri dishes. Cultures were incubated at room temperature

(24-26 C) in clear plastic boxes with no supplemental light.

The experimental design was a 2 X 6 factorial with 5

replicates. Treatments were scored 45 days after inoculation

by counting the total number of somatic cotyledonary

embryos.



Experiment 4-4: Callus Production from Nonembryogenic
'James Saigon' Nucellar Callus in Response to the Major
Salts Formulation of the Callus Maintenance Medium

One-year-old nonembryogenic 'James Saigon' callus was

initiated and maintained on a solid modified MS medium

(callus initiation medium, Table 4-3). Callus was then

multiplied in liquid BM of the same formulation but without

any supplemental PGRs. Only major salts were altered between

treatments. All callus maintenance media contained MS minor

salts, 5% sucrose, 2.05 mM (300 mg 1-1) glutamine, 0.55 mM

(100 mg 1-1) inositol, 53.3 uM (4 mg 1-1) glycine, 3.0 uM (1

mg 1-1) thiamine HC1, 4.1 uM (0.5 mg 1-1) nicotinic acid,

and 2.4 uM (0.5 mg 1-) pyridoxin HC1 (callus maintenance

medium C, Table 4-3).

One gram of callus was used to inoculate 50 ml of

liquid callus maintenance medium in 125 ml Erlenmeyer

flasks. Cultures were incubated at room temperature (24-26

C) in a transparent plastic box without supplemental light

at 100 r.p.m. on rotary shakers. The callus was subcultured









every 2 weeks during the first month and every week during

the second month. The experiment was of a completely

randomized design with 4 treatments and 5 replicates.

Treatments were scored 60 days after inoculation by weighing

the total callus production (fresh weight) per flask.



Experiment 4-5: In Vitro Somatic Embryogenesis from
'James Saigon' Nucellar Callus in Response to the
Solidifing Agent Used in the Embryogenesis Medium

Two year old embryogenic 'James Saigon' callus that was

initiated on a solid modified MS medium (callus initiation

medium, Table 4-3) was maintained on a solid modified MS

medium. The solid callus maintenance medium consisted of 1/4

MS major salts, 1/2 MS minor salts, 2.5% sucrose, 1.0 mM

(150 mg 11) glutamine, 0.27 mM (50 mg 1-1) inositol, 100 uM

(27.8 mg 1-1) FeSO4 7H20, and 1.2 uM (0.4 mg 1-1) thiamine

HC1. Embryogenic callus was transferred to a liquid modified

B-5 medium for callus multiplication (callus maintenance

medium C, Table 4-3).

Two gelling agents, Difco Bacto-agar at 0.7% and

Gelrite gellan gum at 0.18%, were tested for their effect on

somatic embryogenesis. Only the gelling agent was altered

between treatments. All embryogenesis media contained the

same BM components [embryogenesis medium (without 2,4-D),

Table 4-3].

Callus (300 mg/inoculum) was inoculated onto 25 ml of

solid sterile embryogenesis medium in 100 X 15 mm plastic

petri dishes. Cultures were incubated at room temperature









(24-26 C) in clear plastic boxes with no supplemental light.

The experimental design was a completely randomized design

with 2 treatments and 8 replicates. Treatments were scored

45 days after inoculation by counting the total number of

somatic cotyledonary embryos.



Experiment 4-6: In Vitro Production of 'James Saigon'
Nucellar Callus in Response to a Liquid or a Solid
Maintenance Medium

Two-year-old embryogenic 'James Saigon' callus that was

initiated on a solid modified MS medium (callus initiation

medium, Table 4-3) was maintained on a solid modified MS

medium consisting of 1/4 MS major salts, 1/2 MS minor salts,

2.5% sucrose, 1.0 mM (150 mg 1-1) glutamine, 0.27 mM (50 mg

1-1) inositol, 100 uM (27.8 mg 1-1) FeSO4 7H20, and 1.2 uM

(0.4 mg 1-1) thiamine HC1. Embryogenic callus was

transferred to a liquid modified B-5 medium (callus

maintenance medium C, Table 4-3) for multiplication.

Two culture regimes, liquid and solid, were tested for

their effects on callus production. Two gelling agents,

Gelrite gellan gum at 0.2% and Sigma agar gum at 0.7% were

used to solidify the callus maintenance medium. All callus

maintenance media contained the same BM components (callus

maintenance medium C, Table 4-3).

Callus (300 mg/inoculum) was inoculated onto 12 ml of

solid sterile callus maintenance medium in 60 X 15 mm

plastic petri dishes or into 50 ml of sterile liquid BM in

125 ml Erlenmeyer flasks. Solid callus cultures were










incubated at room temperature (24-26 C) in clear plastic

boxes without supplemental light. Liquid callus cultures

were also maintained at room temperature without

supplemental light on a rotary shaker at 100 r.p.m.. Solid

cultures were subcultured twice at 3 week intrervals

supplemental light on a rotary shaker at 100 r.p.m.. Solid

cultures were subcultured twice at 3 week intervals, while

liquid shaker cultures were subcultured twice at the

beginning of the 4th and 6th week after inoculation. The

experimental design was a completely randomized design with

3 treatments and 10 replicates. Treatments were scored 60

days after inoculation by weighing the total callus

production (fresh weight) per flask.



Results and Discussion


Experiment 4-1: In Vitro Somatic Embryogenesis from Mango
Nucellar Callus in Response to Genotype and Plant
Growth Regulators

The results are shown in Tables 4-6 through 4-9 and A-3

through A-6. The total number of cotyledonary embryos

produced per plate (from 200 mg of inoculum on 12 ml of BM),

when averaged within genotypes over all 130 treatments

ranged from 31.0 ('James Saigon') to 0.0 ('Gadong')[Table

4-8]. Treatment responses were highly genotype-dependent

with no single treatment or treatment combination giving the

optimal response for all genotypes tested. 'James Saigon'

produced embryos on almost all treatment combinations

tested, even on treatments lacking PGRs (Table 4-4). Trends





















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Full Text
IN VITRO SOMATIC EMBRYOGENESIS AND PLANT REGENERATION
FROM MANGO (Manqifera indica L.) NUCELLAR CALLUS
BY
STEPHEN GREGORY DEWALD
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

This dissertation is dedicated to my cherished
and loving wife Maria, who has served as a constant
of inspiration and support.
friend
source

ACKNOWLEDGEMENTS
The author expresses his sincere thanks and gratitude
to the members of his advisory committee, Drs. Gloria A.
Moore, Wayne B. Sherman, Robert J. Knight, Jr., Prem S.
Chourey, and especially to his major advisor Dr. Richard E.
Litz.
Many thanks go to his parents Stephen and Ruth Anne,
brother Joseph, and the rest of the DeWald family for their
support and encouragement.
Finally, the author extends his appreciation to the
Florida Mango Forum for their financial support and to the
graduate students, the service staff, and the faculty of the
University of Florida Fruit Crops Department, Gainesville,
and the Tropical Research and Education Center, Homestead,
for the many ways in which they have helped.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES viii
LIST OF FIGURES xi
KEY TO ABBREVIATIONS xiii
ABSTRACT xiv
CHAPTER
I INTRODUCTION 1
II LITERATURE REVIEW 3
Introduction 3
Mango: Taxonomy, Distribution, and
Breeding Efforts.. 3
Polyembryony 9
Histological Studies of Mango Embryology 12
Factors Affecting Polyembryony 15
The Genetics of Polyembryony 17
The Evolutionary Significance of Apomixis 20
Plant Tissue and Cell Culture 21
Somatic Embryogenesis 24
Factors Affecting Iri Vitro
Somatic Embryogenesis 28
Tissue Culture of Mango 37
III SOMATIC AND ADVENTIVE NUCELLAR EMBRYONY
IN MANGO 41
Introduction 41
Materials and Methods 42
Results and Discussion 43
IV CALLUS MAINTENANCE AND SOMATIC
EMBRYOGENESIS IN MANGO 7 2
Introduction 72
Materials and Methods 73
iv

CHAPTER
Page
Experiment 4-1: rn Vitro Somatic
Embryogenesis from Mango Nucellar
Callus in Response to Genotype and
Plant Growth Regulators 77
Experiment 4-2: I_n Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium and Major Salts
Formulations 7 8
Experiment 4-3: In. Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium Formulation
and Sucrose Concentration 79
Experiment 4-4: Callus Production from
Nonembryogenic 'James Saigon'
Nucellar Callus in Response to the
Major Salts Formulation of the
Callus Maintenance Medium 80
Experiment 4-5: I_n Vitro Somatic
Embryogenesis from 'James Saigon'
Nucellar Callus in Response to the
Solidifying Agent Used in the
Embryogenesis Medium 81
Experiment 4-6: In. Vitro Production
of 'James Saigon' Nucellar Callus in
Response to a Liguid or a Solid
Maintenance Medium 82
Results and Discussion 83
Experiment 4-1: rn Vitro Somatic
Embryogenesis from Mango Nucellar
Callus in Response to Genotype and
Plant Growth Regulators 83
Experiment 4-2: In Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium and Major Salts
Formulations 92
Experiment 4-3: I_n Vitro Somatic
Embryogenesis from 'Parris' Nucellar
Callus in Response to the Callus
Maintenance Medium Formulation
and Sucrose Concentration 96
Experiment 4-4: Callus Production from
Nonembryogenic 'James Saigon'
Nucellar Callus in Response to
the Major Salts Formulation of the
Callus Maintenance Medium 98
V

CHAPTER
Page
Experiment 4-5: Ln Vitro Somatic
Embryogenesis from 'James Saigon'
Nucellar Callus in Response to the
Solidifying Agent Used in the
Embryogenesis Medium 98
Experiment 4-6: In. Vitro Production
of 'James Saigon' Nucellar Callus in
Response to a Liquid or a Solid
Maintenance Medium 100
Conclusions 102
V SOMATIC EMBRYO MATURATION, GERMINATION,
AND PLANTLET FORMATION 105
Introduction 105
Materials and Methods 106
Experiment 5-1: Ln Vitro Somatic
Embryo Production and Maturation
from 'James Saigon' Nucellar Callus
in Response to Culture Regime 106
Experiment 5-2: Ln Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and ABA in
a Liquid Embryo Maturation Medium 107
Experiment 5-3: Ln Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and
Supplements to the Embryo Maturation
Medium 108
Experiment 5-4: _In Vitro 'Parris'
Somatic Embryo Maturation in Response
to Solidifying Agent and ABA in the
Embryo Maturation Medium Ill
Experiment 5-5: I_n Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation 113
Experiment 5-6: I_n Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation 114
Results and Discussion 116
Experiment 5-1: Ln Vitro Somatic
Embryo Production and Maturation
from 'James Saigon' Nucellar Callus
in Response to Culture Regime 116
Experiment 5-2: Ini Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and ABA in
a Liquid Embryo Maturation Medium 118

CHAPTER Page
Experiment 5-3: In. Vitro 'Parris'
Somatic Embryo Maturation in Response
to Sucrose Concentration and
Supplements to the Embryo Maturation
Medium 120
Experiment 5-4: Ln Vitro 'Parris'
Somatic Embryo Maturation in Response
to Solidifying Agent and ABA in the
Embryo Maturation Medium 122
Experiment 5-5: In Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation 124
Experiment 5-6: I_n Vitro 'Parris'
Somatic Embryo Germination and Shoot
Formation in Response to the Embryo
Germination Medium Formulation 126
Conclusions 128
VI SUMMARY AND CONCLUSIONS 134
Histological Investigations 134
Tissue Culture Studies 136
Conclusions 138
APPENDIX 140
LITERATURE CITED 148
BIOGRAPHICAL SKETCH 162
vii

LIST OF TABLES
Table Page
4-1 Basal media major salts formulations used with
in vitro culture of mango 74
4-2 Murashige and Skoog minor salts formulation 74
4-3 Basal medium components and supplements used for
in vitro culture of mango 76
4-4 III vitro somatic embryogenesis from
'James Saigon' nucellar callus in response to
various plant growth regulators 84
4-5 Ln vitro somatic embryogenesis from 'Parris'
nucellar callus in response to various plant
growth regulators 85
4-6 I_n vitro somatic embryogenesis from
'Tommy Atkins' nucellar callus in response to
various plant growth regulators 86
4-7 I_n vitro somatic embryogenesis from 'Heart'
nucellar callus in response to various plant
growth regulators 87
4-8 In. vitro somatic embryogenesis from mango
nucellar callus for cultivars in experiment 1 88
4-9 Ln vitro somatic embryogenesis in response
to various complex organic addenda 88
4-10 I_n vitro somatic embryogenesis from 'Parris'
nucellar callus in response to the callus
maintenance medium and major salts formulations..•.93
4-11 In. vitro somatic embryogenesis from 'Parris'
nucellar callus in response to the callus
maintenance medium formulation and sucrose
concentration 9 7
4-12 Callus production from nonembryogenic
'James Saigon' nucellar callus in response to
the major salts formulation of the callus
maintenance medium 99
viii

Table Page
4-13 Ln vitro somatic embryogenesis from
'James Saigon' nucellar callus in response to
the gelling agent used in the embryogenesis
medium 99
4-14 Iri vitro production of 'James Saigon' nucellar
callus in response to liquid or solid callus
maintenance medium 101
5-1 I_n vitro somatic embryo maturation rating
scale used in experiment 5-3 110
5-2 Ln vitro somatic embryo maturation rating
scale used in experiment 5-4 112
5-3 Ln vitro somatic embryo production
and maturation from 'James Saigon' nucellar
callus in response to the culture regime 117
5-4 Ln vitro 'Parris' somatic embryo maturation
in response to sucrose concentration and ABA
in liquid embryo maturation medium 119
5-5 I_n vitro 'Parris' somatic embryo maturation
in response to sucrose concentration and
supplements to the embryo maturation medium 121
5-6 Iri vitro 'Parris' somatic embryo maturation
in response to the solidifying agent and ABA in
the embryo maturation medium 123
5-7 Iri vitro 'Parris' somatic embryo germination
and shoot formation in response to the embryo
germination medium formulation 125
5-8 I_n vitro 'Parris' somatic embryo germination
and shoot formation in response to the embryo
germination medium formulation 125
A-l Plant growth regulators (PGRs) used with
in vitro culture of mango 140
A-2 List of mango cultivars with successful
in vitro nucellar callus initiation 141
A-3 Ln vitro somatic embryogenesis from 'Simmonds'
nucellar callus in response to various plant
growth regulators 142

Table Page
A-4 Ln vitro somatic embryogenesis from 'Florigon'
nucellar callus in response to various plant
growth regulators 143
A-5 Ln vitro somatic embryogenesis from
callus of 'Cambodiana' in response to
various plant growth regulators 144
A-6 I_n vitro somatic embryogenesis from
callus of 'Irwin' in response to various plant
growth regulators 145
A-7 Peach embryo germination medium 146
x

LIST OF FIGURES
Figure Page
3-1 A mango fruitlet approximately 75 days-post-
bloom bisected longitudinally with intact
ovule (megasporangium) 49
3-2 Longitudinal section through a polyembryonic
ovule approximately 30 days-post-bloom 50
3-3 Micropylar region of a polyembryonic ovule 51
3-4 Longitudinal section through the micropylar
half of a polyembryonic ovule approximately
45 days-post-bloom 52
3-5 Globular stage adventive nucellar embryos 53
3-6 Late globular stage adventive nucellar embryo 54
3-7 The initiation of embryogenic nucellar callus 55
3-8 Close-up view of embryogenic mango nucellar
callus initiation 56
3-9 Suspension culture of rapidly proliferating
mango nucellar callus 57
3-10 The epidermis of a late globular stage somatic
embryo prior to redifferentiation and somatic
embryogenesis 58
3-11 The epidermis of a late globular stage somatic
embryo begining redifferentiate 59
3-12 Early stages of epidermal somatic embryogenesis.... 60
3-13 Section through the periphery of a redif¬
ferentiating globular somatic embryo or callus 61
3-14 Four early globular stage somatic embryos 62
3-15 A globular stage somatic embryo beginning to
differentiate an epidermis and showing signs
of polarity
63

Figure Page
3-16 A suspension culture of budding 'Parris'
somatic embryos 64
3-17 Close-up of a cluster of budding 'Parris'
somatic embryos grown in suspension culture 65
3-18 Cross section, at low magnification through a
cluster of budding somatic embryos grown in
suspension culture 66
3-19 A section through the periphery of the
central core of a budding cluster of somatic
embryos grown in suspension culture 67
3-20 Globular stage somatic embryo grown in
suspension culture exhibiting aberrant
development 68
3-21 Globular stage somatic embryos grown in
suspension culture with a poorly formed
epidermis 69
3-22 Scanning electron micrograph of a cluster
of 'Parris' somatic embryos grown on solid
medium 7 0
3-23 Longitudinal section through the base
of a somatic embryo grown on solid medium 71
4-1 'Parris' somatic embryogenesis in response to
major salts formulations of experiment 4-2 104
5-1 'Parris' somatic embryo maturation in
response to solidifying agents and ABA of
experiment 5-4 130
5-2 A culture of germinating 'Parris' somatic
embryos from experiment 5-6 131
5-3 Germinated 'Parris' somatic embryos ready
to be transferred to the soil 132
5-4 Young 'Parris' plants regenerated via
somatic embryogenesis growing in the soil 133
xii

KEY TO ABBREVIATIONS
ABA:
abscisic acid
BA:
6-benzylaminopurine (N® benzyladenine)
BM:
basal medium
B-5
Gamborg et al., B-5 basal medium
CH:
casein hydrolysate
GA:
gibberellic acid
IAA:
indole-3-acetic acid
IBA:
indole-3-butyric acid
LCE:
liquid coconut endosperm (coconut water)
M:
monoembryonic seed
MS:
Murashige and Skoog basal medium formulation
NAA:
2-naphthaleneacetic acid
Na2EDTA:
disodium ethylenediaminetetraacetic acid
P:
polyembryonic seed
YE:
yeast extract
WPM:
Lloyd and McCown woody plant medium
2iP:
(2-isopenteny1)adenine
2,4-D:
(2,4-dichlorophenoxy)acetic acid
xiii

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
IN VITRO SOMATIC EMBRYOGENESIS AND PLANT REGENERATION
FROM MANGO (Mangifera indica L.) NUCELLAR CALLUS
BY
STEPHEN GREGORY DEWALD
May, 1987
Chairman: Richard E. Litz
Major Department: Horticultural Science (Fruit Crops)
Histological studies were conducted on polyembryonic
mango ovules and embryogenic nucellar callus to elucidate
the patterns of embryogenesis in these two systems. The
nucellar origin as well as the variable location of
adventive embryos within the micropylar portion of the
ovules was demonstrated. Adventive nucellar embryos,
characterized by densely staining cytoplasm and prominent
nuclei, were formed prior to the cellularization of the
endosperm.
Embryogenic callus was initiated from both the
micropylar and chalazal portions of excised nucelli. In
suspension culture this callus grew as compact, nonfriable
globular callus. Histological preparations of this callus
revealed that no true, undifferentiated, subculturable
tissue is produced. Proliferation occurs via secondary
budding from the epidermis of preformed somatic embryos.
xiv

Prior to somatic embryogenesis the epidermis
dedifferentiates and forms a layer, several cells thick, of
cytoplasmically dense cells with prominent nuclei. In
suspension culture, somatic embryos exhibited numerous
developmental abnormalities including poorly formed
epidermal layers, vascular system and meristems, noncompact
morphology, and secondary budding. More normal embryo
development occurred on solid medium. Somatic embryos were
shown to possess bipolar meristems with closed vascular
systems, thus uneguivocally demonstrating the embryogenic
nature of plant regeneration from mango nucellar callus.
The morphogenic response of mango nucellar callus was
also investigated with emphasis on factors influencing
callus production, somatic embryogenesis, embryo maturation,
and germination. The addition of 2 mM glutamine, 5 uM 2,4-D,
5 uM kinetin, and vitamins to suspension multiplication
cultures improved the embryogenic efficiency of callus upon
transfer to a solid embryogenesis medium. Efficient somatic
embryogenesis occurred on a modified B-5 medium [lacking
(NH^)2^0.] supplemented with 5% (w/v) sucrose, 2 mM
glutamine, 11 uM NAA, 5 uM 2iP, vitamins, and amino acids.
The use of Gelrite gellan gum (0.19%, w/v) improved the
efficiency of somatic embryogenesis and maturation in
comparison to medium solidified with Difco Bacto-agar or
Sigma agar gum. Maturation and germination occurred on solid
media in which the sucrose concentration was sequentially
lowered and the synthetic plant growth regulators were
XV

0.025%
replaced with 20% (v/v) liquid coconut endosperm and
(w/v) casine hydrolysate. Plant's were established in
soil by watering with dilute basal salts.
the
xvi

CHAPTER I
INTRODUCTION
Mango is one of the world's most important tropical
fruit crops. At the present time, production in Florida is
restricted to approximately 1000 ha of mature mango groves
(IFAS, 1983). However, despite its relatively small size in
Florida's fruit industry, mango production, both here and
abroad, should continue to expand (Bondad, 1980). One of the
major factors limiting the distribution and production of
this delicious tropical fruit crop is the lack of well
adapted, high yielding, disease resistant cultivars with
good quality fruit. Most of the industry throughout the
world is based on the use of chance seedlings maintained
clonally by means of various asexual propagation techniques.
Breeding attempts have been particularly discouraging in
comparison to many other agronomic crops.
Recent advances in biotechnology offer the plant
breeder new and exciting opportunities in cultivar
improvement and development. One of these new
biotechnologies is the field of plant cell and tissue
culture. Plant cell and tissue culture has been used
successfully to propagate large numbers of plants, eliminate
persistent pathogens, create new and economically useful
1

2
variation in existing well established genotypes, rescue
hybrid embryos from wide crosses, create homozygous breeding
lines via haploidy, produce economically important primary
and secondary plant metabolites, and elucidate complex
processes at both the cell and whole plant level. Plant
tissue culture and its associated technologies should be
particularly useful to breeders working with perennial
crops, where cultivar improvement has been slow and
difficult. Unfortunately, the woody perennials as a whole
have proved to be recalcitrant _in vitro.
In vitro somatic embryogenesis from mango nucellar
cultures was first reported by Litz et al_. (1982); however,
the recovery of plants from mango somatic embryos has not
been reported. The first objective of this research was to
examine histologically the processes of in vitro somatic
embryogenesis and adventive nucellar polyembryony in order
to determine more clearly the regeneration pathway in mango.
The second objective of this research was to investigate and
optimize in vitro parameters that affect somatic
embryogenesis, embryo development, and germination, thereby
developing an efficient in vitro plant regeneration system.

CHAPTER II
LITERATURE REVIEW
Introduction
The purpose of this literature review is to introduce
the reader to the mango, its embryology, and in vitro
somatic embryogenesis. Polyembryony and somatic
embryogenesis are of particular importance to this
dissertation and will be discussed in detail. Somatic
embryogenesis in Citrus, is similar to that in mango. Citrus
In vitro culture is perhaps the best understood system for
somatic embryogenesis in woody plant species, and numerous
articles have been published since embryogenic cultures were
established by Stevenson in 1956. For these reasons the
literature pertinent to Citrus is emphasized throughout this
review.
Mango: Taxonomy, Distribution, and Breeding Efforts
Mango is one of the choicest and most popular of the
tropical fruits. With an annual world production of nearly
14 million metric tons, mango is the fifth most important
fruit crop after grape, Musa, Citrus, and apples (FAO
Production Yearbook, 1984).
3

4
The culture of mango is most intensive in India, which
accounts for 2/3 of the total world production. Singh (1960)
reports that it has been under cultivation there for almost
6,000 years. Mangos are particularly well suited to India
where they .thrive in almost every region except altitudes
above 3,000 feet (Singh, 1960). In India, the mango is
referred to as the "king of fruits" and has played an
integral part in India's folklore and religions.
Mango, Mangifera indica L., is a member of the
Anacardiaceae, a large family of mostly tropical woody
perennial plants with inconspicuous flowers often produced
in large clusters, frequently bearing attractive and edible
fruit (Purseglove, 1968). The family contains some 64 genera
and includes several economically important fruit and nut
trees, e.g., cashew (Anacardium occidentale L.), a flesh
fruit (Bouea macrophy1la Griff.), pistachio (Pistacia vera
L.), several edible spondias (Spondias cythera Sonn., S.
mombin L., S. purpurea L.), and some important plant pests,
e.g., poison ivy (Rhus toxicodendron L.) and the Brazilian
pepper tree (Schinus terebinthifolius Radd.) (Popenoe, 1920;
Purseglove, 1968).
The genus Mangifera contains some 41-62 invariably
arborescent species (Mukherjee, 1972; Singh, 1969)
characterized by long, leathery leaves with a
fiberous-resinous fruit. The trees are evergreen with
several distinctive annual growth cycles or flushes. The
brownish-red to red vegetative flushes are unique in that

5
they occur in sectors rather than all over the tree during
any one cycle (Popenoe, 1920) .
Mango flowers are small and generally borne on terminal
panicles. The inflorescence is polygamous producing up to
4,000 staminate and perfect flowers per panicle. The fruit
is a large, fleshy drupe consisting of an edible mesocarp
and a fiberous, stony endocarp. Mango is often grouped into
2 major categories based on embryology: monoembryonic types
contain only a single embryo per seed, while the
polyembryonic types produce multiple (usually asexual)
embryos per seed. Almost without exception the Indian mangos
are monoembryonic, while the Indochinese and Philippines
types are polyembryonic (Singh, 1976). Young and Sauls
(1979) state that many monoembryonic Indian types produce
brightly colored fruit with an attractive blush and are
often susceptible to anthracnose, [Glomerella cingulata
Stonem (S. and V.S.)]. Polyembryonic types more commonly
produce a pale green to yellow, low fibered fruit and
generally possess higher levels of resistance to
anthracnose.
Mukherjee (1950, 1972) determined that the basic
chromosome number in mango and its related species is 2n=40.
Mukherjee hypothesized that mango is a polyploid because it
has a high chromosome number with many nucleolar
chromosomes. Citing secondary meiotic associations, he
further hypothesized that a certain primitive mango type or

6
types probably arose through allopolyploidy and most likely
via amphidiploidy.
Most authors seem to agree that the center of origin
for mango is the Assam-Burma region (Indo-Burma), where
mangos can still be found growing wild (Singh, 1969). The
Mangifera genus probably originated in Burma, Thailand,
Indo-China, and the Malay Peninsula (Singh, 1960). A marked
center of diversity occurs in the region of
Indochina-Malaysia-Indonesia (Singh, 1976) . Wild mangos
generally have fruit that is extremely fiberous-resinous and
may even be poisonous in some species of Mangifera.
Selection has been for fruits with succulence, low fiber,
high sugars, small stones, and low resin content (Singh,
1976).
Mangos were probably introduced from southern India to
Malaysia around 500 B.C., and to eastern Africa in the 10th
century by Arab merchants (Purseglove, 1968). Mukherjee
(1972) believes that mangos did not reach the Pacific
islands until the sixteenth century when the Portuguese
began opening trade routes. They are often given credit for
the extensive spread of mangos throughout the tropics
(Mukherjee, 1972; Popenoe, 1920). Their far-reaching trade
routes linked the Indian ports with the Persian gulf,
Pacific islands, and the African coast. Although the date is
uncertain, the Portuguese are reported to have brought the
Indian, monoembryonic type mangos to Rio de Janeiro around
1700 (Mukherjee, 1972). In Mexico, the earliest mango

7
introductions were probably polyembryonic, Indochinese
seedlings that arrived via the Spanish trade routes between
Manila and the Pacific ports of Mexico (Malo, 1977) .
Henry Perrine probably first introduced mangos into the
United States from Campeche, Mexico in 1833 (Popenoe, 1920).
Rolfs (1915) divides the early history of mango cultivar
development in Florida into 2 periods. The first period is
characterized by the growing of seedling mangos by a few
dedicated and ingenious horticulturists and lasted from 1867
until the end of the 19th century (Knight, personnal
communication). These mangos were polyembryonic types such
as 'Turpentine' and 'No. 11' (Young and Sauls, 1979). The
second period is characterized by the planting of grafted or
budded trees and began with the noteworthy introduction of
the monoembryonic 'Mulgoba' in 1889 (Rolfs, 1915).
'Mulgoba', an Indian cultivar brought to Florida by Van
Deman of the U.S. Department of Agriculture, was the female
parent of the open pollinated seedling 'Haden'. The
commercial potential of 'Haden' was at once realized and it
has served as the standard by which all other Florida
cultivars have been compared.
Recent surveys indicate close to 1,000 ha of commercial
mango orchards in South Florida (IFAS, 1983). In 1977 over
50% percent of the total acreage was devoted to 'Tommy
Atkins' and 'Keitt' (Malo, 1977). Other imporatant cultivars
include 'Irwin', 'Kent', 'Van Dyke', 'Jubilee', 'Sensation',
'Palmer', and 'Haden'.

8
Mango breeding was initiated early in this century in
India and has been reviewed by Mukherjee et a_l. (1968),
Singh (1959), and Singh (1969). Unfortunately, the results
from these breeding efforts have been rather discouraging.
Singh (1959) lists some of the major problems associated
with the breeding of mango in India, e.g., long generation
times (6 years or more), one seed per fruit, high levels of
heterozygosity which makes hybrid performance unpredictable,
unique floral morphology and excessive fruit drop which
makes controlled hybridizations extremely difficult, and the
large field plots needed to grow segregating seedling
populations.
Mukherjee (1976) outlined 3 major methods for cultivar
improvement in mango. The first method is selection from
natural seedlings. This was the way that most of the early
Indian cultivars arose and it is still widely practiced in
developing nations. The second method is selection from
open-pollinated seedling progenies. This technique has been
successfully employed in Florida and Hawaii. Mukherjee
considers that this is still the most promising method for
cultivar development. The final method of improvement is
through controlled hybridization. The major problem seems to
be the overall difficulty in producing large populations of
hybrid seedlings. Hand pollinations have very low success
rates (0.3%) (Singh, 1969). These rates can be improved
somewhat if a relatively small number (40-50) of freshly
opened flowers per panicle are pollinated and the rest

9
removed (Mukherjee, 1976). Singh (1969) states that the most
important variable in obtaining large numbers of hybrid
seeds is the total number of panicles worked per tree and
not the total number of flowers. One method to allow
mass-pollination that has been employed in Florida with some
success is the "cage method." Parents are enclosed within a
screen tent, and pollination is accomplished by placing a
fly infested animal carcass within the enclosure (personal
communication, Knight).
Polyembryony can also restrict gene flow in controlled
hybridizations. The problem is not as pronounced in mango as
it is in Citrus, because most of the high quality Indian and
Florida cultivars are monoembryonic. Knight (1970)
recommends the increased usage of polyembryonic types and
cites favorable characteristics of fruit quality, higher
levels of disease resistance, regular high yields, and ease
of propagation. Spontaneous somatic mutations are another
source of improved cultivars and have given rise to
1Davis-Haden'.
Polyembryony
Polyembryony, the production of multiple embryos within
a single seed, is widespread but sporadically distributed
among the spermatophytes (Gustaffson, 1946). It was first
recorded by Leeuwenhoek in 1719 when he observed orange
seeds containing multiple embryos (Maheshwari, 1950).
Polyembryony is quite common in the gymnosperms but usually

10
involves some form of cleavage polyembryony, i.e., division
of the zygote or proembryo into 2 or more units (Maheshwari,
1950). It is less widespread in the angiosperms, but
exhibits more diversity in its expression. In the
angiosperms adventive embryos may originate from the egg
cell or synergids. This may occur within a single reduced or
unreduced embryo sac of an ovule, either following or
without fertilization. It may also occur from a fertilized
egg cell through the cleavage of the zygote or proembryo
(cleavage polyembryony) or directly from the cells of the
nucellus or inner integument of an ovule, i.e., adventitious
polyembryony (Brizicky, 1964). If the embryos are not a
result of the normal sexual process then polyembryony may be
correctly referred to as a form of apomixis, termed
agamospermy (Stebbins, 1941). Apomictic polyembryony can be
either gametophytic (diplospory or apospory, parthenogenesis
or apogamety) or sporophytic (adventitious embryony)
(Webber, 1940; Maheshwari and Sachar, 1963).
Ernst (1918) and Schnarf (1929) (cited in Webber, 1940)
reviewed the old literature on polyembryony and
distinguished 2 main types: true and false. Their
distinction was based on whether or not the embryos arise
from or protrude into the same or different embryo sacs.
Webber (1940) considered these distinctions purely arbitrary
with no physiological or natural basis.
Polyembryony has been most widely studied in the genus
Citrus. Early reports indicated that the adventive embryos

11
arise in the nucellus shortly after the first division of
the zygote or sometime later (Bacchi, 1943; Rangan et ad.,
1969). Substantial evidence now seems to indicate that
proembryos or proembryo initials are present in the
peripheral cells of polyembryonic nucelli prior to anthesis
(Esen and Soost, 1977; Kobayashi et al., 1979). Kobayashi et
al. (1981), studying some 74 Citrus cultivars, found
distinct primordial cells to be present at the time of
flowering in all of the polyembryonic cultivars, but did not
observe these cells in any of the monoembryonic cultivars
studied. Esen and Soost (1977) concluded that although
adventive nucellar embryogenesis seems to be independent of
pollination or fertilization, subsequent embryo development
and maturation require at least fertilization and division
of the polar nuclei (endosperm). They added that previous
reports of adventive embryo development in the absence of
fertilization (Webber, 1930; Wright, 1936) were probably in
error.
Survival is a function of the relative vigor of one
embryo and its location in respect to available nutrients
within the embryo sac (Esen and Soost, 1977). Numerous
studies in Citrus have shown that, in general, the location
of the nucellar embryos provides a competitive advantage for
their survival relative to that of the zygotic embryo (Ohta
and Furusato, 1957; Frost and Soost, 1968). Similarly, it
has been found that the more nucellar embryos initiated per
ovule the less likely it is that the sexual embryo will

12
survive to maturity (Frost, 1926). Frost (1926) indicated
that there is evidence of heterotic vigor in zygotic embryos
from wide crosses and that selection of parents can
significantly influence the survival chances of the zygote
relative to that of its asexual neighbors.
Histological Studies of Mango Embryology
Polyembryony in mango was first recorded by Schact in
1859 (Belling, 1908). The nucellar origin of the adventive
embryos in both Citrus and mango was elucidated by
Strasburger in 1878 (cited by Belling, 1908) . Juliano and
Cuevas (1932) described megasporogenesis in the mango cv.
Pico. In 'Pico', an important polyembryonic Philippine
cultivar, very few perfect flowers of the panicle contain
functional megasporangia at the time of flowering. The
megasporangium (ovule) is formed from an outgrowth of the
inner carpellary wall. It grows at approximately the same
rate as the carpel until fertilization, at which time there
is a dramatic enlargement of the carpel walls. Unilateral
growth patterns give rise to a bitegmic, crassinucellate,
anatropous ovule contained within a single unicarpous pistil
(Juliano and Cuevas, 1932). A single hypodermal cell in the
nucellus functions as the archesporial cell. This
archesporial cell does not undergo meiosis directly, but
rather divides periclinally to form an outer primary
parietal cell and an inner primary sporagenous cell
(megaspore mother cell). The primary parietal cell

13
subsquently forms an extensive nucellus while the megaspore
mother cell is pushed deeper into the developing ovule. The
megaspore mother cell differentiates and becomes distinct
from the rest of the nucellus, with its densely staining
cytoplasm and large polygonal shape (Juliano and Cuevas,
1932) .
It is at this time, apparently, that certain cells of
the nucellus, especially in the region of the micropyle
surrounding the megaspore mother cell, become
distinguishable as proembryo initials by their densely
staining cytoplasm (Belling, 1908). The megaspore mother
cell enters into meiosis that results in the formation of a
linear tetrad of daughter cells. The chalazal-most megaspore
daughter cell becomes functional (thereby pushing itself
deeper into the nucellus) and undergoes megagametogenesis to
give rise to the normal 8-celled embryo sac. The polar
nuclei, at maturity, occupy a position very close to the egg
cell apparatus.
Belling (1908) reports that at the time of
fertilization, in the polyembryonic mango 'No. 11', dense
protoplasmic nucellar cells, separated from the embryo sac
by a layer of flattened cells, can be distinguished.
Immediately following double fertilization, while still in
the flower stage, the triploid endosperm nucleus starts
dividing. These divisions take place well before any
divisions of the zygotic or adventive embryos. In fact, the
first embryonic divisions cannot be detected until the

14
ovules are more then 3 mm long (fruit 7 mm). As the
endosperm develops, the nucleated protoplasm becomes
appressed to the embryo sac wall opposite the places where
the adventive embryos are forming. Belling (1908) contrasts
the cellularization of the endosperm in Citrus and mango
stating that it occurs much earlier in Citrus than in mango.
Sachar and Chopra (1957) studied endosperm development
in a large number of monoembryonic and polyembryonic mangos,
concluding that the endosperm is of the nuclear type.
Numerous free nuclei are formed and distributed along the
periphery of the embryo sac. The nuclei often fuse and
exhibit large variation in size and shape. Cellularization
occurs quite late and proceeds from the micropyle to the
chalazal end of the embryo sac. They found an extremely wide
range of variation with respect to the time of adventive
embryo initiation and development in polyembryonic mangos.
It waa not uncommon to observe proembryos adjacent to
cotyledonary embryos. The location of the adventive embryos
is also quite variable, but is generally more prevalent in
the micropylar half of the ovule. Nucellar embryos are
commonly observed fused at their radical, resulting in the
production of multiple shoots on a single root.
Sachar and Chopra (1957) also described embryo
development in the monoembryonic Indian cv. Desi. The zygote
remains in a resting state until hundreds of endosperm
nuclei are produced. Two fairly rapid divisions form a
3-celled proembryo. Both the apical and the basal cells

15
contribute to the formation of the embryo proper with no
organized suspensor.
Several studies have been conducted in mango to
ascertain the fate of the zygotic embryo in the
polyembryonic genotypes (Belling 1908; Juliano, 1934; 1937;
Sachar and Chopra, 1957). In some cultivars, the zygotic
embryo is more or less persistent, but in most cases it
eventually aborts (Sachar and Chopra, 1957). This may be due
to its inferior location within the embryo sac.
Factors Affecting Polyembryony
Because of the economic importance of polyembryony in
Citrus breeding, numerous studies have been conducted on
ways to influence agamospermy in this genus (Traub, 1936;
Furusato et al., 1957; Furusato and Ohta, 1969; Watanabe,
1985a; 1985b). Considerable year to year variation has been
documented and a number of enviromental factors have now
been shown to significantly affect the percentage of zygotic
seedlings produced by polyembryonic Citrus cultivars.
Information concerning polyembryony in general is
complicated by the fact that monoembryony does not
necessarily imply sexual reproduction (Ozan et ad., 1962).
Particularly in cases where the degree of polyembryony is
low, i.e., the mean number of embryos per seed is only
slightly greater than one, a monoembryonic seed may well
contain a single asexual embryo. To avoid this problem the
more recent studies involving polyembryony in Citrus are

16
performed using Poncirus trifoliata L. as the pollen parent,
since this species contains a single dominant gene coding
for the easily recognizable trifoliate leaf character.
Traub (1936) was one of the first researchers to report
the artifical control of polyembryony. He indicated that by
decreasing the food supply to fruit-bearing twigs he was
able to decrease the number embryos per seed. Furusato £t
al. (1957) found that the mean number of embryos per seed
was significantly higher on the north side of the tree than
on the south side, in older trees than in younger (30 vs 5
years), and in years of high yields than in off years. The
percentage of zygotic to nucellar seedlings has also
reportedly been increased by high temperature greenhouse
incubations after fertilization (Nakatani et aJ. , 1978) and
by flower and fruit bud treatments with plant growth
regulators (PGRs) including: maleic hydrazide (Furusato and
Ohta, 1969), GA (DeLange and Vincent, 1977), and
2,3,5-triiodobenzoic acid (Yoshida, 1979). Watanabe (1985a;
1985b) was unable to significantly reduce the number of
embryos per seed by use of X-rays, NAA, coumarin, or 2,4-D
in addition to any of the previously mentioned PGRs.
Furthermore, the use of high temperatures to reduce embryo
number has not been reproduced (Moore, personal
communication). There have been several references
concerning the use of radiation to reduce polyembryony
(Spiegel-Roy et al_. , 1972; Ikeda, 1981; Watanbe, 1985a;
1985b). Ikeda (1981) found that a low dose of gamma rays

17
(1-2 kR) applied for 20 hours to small floral buds (20-30
days pre-anthesis) selectively reduced the number of
nucellar embryos per seed. The developmental period at which
the radiation was supplied was a critical factor. Watanabe
(1985b) obtained a high percentage (86-100%) of
interspecific hybrids by growing artifically pollinated
Citrus trees in a gamma field. In another study Watanabe
(1985a) demonstrated histologically that adventive embryos
were preferentially inhibited by exposure to continuous
gamma irradiation (500 R/day) and that they seldom developed
past the guartet cell stage.
Tisserat and Murashige (1977a; 1977b) isolated a
"graft-transmissible" and "diffusable embryogenic repres-
sant" from monoembryonic citrus cultivars. They found that
the chalazal half of monoembryonic C. medica L. ovules
suppressed embryogenesis in several embryogenic plant
cultures. High levels of ethanol, IAA, ABA, and GA all
suppressed embryogenic activity.
In conclusion, from these studies it is quite clear
that the expression of polyembryony can be significantly
altered by environmental conditions (Furusato et al., 1957;
Watanabe 1985a; 1985b). Generally, conditions which stress
the tree have reduced the degree of polyembryony.
The Genetics of Polyembryony
Several studies attempting to elucidate the inheritance
of polyembryony in Citrus have been conducted (Parlevliet

18
and Cameron, 1959; Ozan et a_l. , 1962; Cameron and Soost
1979). Parlevliet and Cameron (1959), utilizing several
segregating seedling populations derived from hand
pollinations, sampled some 700 Citrus hybrids. The
monoembryonic cultivars produced only monoembryonic progeny,
whereas the monoembryonic X polyembryonic (MxP) crosses
produced seedling populations exhibiting a wide range of P:M
offspring (8:19-10:0). They postulated a rather simple mode
of inheritance for polyembryony, stating that 1 or possibly
2 dominant or semidominant genes along with possible
modifying genes are responsible for polyembryony. Ozan et
al. (1962) analyzed some 2,000 hybrid seedlings derived from
crosses involving three apparently obligate monoembryonic
Citrus cultivars crossed with the polyembryonic P.
trifoliata. The total percentage of P seeds ranged from
2-8%, while the range of embryos per seed was 2-6. When the
P seeds were grown out, all evidence indicated that the
seeds were not apomictic in origin but rather the result of
cleavage of the zygotic embryo. In a more recent study,
Cameron and Soost (1979) analyzed a population of 121
seedlings derived from 14 crosses involving both M and P
parents. In their discussion, Cameron and Soost, clearly
indicate that their data are in contrast to previous reports
in which generally higher proportions of P offspring are
found. To account for this, they proposed that polyembryony
in Citrus is controlled by two complementary, dominant
genes.

19
In summary, the mode of inheritence for polyembryony in
Citrus is more complex than was first suspected and does not
appear to be controlled by a single or even two dominant
genes. Numerous crosses have shown that the genetic
background can significantly influence the expression of
polyembryony, implicating the possible role of modifying
genes. Some crosses of P x M Citrus produce only P
offspring, while another cross between monoembryonic parents
appears to have given rise to a polyembryonic offspring
(Cameron and Soost, 1979).
The situation in mango is also unclear due to the lack
of critical genetic studies. LeRoy (1947) proposed that
polyembryony in mango is controlled by 1 or more recessive
genes. Sturrock (1968) also concluded that polyembryony in
mango is recessive and probably controlled by a single gene,
but his conclusions were based on suspected cross
pollinations rather than controlled crosses. Litz and
Schaffer (1987) found that putrescine concentration is much
higher in non-embryogenic nucellar callus of monoembryonic
mangos than in non-embryogenic or embryogenic callus of
polyembryonic cultivars. Litz (1987) speculates that
blocking of S-adenosy1-L-methionine decarboxylase (SAMDC)
activity, which has been found associated with the
accumulation of putrescine and the production of ethylene
may cause ethylene production in mango nucelli. The ethylene
production, which has been shown to inhibit somatic
embryogenesis (Tisserat. and Murashige, 1977c), would then

20
play an important regulating role in the control of
polyembryony in mango.
The Evolutionary Significance of Apomixis
The question often arises as to the evolutionary
significance of adventive embryony and other apomictic forms
of reproduction. Several hypotheses have been proposed
regarding the adaptive significance of apomictic plants
(Marshall and Brown, 1981). One hypothesis referred to as
the "escape from sterility" hypothesis proposes that there
is no selective advantage in agamospermous reproduction over
sexual reproduction. This view stresses that the principal
advantage of apomixis is to restore fertility to sexually
sterile individuals, generally derived from polyploidy and
wide hybridization. In the past this theory was widely
accepted because it was compatible with the theory that
sexual reproduction maximizes the rate of adaptive
evolution. It also provided a ready cause for the close
association of apomixis with polyploidy and interspecific
hybridization. Clausen (1954) was one of the first to
recognize formally that apomixis, particularly facultative
apomixis, does not automatically limit an individual's
capacity for adaptive evolution. He compared the adaptive
advantage of facultative agamospermy to the mass production
of automobiles, in which the production of new genotypes is
permitted at the same time that reproductive fidelity of the
best genotypes can be faithfully maintained. The hypothesis

21
often referred to as the "Henry Ford" or "Model T"
hypothesis has gained wide acceptance (Marshall and Brown,
1981). Williams (1975) has cited one shortcoming associated
with this hypothesis, i.e., it ignores the cost of meiosis.
Williams explains that the cost of meiosis is incurred for
sexual reproduction at both the individual and at the group
level. At the individual level only one half of the total
genomic complement is passed on to the offspring in sexual
reproduction, while at the population or group level
valuable resources are spent on the production of male
gametes. A third hypothesis has evolved out of recent
quantitative studies which consider the cost of meiosis
(Maynard-Smith, 1978). It has been called the "automatic
advantage" hypothesis because it proposes that apomictic
plants have a twofold advantage over sexual reproduction.
The first advantage is that the apomictic offspring carry
the full genetic complement of their mother. The second
advantage is that no resources are wasted on male gametes;
hence, the cost of meiosis is taken into consideration.
Plant Tissue and Cell Culture
Plant cell and tissue culture has evolved over the past
2 centuries as a result of observations concerning the
response of excised plant parts. Several botanists in the
mid-nineteenth century observed that plants often produce
callus, in response to wounding. The initiation and growth
of this dedifferentiated tissue has proved to be of

22
particular importance to in vitro culture of plant tissues
and cells.
Some of the first attempts to culture excised plant
tissues were made by the German botanist, Gottlieb
Haberlandt (1854-1945). Haberlandt is credited with the
theory of totipotency, which states that every plant cell,
under the proper conditions, is capable of giving rise to a
whole plant (Haberlandt, 1902). The first successful plant
tissue cultures were derived from excised root tips
(Knudson, 1916). Using a tomato root with an intact
meristem, White (1934) was able, by subculture, to establish
an in vitro continuous culture.
Further advances in the culture of excised plant parts
awaited the discovery of a complex group of natural and
synthetic plant growth substances. Went (1926), using
detached oat coleoptiles, discovered the first plant growth
regulator, indoleacetic acid (IAA). Its subsequent isolation
and chemical characterization (Kogl et al. , 1934), led Snow
(1935) to recognize the importance of this naturally
occurring auxin for cell proliferation. The incorporation of
IAA in the culture medium resulted in the first continuous
plant tissue (not organs) cultures in 1939 by White (1939),
Nobecourt (1939), and Gautheret (1939).
Using a medium enriched with liqud coconut endosperm
(LCE) , Van Overbeek et a_l. (1941) were able to culture
immature hybrid embryos of Datura species. The isolation of
the cytokinin, kinetin by Miller et a_l. (1955) lead Skoog

23
and Miller (1957) to the recognition that the
differentiation of shoots or roots in tobacco callus was
determined by the ratio of auxin:cytokinin in the culture
medium. Although differentiation in all plant tissue and
cell cultures is apparently not controlled so simply as in
the tobacco system, its dependence on the interplay of
complex plant growth substance and nutrients does still
appear to be valid.
Steward et al. (1958a) and Reinert (1958) were able to
regenerate whole plants via somatic embryogenesis from
carrot suspension cultures. The inherent uncertainty of the
totipotency concept as observed in liquid suspensions
cultures was overcome by Vasil and Hildebrandt (1965) using
tobacco hybrid cells cultured in a suspended drop of medium
enriched with LCE and NAA. Thus, the concept of totipotency,
as envisioned by Haberlandt almost 60 years earlier had been
verified.
Two major pathways are distinguished in the de novo
regeneration of plants in vitro, organogenesis and somatic
embryogenesis. In organogenesis, plants are regenerated via
the separate formation of shoots and roots, without
exhibiting the distinct developmental stages observed in
zygotic embryos. The organs are formed via meristems with
vascular connections to the explant, and are probably not of
single cell origin. The ratio of auxin:cytokinin in the
culture medium is often very important in determining root
or shoot production (Skoog and Miller, 1957). Typically, a

24
shoot is formed followed by a root, which may or may not
require subculture to a different nutrient medium (Flick et
al. , 1983). In contrast, regeneration via somatic
embryogenesis is characterized by the absence of vascular
connection to the maternal tissue of the explant and has
been demonstrated to have a single cell origin. Somatic
embryos pass through developmental stages similar to those
of zygotic embryos, germinate and form plantlets (Ammirato,
1983) .
Somatic Embryogenesis
Somatic embryogenesis is the process of embryo
initiation from cells that are not the products of gametic
fusion, i.e., somatic cells (Tisserat et al., 1979).
Tisserat et a_l. (1979) suggested that adventive and somatic
embryogenesis are valid and useful synonyms for asexual
embryogenesis when referring to the general case, but
apomixis and nucellar embryony should only be used to
describe specific naturally occurring events. In this
manuscript somatic embryogenesis will be used when referring
to the in vitro situation and adventive nucellar
embryogenesis will be used to refer to the in vivo event.
An embryo is an early stage in a plant's development.
It is a bipolar structure with a closed, discrete vascular
system. The closed vascular system implies no maternal
vascular connection, while the bipolarity arises from a
shoot and root meristem at opposite ends. Ammirato (1983)

25
cautions against the indiscriminate use of the term _in vitro
somatic embryogenesis to describe cultures where no
histology has been performed and plantlets have not been
regenerated. Although many systems superficially resemble
somatic embryogenesis, histological studies are sometimes
needed to confirm the lack of vascular connections in the
somatic embryos.
Haberlandt's theory of totipotency (1902) was verified
by Reinert (1958) and Stewart et al. (1958a) who
independently reported somatic embryogenesis in tissue
cultures of carrot (Daucus carota). Since that time in vitro
somatic embryogenesis has been reported in a number of
monocots, dicots, and gymnosperms (Tisserat et al., 1979).
Several theories have been advanced to explain the
phenomenon of in vitro somatic embryogenesis based upon
observations of embryogenic carrot cultures, the model
system for somatic embryogenesis. Sharp et aK (1980) have
arbitarily distinguished 6 theories.
One of the most widely accepted theories postulates
that cells must undergo dedifferentiation before they can
attain embryological competency (Halperin, 1970). Indeed
this seems to be the case in carrot, where a recognizable
callus stage is a prerequisite to somatic embryogenesis.
However, not all embryogenic systems have a distinguishable
dedifferentiated stage, e.g., Citrus (Button et ad., 1974)
and Ranunculus (Konar et al.,
1972).

26
Another theory postulates that a cell must be
physiologically isolated from repressive factors of its
neighboring cells. This theory evolved mainly from
observations with the meiocytes of higher plants and has
been corroborated by observation with Citrus (Button et a_l. ,
1974).
A third theory has been developed primarily to account
for observations which are not readily explained by the
theory of dedifferentiation. Street (1978) proposed that
dedifferentiation, i.e., callus formation, itself does not
guarantee embryological competence, but rather the
physiological status of the individual explant cells at the
time of culturing together with the subsequent culture
environment are the primary determinants. This theory is
strongly supported by the embryogenic qualities of nucelli
and other reproductive tissue in general.
A fourth theory emphasizes the importance of
intercellular communication and subsequent cytodifferentia-
tion for somatic embryogenesis (Halperin, 1967). It evolved
from observations on the importance of callus formation
prior to somatic embryogenesis, and has been supported by
observations with cultured protoplasts in which a cell mass
is formed prior to somatic embryogenesis.
The fifth theory hypothesizes that the physiological
status of the explant itself is the primary determinant of
embryological competence (Tisserat et al.,
1979) .
The

27
culture environment only enhances or represses embryogenic
determination.
Finally, Sharp et al_. (1980) have developed a working
hypothesis which attempts to integrate many aspects of the
previously discussed theories. They propose that in vitro
somatic embryogenesis follows 2 major developmental
patterns. In the first pattern somatic embryogenesis occurs
directly from cells in the explant that have been
predetermined to form somatic embryos. The culture
environment can be envisioned as permissively allowing these
predetermined cells to undergo the process of embryogenesis.
Examples of this pattern would be Citrus and mango nucellar
cultures. In contrast, the second pattern of somatic
embryogenesis hypothesizes that the explant must
dedifferentiate before it can be induced in a directed
manner to undergo embryogenesis. An example of indirect
somatic embryogenesis would be the carrot pith cultures. In
both cases the cultured cells are induced to undergo
embryogenesis, but in the first case it is a permissive
induction, while in the latter it is a directive induction.
Sharp's hypothesis appears to delineate a possible role
for plant growth regulators as agents that contribute to the
determination of the cells but that do not directly alter
their embryogenic competence. It also provides some
explanation for the disparities observed in the effects of
auxins on somatic embryogenesis in various plant culture
systems. In the direct pattern of embryogenesis, auxins are

28
viewed as merely cloning agents which produce multiple
copies of the predetermined cells while in indirect
embryogenesis auxins are envisioned as mitogenic substances
which redetermine the callus cells to an embryogenic state.
Factors Affecting In Vitro Somatic Embryogenesis
Somatic embryogenesis is a dynamic phenomenon
involving many genes and gene complexes, all controlled in a
highly coordinated fashion (Sung et al., 1984). Several
factors have a direct or indirect effect on somatic
embryogenesis, and a number of reviews have addressed this
(Raghavan, 1976a and 1976b; Reinert et al., 1977; Kohlenbach
1977; 1978; Tisserat et a^. , 1979; Ammirato, 1983 ; 1984;
Bhojwani and Razdan, 1983; Ozias-Akins and Vasil, 1985).
Plant genotype. Certain taxa have been more amenable to
in vitro somatic embryogenesis, e.g., Umbel1iferae, but
within a given species or taxonomic group, individual
genotypes often show pronounced differences in their
embryogenic potential. In alfalfa (Kao and Michaluk, 1980),
clover (Keys et al^. , 1980) , corn (Lu et a_l. , 1982) , carrot
(Steward et a_l. , 1975), and indeed almost every culture
system where somatic embryogenesis has been investigated,
significant differences have been found among individual
cultivars (Ammirato, 1983).
Explant source. The explant source and its
physiological qualities are perhaps the most significant
factors in determining whether an embryogenic culture can be

29
initiated. Other in vitro influences merely enhance or
repress the embryogenic response (Tisserat et a_l. , 1979).
For some plant species, e.g., D. carota, almost any explant
taken at any stage of development can be used to establish
embryogenic cultures. However, for other, more recalcitrant
plant species, choice of explant at an appropriate
developmental stage may be critical, e.g., sweet potato (Liu
and Cantliffe, 1984). Floral tissues, in general, have been
very useful for initiating embryogenic cultures in a number
of plant species (Ammirato, 1983). Ovules of several fruit
species have been successfully cultured, e.g., Citrus
(Kochba and Button, 1974), grape (Srinivasan and Mullins,
1980), apple (Eichholtz, 1979), mango (Litz et al., 1982),
papaya (Litz and Conover, 1982), jaboticaba (Litz, 1984c),
and Eugenia spp. (Litz, 1984a). Zygotic embryos have also
proved to be very useful for initiating embryogenic
cultures, e.g., cotton (Joshi and Johri, 1972), Ranunculus
sceleratus (Sachar
and Guha,
1962) ,
and
barley
(Norstog,
1970). Anthers and
microspores
are
also
frequently used,
e.g., Triticale (Sun et al.,
1973) ,
Ranunculus
sceleratus
(Nataraja and Konar, 1970), Prunus avium (Zenkteler et al.,
1975) , Coffea arabica (Sharp et al., 1973) , and Datura (Guha
and Maheswari, 1964) . The use of nonmaternal tissues, e.g.,
microspores and zygotic embryos, has limited the application
of somatic embryogenesis for propagation and for improvement
of clonally propagated crops. Meristems, e.g., coffee (Sharp
et al., 1973), Ranunculus (Nataraja and Konar, 1970), and

30
immature vegetative tissue, e.g., millets (Vasil and Vasil,
1981), sorghum (Brettell et al. , 1980) are other important
sources of explant material.
The physiological condition of the explant at time of
culturing also significantly affects its embryogenic
potential. In many recalcitrant plant species there appears
to be a rather discrete developmental window, during which
the cells of the explant are receptive to outside influences
and somatic embryogenesis can be induced. With Citrus
Gmitter (1985) found that ovules of C. sinesis Osb. cv.
Hamlin became more responsive with increased fruit maturity.
The size and shape of the explant can also be important.
Explants below a certain minimum size may not have enough
viable cells in contact with the medium (Flick et ad.,
1983). The polarity of the explant can also influence its
responsiveness. In Pennisetum, Vasil and Vasil (1981) found
that excised zygotic embryos were most responsive when both
the root and the shoot were in direct contact with the
culture medium. The ploidy level of the explant does not
seem to be a limiting factor, and somatic embryogenesis has
occurred from haploid, diploid, and polyploid tissues.
Plant growth regulators. Perhaps the most widely
studied variable affecting in vitro somatic embryogenesis
has been the kind and quantity of PGRs used in the culture
medium. Of the PGRs, auxin, particularly 2,4-D, has been the
most often cited as being useful for in vitro somatic
embryogenesis. In carrot, callus is maintained on

31
2.4-D-supplemented media. The callus is transferred to an
auxin-free medium in order to induce somatic embryogenesis
(Halperin and Wetherell, 1964; Halperin, 1966). In general,
the presence of auxin or of an auxin-like substance is
required for the initiation of embryogenic cultures, while
the quantitative lowering or qualitative changing of the
auxin source generally allows somatic embryo growth and
maturation to proceed (Ammirato, 1983). When _in vitro
somatic embryogenesis has occurred without auxin in the
culture medium, it has involved tissues that were embryonic,
e.g., cotyledons of Ilex (Hu and Sussex, 1971) or
reproductive, e.g., ovules of Citrus (Kochba and
Spiegel-Roy, 1973). In Citrus and mango (Litz and Schaffer,
1987) it appears that somatic embryogenesis can occur
without the addition of PGRs, but the addition of exogenous
auxins does increase callus proliferation. The addition of
2.4-D to the culture medium is more effective for callus
proliferation in Citrus than either NAA or IAA (Murashige
and Tucker, 1969); however, Moore (1985) and Kochba and
Spiegel-Roy (1977) found that the addition of various auxins
significantly reduces somatic embryogenesis in several
Citrus tissue culture systems even at relatively low
concentrations (0.5 uM). Raghavan (1984a) points out that
many of the confusing results concerning the role of auxins
in somatic embryogenesis are related to the use of diverse
tissues (freshly isolated vs. habituated callus), the size
of the inoculum, and the concentration and type of auxin.

32
Sharp et al_. (1980) make a strong distinction between the
auxin requirement for cultures that undergo indirect somatic
embryogenesis and those that undergo direct embryogenesis,
e.g., Citrus. They concluded that there is an absolute
requirement for exogeneous application of auxin in those
cultures undergoing indirect embryogenesis, and its role is
hypothesized to be one of a directive inducer, while in
systems undergoing direct embryogenesis, there does not seem
to be a strict auxin requirement. The role of auxin in these
systems is thought to be as a cloning agent for
pre-embryogenic determined cells.
Cytokinins are another class of PGRs routinely used in
embryogenic culture systems. Their quantitative interaction
with auxins and their role in developmental control are well
documented in organogenesis (Skoog and Miller, 1957). The
type and concentration of cytokinin often differ in media
used for callus multiplication, embryo induction, and embryo
maturation media. Evans et al_. (1981) reported that
cytokinins have been employed in the initiation medium of
over 65% of all crop plants that have been cultured.
Abscisic acid (ABA) has been used successfully in a
large number of embryogenic plant culture systems. It is an
inhibitor of embryo development, whereby it allows embryo
maturation to proceed in a more normal fashion. This
normalizing effect has been reported in caraway (Ammirato,
1974), carrot (Kamada and Harada, 1981), Pennisetum (Vasil
and Vasil, 1981), and Citrus (Kochba et al.,
1978) .

33
According to Ammirato (1983) ABA also suppresses secondary
embryo proliferation and precocious germination.
Gibberellins have been used successfully in a few
instances to induce a normal pattern of embryo development
and stimulate germination and shoot elongation, e.g., Citrus
(Kochba et al., 1974), Panicum (Lu and Vasil, 1981), and
corn (Lu et al♦, 1982). Ethylene and other volatile gases
may exert a significant effect on closed tissue culture
systems but little research has been done to establish these
effects (Ozias-Akins and Vasil, 1985). Ethephon, which
liberates ethylene, was reported to stimulate embryogenesis
in Citrus cultures (Kochba et al. , 1978).
Nitrogen. Elemental nitrogen occurs in the highest
concentration of all the macroelements included in most
culture media. The total nitrogen concentration of the
various nutrient media ranges from 3.3 mM in White's to 60.0
mM in MS (sulfur ranges from 1-5 mM while sodium ranges from
0.2-8.0 mM). Of all the mineral nutrients, the form in which
nitrogen (oxidized or reduced, organic or inorganic) is
supplied, probably has the most dramatic effect on both
growth and differentiation of cultured cells (Ozias-Akins
and Vasil, 1985). The importance of total nitrogen and the
form in which it is supplied to in vitro somatic
embryogenesis has been well established (Halperin and
Wetherell, 1965). A direct requirement for nitrogen either
as ammonium or in another of its reduced forms is necessary.

34
The time at which nitrogen is applied also appears to
be important. Halperin and Wetherell (1965) found that
cultures initiated on a medium supplied with nitrogen in the
nitrate form were less embryogenic than cultures initiated
on media containing the same total nitrogen, but supplied in
the ammonium form. Gamborg et al_. (1968) found that when the
total nitrogen reguirement of a suspension culture medium is
supplied in
the form of
the
ammonium ion,
the pH has a
tendency to
drop and
may ultimately
restrict
the
availability
of nitrogen.
Although the
importance
of
nitrogen and
the form in
which it is supplied
has been
well
established,
there seems
to
be no general
formula
which
optimizes embryogenesis in all plant tissue culture systems.
Cultured cells can differ in their ability to utilize the
various forms of nitrogen. Carrot cells in vitro
characteristically have a very well developed glutamine
synthetase system (Caldas, 1971). Thus, the reduced forms of
nitrogen may not be as important as in other plant cultures.
The concentration of 2,4-D in the medium can also
significantly alter the culture's ammonium reguirement
(Halperin and Wetherell, 1965; Halperin, 1966).
Many plant culture systems respond well to the addition
of reduced forms of nitrogen, i.e., amino acids, to the
culture medium. In general, glutamine and to a lesser
extent, glycine and aspartic acid have been the most useful
amino acids for increasing the efficiency of somatic
embryogenesis (Ammirato, 1983). Complex organic addenda such

35
as endosperm extracts, casein hydrolysate (CH), and malt and
yeast extracts serve as important sources of amino acids and
other reduced forms of nitrogen (Ozias-Atkins and Vasil,
1985) .
Carbohydrates. Carbohydrates serve as a carbon source
for plant cultures, and because they are often present at
such high concentrations (1-12%) they are effective as an
osmoticum. The disaccharide sucrose is usually the most
effective sugar (Verma and Dougall, 1977), but other sugars
are also used. Maltose increases the germination of Citrus
aurantifolia somatic embryos (Miller, 1986). Lactose and
galactose increase somatic embryogenesis in various Citrus
spp. (Kochba et al_. , 1982), and sorbitol is effective in
Malus cultures (Chong and Taper, 1974). Inositol
(myo-inositol) has only rarely been shown to be essential in
plant tissue culture, e.g., in tobacco (Linsmaier and Skoog,
1965), but it does serve an important role in carbohydrate
metabolism (Loewus and Loewus, 1983) and is routinely
included in the culture medium.
Complex organic addenda. Complex, undefined organic
substances not only are important source of nitrogen, but
also are sources of PGRs, carbohydrates, and vitamins (Dix
and van Staden, 1982). They have also been useful for
somatic embryogenesis. In the early studies with carrot,
Steward et al. (1958a) thought that the addition of LCE to
the culture medium was essential for somatic embryogenesis;
however, efficient somatic embryogenesis has been achieved

36
on completely defined media (Steward et al.., 1964). Many
early plant tissue culture systems using low salt media
relied on the addition of complex, undefined naturally
occurring substances to supplement the simple inorganic salt
formulations being used (Ozias-Atkins and Vasil, 1985).
Various combinations of PGRs, particularly auxins and
cytokinins, can often replace the requirement for complex
organic addenda. Casein hydrolysate was shown to increase
somatic embryogenesis and development in one of the earliest
report of Citrus tissue culture (Maheswari and Rangaswamy,
1958). Malt extract, which has been shown to contain
cytokinins, giberellins, and auxins (Dix and van Staden,
1982), is commonly added to embryogenic cultures of Citrus
(Kochba and Spiegel-Roy, 1973; Moore, 1985).
Other factors. Several undefined gelling agents have
been used to solidify culture media, the most common of
which is agar. The impurities present in agar often are an
important source of trace elements (Heller, 1953). Many
different commercial preparations of agar are now available
which vary significantly in quality. Gelrite gellan gum is a
self-gelling hydrocolloid that forms a transparent gel in
the presence of soluble solids (Pasqualetto et al., 1986).
A number of vitamins have also been used in plant
cultures. Thiamine is the one vitamin which has been
demonstrated repeatedly to significantly affect plant
regeneration (White, 1951; Murashige and Skoog, 1962;
Linsmaier and Skoog, 1965; Gamborg et al., 1968). Other

37
vitamins included in culture media are pyridoxine, nicotinic
acid, and glycine (Ozias-Akins and Vasil, 1985).
The osmolarity of the culture medium can also affect
somatic embryogenesis and embryo development. Litz (1986)
working with Carica and Wetherell (1984) with carrot found
that pretreatment exposures of embryogenic suspension
cultures to media with high osmolarities (0.3-1.0 M)
followed by subculture to media with lower osmolarities,
resulted in increased levels of somatic embryogenesis. These
results are thought to mimic the changes in osmolarity found
in the endosperm of developing ovules, in which high
concentrations are associated with the earliest stages of
embryogenesis and development (Raghavan, 1976b). High
osmotic concentrations are also thought to suppress organ
differentiation and prevent precocious germination in young
somatic embryos (Ammirato, 1983).
In Citrus, various culture stresses can increase
somatic embryogenesis. 'Shamouti' cultures become more
embryogenic when relatively long culture intervals are used
and when callus is briefly subcultured onto a medium devoid
of sucrose (Kochba and Button, 1974).
Tissue Culture of Mango
The in vitro potential of mango was clearly recognized
by Maheshwari and Rangaswamy (1958); however, no cultures
were actually initiated. The first successful reports of
tissue culture in mango were by Rao et al. (1982) and Litz

38
et al. (1982). Rao et al_. (1982) reported callus initiation
from mango cotyledons on MS medium supplemented with 15%
LCE, 4% sucrose, 27 uM NAA, and 11.5-23 uM kinetin. Cultures
consisted of a dark compact callus that differentiated
prolific roots. Root proliferation continued with subsequent
subcultures, but no shoots or plantlet formation was
observed.
Litz et a_l. (1982) obtained callus initiation and
somatic embryogenesis from cultured ovules of polyembryonic
mangos collected approximately 2 months post-pollination.
They initiated cultures on a modified solid MS medium with
1/2 strength MS major salts, 6% sucrose, 0.8% Difco-Bacto
agar, 2.73 mM glutamine, 0.57 mM ascorbic acid with or
without 20% filter-sterilized LCE or 4.4-8.8 uM BA. The
nucelli were excised from the cultured ovules after 1-3
weeks and subcultured to fresh media. Embryogenic calli were
transferred to either solid or liquid media containing 20%
LCE. Cultures were incubated in a growth chamber at 25 C
with a 16 hr photoperiod (1000 lux). Liquid cultures were
maintained on a rotary shaker at 100 r.p.m. The most
efficient media for somatic embryogenesis was reported to be
liquid media with 20% LCE. The embryogenic response was
cultivar-dependent with 5 of the 9 polyembryonic cultivars
producing embryogenic callus. No root, shoot, or plantlet
production was reported from somatic embryos transferred to
LCE-free media.

39
There have been additional reports on in vitro somatic
embryogenesis of mango (Litz, 1984b; Litz et al., 1984; Litz
and Schafer, 1987; Litz, 1987). Nucelli and somatic embryos
were excised from cultured mango ovules 1-2 weeks after
culturing and transferred to a liquid medium containing 9 uM
2,4-D instead of LCE. Cultures were maintained in liquid for
3 months with monthly subcultures, and then transferred to
liquid MS medium without PGRs and with or without 0.5%
activated charcoal. Somatic embryos were transferred to a
solid germination medium supplemented with 0-22 uM BA
together with 0.01% CH, 0.01% ME, or 10% LCE. Activated
charcoal was reported to be deleterious to embryo
maturation. Limited root formation was observed on solid
media supplemented with 4.4-8.8 uM BA (Litz et jrl. , 1984).
Excised nucelli from monoembryonic mango ovules when
cultured on a modified solid MS media (described previously)
supplemented with either 4.5-13.5 uM 2,4-D or to a lesser
extent 5.4-27.0 uM NAA, gave rise to a "loose and friable"
slowly growing callus (Litz, 1984b). Somatic embryos were
reported to have arisen indirectly from the nucelli via
callus formation. This observation contrasted with reports
of bn vitro somatic embryogenesis in monoembryonic Citrus
where embryos were reported to have arisen directly from the
nucellus without intermediate callus formation (Rangan et
al. , 1968). The cytokinins BA and 2iP were both reported by
Litz (1984b) to be ineffective in initiating callus. The
addition of 20% filter-sterilized LCE to the culture media

40
somewhat delayed a gradual necrosis that was observed in all
the embryos, while casein hydrolysate (CH), malt extract
(ME), and reducing agents were found to be ineffective.
Alterations in physical culture conditions such as light vs.
dark and solid vs. liquid were also reported to be
ineffective in the alleviation of the gradual necrosis.
Limited germination (root formation) was observed in liquid
media without PGRs. Litz and Schaffer (1987) reported that
although 2,4-D enhances callus formation from mango nucelli,
it does not significantly affect somatic embryogenesis.
There have been no histological studies to verify that
regeneration from mango nucellar callus is via somatic
embryogenesis, although the evidence is persuasive. Somatic
embryogenesis is most efficient in liquid cultures
supplemented with 20% LCE or 2,4-D. Polyembryonic and to a
lesser extent monoembryonic mangos both are capable of
somatic embryogenesis. The response is genotype-dependent.
Limited root growth has been reported but no plantlet or
shoot formation. Embryos exhibited a gradual and progressive
terminal necrosis.

CHAPTER III
SOMATIC AND ADVENTIVE NUCELLAR EMBRYONY IN MANGO
Introduction
The term somatic embryogenesis has been used throughout
this dissertation to refer to the initiation of embryos from
somatic tissues under _in vitro conditions. The terms
adventive embryogenesis and adventive nucellar embryogenesis
have been used only to describe the natural in vivo
production of adventive embryos from cells of the nucellus.
Somatic embryos are generally thought to pass through all
the developmental stages exhibited by zygotic and adventive
embryos in vivo (Tisserat et al_. , 1979 ; Ammirato, 1983),
although minor differences in the first series of cell
divisions have been reported with carrot somatic embryos in
comparison to zygotic embryo development (McWilliams et al.,
1974). The most notable difference between somatic embryo
and zygotic embryo development in most plant cultures is the
lack of suspensors in the somatic embryos, e.g., Citrus
(Esan 1973), Daucus (Halperin and Jensen, 1967), and
Ranunculus (Konar et al., 1972). Cotyledon abnormalities
have often been associated with the development of somatic
embryos and include polycotyledony (Halperin, 1964), fused
41

42
cotyledons (Rashid and Street, 1973), unequally sized pairs
(Rao 1965) , and fasciation (Rao 1965) .
Materials and Methods
Flowers and fruitlets were gathered during the 1984 and
1985 season from the mango germplasm collection of the
University of Florida, Tropical Research and Education
Center in Homestead. Ovules over 1 cm in length were
dissected from the fruit and placed directly into the
fixative. The material was fixed and stored in FAA [90:5:5
of (95%) ethanol, formalin, glacial acetic acid].
Embryogenic calli were initiated from excised nucelli
of the polyembryonic 'Parris' on solid, modified MS medium
(callus initiation medium, Table 4-3). Embryogenic nucellar
callus was multiplied in suspension culture on a modified
B-5 medium supplemented with kinetin and 2,4-D (callus
maintenance medium C, Table 4-3). Embryo production,
maturation and germination occurred on a modified B-5 media
solidified with 0.19% Gelrite gellan gum and supplemented
with NAA and 2iP (for embryo production) or 20% liquid
coconut endosperm and 0.025% casein hydrolysate (for
maturation and germination). Callus and somatic embryos used
for histology were killed, fixed, and stored for 48 hours in
FAA (prepared with 70% ethanol).
All material used for light microscopy was removed from
FAA fixative, dehydrated with tertiary butyl alcohol, and
embedded in paraffin (Paraplast) according to Johansen

43
(1940). Sections were cut at 8-9 uM using an American
Optical rotary microtome and stained in safranin and fast
green.
Clusters of cotyledonary somatic embryos produced on
solid media were used for scanning electron microscopy.
Embryos were harvested and fixed for 1 hour in a solution of
4.25% gluteraldehyde with 0.05 M sodium cacodylate buffer.
The material was then rinsed 3 times for 20 minutes in the
cacodylate buffer. Material was postfixed for 1 hour in a 2%
OsO^ solution with 0.05 M cacodylate buffer. Specimen
fixation was followed by three 20 minute rinses in distilled
H20 and dehydration was accomplished using a 6 step series
of ethanol solutions (25, 50, 75, 95, 100, and 100%). Any
remaining moisture was removed by critical point drying.
Material was gold coated under vacuum and observed with a
Philips scanning electron microscope.
Results and Discussion
Adventive Nuce liar Embryony. The mango fruit is a drupe
which contains a large fibrous, hard endocarp enclosing the
seed. Figure 3-1 shows a young mango fruit approximately 75
days post-bloom. At this stage the tough, fiberous endocarp
has not yet developed around the ovule (megasporangium). The
fruit is easily cut open to expose the ovule which is borne
on a funiculus or stalk. The micropyle is just below the
point where the funiculus joins the ovule proper. The
chalazal region is at the opposite end of the ovule.

44
Mangos possess crassinucellate ovules which are
characterized by having well developed and persistent
nucelli. Figure 3-2 is representive of a polyembryonic mango
ovule approximately 30 days post-bloom. In this 'James
Saigon' ovule (3 mm length) the nucellus has already
undergone considerable degeneration. The nucellus appears as
a layer, several cells thick, consisting of mostly flattened
cells surrounding the entire embryo sac cavity. In this
ovule the nucellus is more developed and persistent near the
micropyle. The endosperm has not become cellularized at this
stage of development and is still in the free-nuclear
condition. It is characteristically a thin line of densely
staining nuclei and cytoplasm which is appressed against the
nucellus by the central vacuole. This free-nuclear endosperm
appears to be more concentrated in the micropylar region of
the ovule. The adventive nucellar embryos are a small
cluster of densely stained cells at the extreme micropylar
end of the embryo sac cavity (Figures 3-2; 3-3). In these
micrographs the micropyle is clearly defined. The epidermal
layer of the the integument lines the micropyle and
delineates the cells of the nucellus. In Figure 3-3 six
adventive embryos can be clearly distinguished. They range
from a two-celled proembryo to a twenty-four-celled early
globular stage adventive embryo. In the earliest stages of
adventive nucellar embryony, the proembryos contain cells
with rich densely staining cytoplasm, thin cell walls,
little vacuolation, and prominent nuclei. At approximately

45
45 days post bloom the adventive nucellar embryos have
enlarged and protrude into the embryo sac cavity as observed
in the 7 mm length 'Parris' ovule (Figure 3-4). At this
stage the endosperm has become fully cellularized. Adventive
nucellar embryos at the early to late globular stage are
demonstrated in Figures 3-5 and 3-6. The 0.5 mm length
adventive embryo in Figure 3-6 possesses a well developed
epidermis; however, no embryonic organs or vascularization
are distinguishable.
In Vitro Somatic Embryony. Embryogenic nucellar callus
can be initiated from both the micropylar and the chalazal
portions of excised nucelli cultured on solid callus
initiation medium (Table 4-3, see Figure 3-7). Whatever
factors limit adventive embryogenesis in the chalazal
portion of the nucellus in vivo are apparently not present
or are inhibited in vitro. This is in contrast with the
initiation of Citrus nucellar cultures as reported by
Kobayashi et a_l. (1979), in which the morphologically
distinct "primordium cells" of the adventive embryos
proliferate to form embryogenic ovular callus in
polyembryonic Citrus cultures.
Embryogenic mango nucellar callus is compact,
nonfriable and globular in form (Figure 3-8). It consists of
numerous light-dark brown globules (0.5-2.0 mm diameter)
with cream-white somatic embryos 2-5 mm in length arising
from the periphery. When this callus is transferred to

46
liquid medium (callus maintenance medium, Table 4-3), it
proliferates rapidly as globular callus (Figure 3-9).
Somatic embryogenesis from globular callus grown in
suspension is restricted primarily to the epidermal layer of
the globular callus or somatic embryos. The epidermis of
globular callus or somatic embryos prior to the initiation
of embryogenesis is a smooth and continous layer of cells
completely surrounding the somatic embryo. The cells are
highly vacuolated without densely staining cytoplasm (Figure
3-10). Eventually the epidermis begins to redifferentiate
and is no longer a continuous single cell layer (Figure
3-11). Numerous embryonic initials, i.e., cells with densely
staining cytoplasm are visible. As dedifferentiation
proceeds the layer of densely staining cells with prominent
nuclei becomes thicker (Figure 3-12), causing the somatic
embryos to have a rough and bumpy appearance at low
magnification. The process of dedifferentiation of somatic
embryos from the epidermal region of a late globular stage
somatic embryos is shown in Figure 3-13. Numerous distinct
somatic embryos from the single cell to the octet stage of
embryo development can be observed. Subsequently, more
advanced stages of somatic embryo development become evident
(Figures 3-14 and 3-15). In Figure 3-15 a globular stage
somatic embryo with a distinct epidermis is shown.
The embryogenic 'Parris' suspension cultures used in
the regeneration experiments of Chapters IV and V have a
rather distinctive growth morphology. Proliferating globular

47
'Parris' callus in suspension appears very differentiated.
Cultures consist of clusters of somatic embryos 0.5-1.5 cm
in diameter which have formed via the budding of secondary
embryos (Figures 3-16 and 3-17). These clusters of budding
somatic embryos contain a distinct central core (Figure
3-18), which consists of a late globular to early torpedo
stage somatic embryo. Numerous somatic embryos at various
stages of development radiate from the outer portions of the
epidermis-derived cell layer. The somatic embryos which form
the central core are still composed of living tissue.
However, the nonembryogenic epidermis-derived regions of the
somatic embryos become necrotic (Figure 3-18). The necrotic
portions of the epidermis along with some of the underlying
cell layers are sloughed off (Figure 3-19).
Somatic embryos produced in suspension culture exhibit
a number of developmental abnormalities, some of which are
evident at very early stages (Figures 3-20 and 3-21).
Globular somatic embryos without a distinct epidermis are
prevalent. Moreover, their overall morphology appears to be
less compact than typical adventive embryos (Figures 3-5 and
3-6) .
Fewer developmental abnormalities occur when somatic
embryos are produced on solid medium even though
polycotyledony and unequal sized cotyledons were present
(Figure 3-22). After the differentiation of cotyledons on
embryogenesis medium (Table 4-3) the bipolar nature of the
regenerated somatic embryos together with their closed

48
vascular system was apparent (Figure 3-23). Thus, the nature
of regeneration via somatic embryogenesis was unequivocally
demonstrated.

ii|iiii|iiii|iiii|iiiimii|imi
2> 3I 41 5I
111111111
CM 1
Figure 3-1 A mango fruitlet approximately 75 days-post-bloom
bisected longitudinally with intact ovule
(megasporangium). f:funiculus, m:micropyle,
c:chalazal region

Figure 3-2 Longitudinal section through a polyembryonic ovule
approximately 30 days-post-bloom. The adventive
nucellar embryos (ae) appear as a cluster of darkly
staining cells near the micropyle (m) end of the
ovule. The nucellus is a thin layer of cells which
completely surrounds the embryo sac cavity (esc)
(bar=l mm), crchalazal region, e:endosperm,
i:integument, f:funiculus

4fco£«í t*4**3r»
7- •£»* *■ ».«■'
’< i' ' /
Figure 3-3 Micropylar region of a polyembryonic ovule. The adventive
embryos (ae) are located within the nucellus (n) directly
adjacent to the free-nuclear endosperm (e) (bar=100 urn).
i:integument, ei:epidermis of the integument, m:micropyle

Figure 3-4 Longitudinal section through the micropylar half of a
polyembryonic ovule approximately 45 days post-bloom
showing adventive nucellar embryos (ae) projecting
into the embryo sac cavity (esc). Note the cellularized
endosperm (e) (bar=l mm).

Figure 3-5 Globular stage adventive nucellar embryos, indicated
by arrows. Each contains approximately 30-40 cells
(bar=100 urn).

Figure 3-6 Late globular stage adventive nucellar embryo. Note
the well developed epidermal layer (bar=100 urn).

Figure 3-7 The initiation of embryogenic nucellar callus. The
nucellus has been excised, cut in half longitudinally
(upper and lower pieces), and separated into micropyle
(rri) , central (cn) , and chalazal (c) portions. Note that
callus has been initiated from all three portions of one
excised nucellus half. The medium is callus initiation
medium Table 4-3.

Figure 3-8 Close-up view of embryogenic ntango nucellar callus
initiation. The callus is light brown-colored, compact,
globular , and nonfriable. Note the presence of
differentiated somatic embryos, indicated by arrows.
Ln
O'

L/i
Figure 3-9 Suspension culture of rapidly proliferating mango
nucellar callus growing in presence of 2,4-D.

Figure 3-10 The epidermis (ep) of a late globular stage
somatic embryo grown in suspension culture prior
to redifferentiation and somatic embryogenesis
(bar=100 urn).

Figure 3-11 The epidermis (ep) of a late globular stage somatic
embryo begining to redifferentiate. Note the epidermis
is now discontinous and contains many cytoplasmically
dense cells (bar=100 urn).

ON
O
Figure 3-12 Early stages of epidermal somatic embryogenesis. The
epidermis has divided to form a layer of actively
dividing cells, 2-6 cells thick (bar=100 urn).

. VI
Section through the periphery of a redifferentiating
globular somatic embryo or callus. Arrows indicate
several distinct stages of somatic embryo development;
diad, tetrad, and octet stages (bar=50 urn).
Figure 3-13

Figure 3-14 Four early globular stage somatic embryos. Each
somatic embryo is composed of from 4-8 cells
(bar=10 urn).

Figure 3-15 A globular stage somatic embryo beginning to differentiate
an epidermis and showing signs of polarity (bar=100 urn).

Figure 3-16 A suspension culture of budding 'Parris' somatic
embryos. Note the prevalence of cotyledonary somatic
embryos and the lack of undifferentiated callus.

Figure 3-17 Close-up of a cluster of budding 'Parris' somatic
embryos grown in suspension culture (callus
maintenance medium C, Table 4-3). Arrow indicates
rudimentary cotyledons (bar=5 mm).

\
Figure 3-18 Cross section, at low magnification through a cluster of
budding somatic embryos grown in suspension culture. Each
cluster is composed of a central core (cc) which is a late
globular stage somatic embryo. Somatic embryogenesis has
occurred from the cells derived from the epidermis. Arrows
indicate nonembryogenic regions that have become necrotic
(bar=l mm).

Figure 3-19 A section through the periphery of the central core (cc)
of a budding cluster of somatic embryos grown in suspension
culture. Somatic embryogenesis (se) is occurring from the
epidermis-derived cells. The dark arrows indicate
nonembryogenic regions of the epidermis which have become
necrotic and are being sloughed off (bar=l mm).

I?
\
1
1
Figure 3-20 An early globular stage somatic embryo grown in suspension
culture exhibiting aberrant development. Note its non-compact
morphology and lack of defined epidermis (bar=100 uM).
00

Figure 3-21 A globular stage somatic embryo grown in suspension culture
with a poorly formed epidermis which appears to be undergoing
secondary budding from its periphery (bar=100 urn).

70
Figure 3-22 A scanning electron micrograph of a cluster
of 'Parris' somatic embryos grown on solid
medium (bar=500 urn).

Figure 3-23 A longitudinal section through the base of a somatic
embryo grown on solid medium. Arrows indicate shoot
(upper) and root (lower) meristems. Note the absence
of a maternal vascular connection (bar = 100 urn) .

CHAPTER IV
CALLUS MAINTENANCE AND SOMATIC EMBRYOGENESIS IN MANGO
Introduction
The efficient regeneration of plants via somatic
embryogenesis is partially dependent on the production of
large quantities of uniform, embryogenica1ly competent
callus. This callus must then be induced by means of the
culture environment to undergo efficent and preferably
synchronized embryogenesis. Control of somatic embryo
maturation and germination is a final prerequisite for
normal plantlet formation.
In chapters IV and V a series of experiments are
described that addresses somatic embryogenesis and plant
recovery in mango. In Chapter IV, callus production and
somatic embryogenesis are considered. In Chapter V
parameters that affect the maturation and germination of the
somatic embryos are considered.
Callus production parameters have been quantified by
both biomass production and by measuring the embryogenic
competence of the callus produced. Somatic embryogenesis
parameters have been quantified by total cotylodonary embryo
production. Ratings have also been used to evaluate the
72

73
somatic embryos based on absence of necrotic or vitrified
regions, large relative size (5-8 mm in length), single
cotyledons without fasciations, and no secondary embryo
proliferation.
Materials and Methods
Immature mango fruits of both monoembryonic and
polyembryonic cultivars were harvested 2-3 months post¬
pollination from the mango germplasm collections of the
University of Florida Tropical Research and Education Center
in Homestead and from the U.S.D.A. Subtropical Horticultural
Research Unit in Miami, Florida. Surface-sterilization of
the fruitlets was accomplished using the following protocol:
10 minutes in 70% ethanol, 30 minutes in a 1% sodium
hypochlorite solution with 2-3 drops of Tween 20 or Tween
80, followed by 2 rinses in sterile distilled water. Ovules
were dissected from the fruitlets using sterile forceps and
scapels. Embryogenic callus was initiated from whole
immature ovules, immature ovule halves without embryo(s),
zygotic embryos, nucellar embryos, and excised nucelli. All
callus was initiated on a modified solid Murashige and Skoog
(MS) medium consisting of 1/2 concentration major salts
(Table 4-1), MS minor salts (Table 4-2), 6% sucrose, 0.8%
Difco Bacto-agar, 2.74 m.M (400 mg 1 ) glutamine, 0.55 mM
(100 mg 1 ''') inositol, 0.57 mM (100 mg 1 ^) ascorbic acid,
1.2 uM (0.4 mg 1 1) thiamine HC1, and 4.5 uM (1 mg 1 1)
2,4-D (callus initiation medium, Table 4-3). All media were

74
Table 4-1. Basal media major salts formulations
used with in vitro culture of mango.
Major Salts
Formulation
mM(mg 1 •*■)
MSZ
1/2 MS
WPM^
B-5X
nh4no3
20.6(1650)
10.3(825)
5.0(400)
-
kno3
18.8(1900)
9.4 (950)
-
25.0(2500)
Ca(N03)2 4H20
-
2.4(556)
-
k2so4
-
-
(990)
-
MgS04 7H20
1.5(370)
1.8(185)
1.5(370)
1.0 (250)
CaCl2 2H20
3.0(440)
1.5(220)
0.7(96)
-
kh2po4
1.3(170)
0.6(85)
1.3(170)
-
NaH2P04 H20
-
-
-
1.1(150)
zMurashige and Skoog, 1962.
^Woody Plant Media, Lloyd and McCown, 1982.
xGamborg B-5, Gamborg, et al., 1968, without
(NH4)2so4.
Table 4-2. Murashige and Skoog minor salts formulation.
Minor
salts2
uM(mg I--'-)
Minor
salts
uM(mg l-"'-)
Na2EDTA
100(37.3)
KI
5.0(0.830)
FeS04
7H20
100(27.8)
Na2Mo04 2H20
1.0(0.250)
MnS04
o
CM
100(22.3)
CoS04
6H20
0.1(0.025)
ZnS04
h3bo3
7H20
30(8.6)
100(6.2)
CuS04
5H20
0.1(0.025)
zMurashige and Skoog, 1962.

75
adjusted to pH 5.75 using HC1, KOH, or NaOH prior to
-2
autoclaving at 121 C and 1.1 kg cm for 18 minutes. Plant
growth regulators (PGRs) were dissolved in IN HC1, KOH, NaOH
or 95% ethanol according to Table A-l.
Explants were cultured on sterile medium in 60 X 15 mm
plastic petri dishes, stored in clear plastic boxes and
incubated either in growth chambers at a constant
temperature of 25 C, with a 16 hr photoperiod (40 umol s"
-2
m ) provided by cool-white fluorescent lamps or at ambient
room temperature without supplemental light. Embryogenic
callus was established from both monoembryonic and
polyembryonic mango cultivars (Table A-2).
Original explants were subcultured onto fresh medium
several days after the initial culturing to remove the
tissues from oxidation products that had accumulated in the
media. They were subcultured again 1 week later and
subsequently at 3-4 week intervals.
One hundred to 500 mg of excised proliferating callus,
visible 1-3 months after culturing, was transferred to
fresh, solid callus initiation medium (Table 4-3) or
directly into liquid medium for multiplication and
maintenance. Liquid cultures consisted of 50 ml of basal
medium (BM) in 125 ml Erlenmeyer flasks maintained at 100
r.p.m. on rotary shakers. Liquid callus cultures were
subcultured at 2 week intervals until the fresh weight of
the callus was approximately 10 g per flask. Cultures were

Table 4-3. Basal medium components and supplements used for
in vitro culture of mango.
Medium
components
callus
initiation
callus
maintenance
A
callus
maintenance
B
callus
maintenance
C
embryogenesis
Major salts
1/2 MS
1/2 MS
1/2 MS
B-5Z
B-sy
Minor salts
MS
MS
MS
MS
MS
Sucrose
6%
5%
5%
5%
5%
Glutamine
2.74 mM
0
2.05 mM
2.05 mM
2.05 mM
myo-Inositol
0.55 mM
0.55 mM
0.55 mM
0.55 mM
0.55 mM
Glycine
0
0
0
53.3 uM
53.3 uM
Ascorbic acid
0.57 uM
0
0
0
0
Thiamine HC1
1.2 uM
1.2 uM
1.2 uM
3.0 uM
3.0 uM
Nicotinic acid
0
0
0
4.1 uM
4.1 uM
Pyridoxine HC1
0
0
0
2.4 uM
2.4 uM
2,4-D
4.5 uM
4.5 uM
4.5 uM
4.5 uM
0
NAA
0
0
0
0
10.8 uM
Kinetin
0
18.6 uM
4.6 uM
4.6 uM
0
2iP
0
0
0
0
4.9 uM
Difco Bacto-agar 0.8%
0
0
0
0.75%
Gelrite gellan
gum 0
0
0
0
0.175-0.2%
Sigma agar gum
0
0
0
0
0.7%
z
Modified Gamborg B-5 major salts (Table 4-1).
y
Modified B-5 or one of 3 major salts formulations MS, 1/2 MS, WPM (Table 4-1).

77
then subdivided into 2 flasks of 5 g callus per flask with
fresh medium.
Experiment 4-1; In Vitro Somatic Embryogenesis from Mango
Nucellar Callus in Response to Genotype and Plant Growth
Regulators
Calli from 10 mango cultivars (8 polyembryonic, 2
monoembryonic), initiated 3 months previously on solid media
(Table 4-3), were multiplied in liquid medium of the
composition. Treatments consisted of 200 mg callus
inoculated onto 12 ml of sterile BM in 60 X 15 mm plastic
petri dishes supplemented with 1 or more combinations of the
following PGRs,
2,4-D, NAA,
IBA, or
IAA
at 0, 5,
10, or
20
uM and kinetin,
2iP, or BA
at 0,
5,
20, or
80 uM.
In
addition to the various synthetic PGRs, 3 complex organic
growth addenda were used, 20% (v/v) liquid coconut endosperm
(LCE), 0.015% (w/v) casein hydrolysate (CH), and 0.05% (w/v)
yeast extract (YE). All PGRs were prepared according to
Table A-l.
A modified 10 X 10 X 13 unreplicated factorial
experimental design was used, with 10 cultivars and 130 PGR
treatment combinations plus 6 treatment combinations with
complex organic addenda. Petri dishes were stacked in clear
plastic boxes and incubated in a growth chamber at a
constant temperature of 25 C, with a 16 hr photoperiod
-1 -2
provided by low intensity illumination (40 umol s m )
from cool-white fluorescent lamps. The total number of

78
somatic cotyledonary embryos per dish was determined 2
months after inoculation.
Experiment 4-2: In Vitro Somatic Embryogenesis from 'Parris'
Nucellar Callus in Response to the Callus Maintenance
Medium and Major Salts Formulations
One-year-old embryogenic 'Parris' callus, initiated and
maintained on a solid modified MS medium (callus initiation
medium, Table 4-3) was transferred to liquid medium for
callus multiplication. Based on the results from Experiment
4-1 2 liquid callus maintenance media formulations were
tested for their effects on somatic embryogenesis. Callus
maintenance medium A (Table 4-3) was supplemented with 4.5
uM (1 mg 1 1) 2,4-D and 18.6 uM (4 mg 1 ^) kinetin. Callus
maintenance medium B (Table 4-3) was supplemented with 4.5
uM (1 mg 1 â– *â– ) 2,4-D, 4.6 uM (1 mg 1 1) kinetin, and 2.05 mM
(300 mg 1 ^) glutamine. Both callus maintenance media
formulations contained the same BM consisting of: 1/2 MS
major salts, MS minor salts, 5% sucrose, 0.55 mM (100 mg
1 "*â– ) inositol, and 1.2 uM (0.4 mg 1 1) thiamine HC1.
Four BM major salts formulations MS, 1/2 MS, Lloyd
McCown Woody Plant Medium (WPM), and a modified Gamborg B-5
(B-5) were tested for their effect on somatic embryogenesis
(Table 4-1). All embryogenesis media contained: MS minor
salts, 5% sucrose, 0.75% Difco Bacto-agar, 2.05 mM (300 mg
1 1) glutamine, 0.55 mM (100 mg 1 ^) inositol, 53.3 uM (4 mg
1 ^) glycine, 3.0 uM (1 mg 1 ) thiamine HC1, 4.1 uM (0.5 mg
1 1) nicotinic acid, 2.4 uM (0.5 mg 1 1) pyridoxine HC1,

79
10.8 uM (2 mg 1 1) NAA, and 4.9 uM (1 mg 1 2iP
(embryogenesis medium, Table 4-3).
Callus (300 mg/inoculum) was inoculated onto 12 ml of
solid sterile embryogenesis medium in 60 X 15 mm plastic
petri dishes. Cultures were incubated at room temperature
(24-26 C) in a clear plastic box with no supplemental light.
The experimental design was a 2 X 4 factorial with 6
replicates. Treatments were scored 45 days after inoculation
by counting the total number of somatic cotyledonary embryos
produced.
Experiment 4-3: In Vitro Somatic Embryogenesis from 'Parris'
Nucellar Callus in Response to the Callus Maintenance
Medium Formulation and Sucrose Concentration
One-year-old embryogenic 'Parris' callus, initiated and
maintained on a solid modified MS medium (callus initiation
medium, Table 4-3), was transferred to liquid medium for
callus multiplication. Two callus maintenance media
formulations, A and B (Table 4-3) were tested for their
effects on somatic embryogenesis.
Six sucrose concentrations: 0, 2, 3, 4, 5, and 6% were
also tested for their effect on somatic embryogenesis. Only
sucrose concentrations and callus maintenance medium
formulations were altered between treatments. All
embryogenesis media contained the same BM components
(embryogenesis medium, Table 4-3) and were solidified with
0.75% (w/v) Difco Bacto-agar.

80
Callus (300 mg/inoculum) was inoculated onto 12 ml of
solid sterile embryogenesis medium in 60 X 15 mm plastic
petri dishes. Cultures were incubated at room temperature
(24-26 C) in clear plastic boxes with no supplemental light.
The experimental design was a 2 X 6 factorial with 5
replicates. Treatments were scored 45 days after inoculation
by counting the total number of somatic cotyledonary
embryos.
Experiment 4-4: Callus Production from Nonembryogenic
'James Saigon1 Nucellar Callus in Response to the Major
Salts Formulation of the Callus Maintenance Medium
One-year-old nonembryogenic 'James Saigon' callus was
initiated and maintained on a solid modified MS medium
(callus initiation medium, Table 4-3). Callus was then
multiplied in liquid BM of the same formulation but without
any supplemental PGRs. Only major salts were altered between
treatments. All callus maintenance media contained MS minor
salts, 5% sucrose, 2.05 mM (300 mg 1 1) glutamine, 0.55 mM
(100 mg 1 1) inositol, 53.3 uM (4 mg 1 glycine, 3.0 uM (1
-1 -1 . . . .
mg 1 ) thiamine HC1, 4.1 uM (0.5 mg 1 ) nicotinic acid,
and 2.4 uM (0.5 mg 1 pyridoxin HC1 (callus maintenance
medium C, Table 4-3).
One gram of callus was used to inoculate 50 ml of
liquid callus maintenance medium in 125 ml Erlenmeyer
flasks. Cultures were incubated at room temperature (24-26
C) in a transparent plastic box without supplemental light
at 100 r.p.m. on rotary shakers. The callus was subcultured

81
every 2 weeks during the first month and every week during
the second month. The experiment was of a completely
randomized design with 4 treatments and 5 replicates.
Treatments were scored 60 days after inoculation by weighing
the total callus production (fresh weight) per flask.
Experiment 4-5: In Vitro Somatic Embryogenesis from
'James Saigon1 Nucellar Callus in Response to the
Solidifing Agent Used in the Embryogenesis Medium
Two year old embryogenic 'James Saigon' callus that was
initiated on a solid modified MS medium (callus initiation
medium, Table 4-3) was maintained on a solid modified MS
medium. The solid callus maintenance medium consisted of 1/4
MS major salts, 1/2 MS minor salts, 2.5% sucrose, 1.0 mM
(150 mg 1 ''â– ) glutamine, 0.27 mM (50 mg 1 ^) inositol, 100 uM
(27.8 mg 1 ^) FeSO^ ^K20, and ^•2 uM (0.4 mg 1 ^) thiamine
HC1. Embryogenic callus was transfered to a liquid modified
B-5 medium for callus multiplication (callus maintenance
medium C, Table 4-3).
Two gelling agents, Difco Bacto-agar at 0.7% and
Gelrite gellan gum at 0.18%, were tested for their effect on
somatic embryogenesis. Only the gelling agent was altered
between treatments. All embryogenesis media contained the
same BM components [embryogenesis medium (without 2,4-D),
Table 4-3].
Callus (300 mg/inoculum) was inoculated onto 25 ml of
solid sterile embryogenesis medium in 100 X 15 mm plastic
petri dishes. Cultures were incubated at room temperature

82
(24-26 C) in clear plastic boxes with no supplemental light.
The experimental design was a completely randomized design
with 2 treatments and 8 replicates. Treatments were scored
45 days after inoculation by counting the total number of
somatic cotyledonary embryos.
Experiment 4-6: In Vitro Production of 'James Saigon1
Nucellar Callus in Response to a Liquid or a Solid
Maintenance Medium
Two-year-old embryogenic 'James Saigon' callus that was
initiated on a solid modified MS medium (callus initiation
medium, Table 4-3) was maintained on a solid modified MS
medium consisting of 1/4 MS major salts, 1/2 MS minor salts,
2.5% sucrose, 1.0 mM (150 mg 1 1) glutamine, 0.27 mM (50 mg
1 1) inositol, 100 uM (27.8 mg 1 â– *â– ) FeSO^ and 1.2 uM
(0.4 mg 1 thiamine HC1. Embryogenic callus was
transferred to a liquid modified B-5 medium (callus
maintenance medium C, Table 4-3) for multiplication.
Two culture regimes, liquid and solid, were tested for
their effects on callus production. Two gelling agents,
Gelrite gellan gum at 0.2% and Sigma agar gum at 0.7% were
used to solidify the callus maintenance medium. All callus
maintenance media contained the same BM components (callus
maintenance medium C, Table 4-3).
Callus (300 mg/inoculum) was inoculated onto 12 ml of
solid sterile callus maintenance medium in 60 X 15 mm
plastic petri dishes or into 50 ml of sterile liquid BM in
125 ml Erlenmeyer flasks. Solid callus cultures
were

83
incubated at room temperature (24-26 C) in clear plastic
boxes without supplemental light. Liquid callus cultures
were also maintained at room temperature without
supplemental light on a rotary shaker at 100 r.p.m.. Solid
cultures were subcultured twice at 3 week intrervals
supplemental light on a rotary shaker at 100 r.p.m.. Solid
cultures were subcultured twice at 3 week intervals, while
liquid shaker cultures were subcultured twice at the
beginning of the 4th and 6th week after inoculation. The
experimental design was a completely randomized design with
3 treatments and 10 replicates. Treatments were scored 60
days after inoculation by weighing the total callus
production (fresh weight) per flask.
Results and Discussion
Experiment 4-1; In Vitro Somatic Embryogenesis from Mango
Nucellar Callus in Response to Genotype and Plant
Growth Regulators
The results are shown in Tables 4-6 through 4-9 and A-3
through A-6. The total number of cotyledonary embryos
produced per plate (from 200 mg of inoculum on 12 ml of BM),
when averaged within genotypes over all 130 treatments
ranged from 31.0 ('James Saigon') to 0.0 ('Gadong') [Table
4-8]. Treatment responses were highly genotype-dependent
with no single treatment or treatment combination giving the
optimal response for all genotypes tested. 'James Saigon'
produced embryos on almost all treatment combinations
tested, even on treatments lacking PGRs (Table 4-4). Trends

Table 4-4. I_n vitro somatic embryogenesis from 'James Saigon' nucellar
callus in response to various plant growth regulators.
Total number of cotyledonary embryos2
uM
0
5
Kinetin
20 80
5
2 iP
20
80
5
BA
20
80
0
35+y
100
30 +
0
30
0
10
40
X
25
5
15
40
40
25
30
10
10
50
35
0
2,4-D
10
13
50
50
10
30
50
20
30
10
20
10
20
20
0
20
25
15
12
5
30
5
30 +
30
50
1
60
25 +
0
25
50
25
NAA
10
75
10
25 +
5
30
30
50
75 +
50 +
20
20
15
75 +
60
1
30
40
75
50
50
15
5
25
50 +
50
50
50
20
10
20
50
10
IAA
10
60 +
15
5
30
0
25
75
15
20
60 +
20
60
7
20
40
12
20
40
50
5
25
30
25
3
50
60
40
75 +
60
20
IBA
10
10
0
3
50 +
10 +
30 +
60
25
20
8
10
30
20
75 +
60
30
60
75
25
200 mg
of callus inoculated onto 12
ml of
callus
initiation
medium
, (Table
4-3)
supplemented
with
PGRs
according to
the
table above.
, + =most
embryos over
6 mm
in length.
Blank
spaces
represent no
data
due
to contamination.

Table 4-5. Iji vitro somatic embryogenesis from 'Parris' nucellar
callus in response to various plant growth regulators.
Tota 1
number of
cotyledonary
embryos
z
Kinetin
2iP
BA
uM
0
5
20
80
5
20
80
5
20
80
0
y
0
0
0
0
0
5
2
50
0
0
40
2,4-D
10
2
0
0
0
0
0
0
20
0
0
0
0
0
0
5
20
5
10
0
75
10
0
0
0
NAA
10
50
40
0
0
100
50
50
30
0
20
0
25
10
0
8
5
0
0
5
5
0
5
0
0
0
0
0
0
IAA
10
0
0
0
0
20
5
0
0
0
5
0
0
25
30
0
IBA
10
0
0
0
0
20
60
0
0
2
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the table above.
yBlank spaces represent no data due to contamination.

Table 4-6. I_n vitro somatic embryogenesis from ’Tommy Atkins' nucellar
callus in response to various plant growth regulators.
uM
0
Tota 1
number
of
cotyledonary
embryo
sz
BA
20
80
Kinetin
5 20
80
5
2iP
20
80
5
0
0
10
6
0
0
6
3
8
y
0
5
1
0
5
0
0
6
0
12
0
0
2,4-D
10
0
0
0
0
0
0
0
0
0
3
20
0
0
0
0
0
0
0
0
0
0
5
0
3
2
0
5
0
0
10
5
NAA
10
2
2
5
3
5
1
2
7
2
20
8
5
3
0
4
3
3
0
0
5
12
20
5
30 + X
3
5
15
15
12
IAA
10
10
30 +
10
0
2
3
3
3
12
5
20
40
0
0
0
6
10
0
15 +
7
7
5
12
0
0
0
30 +
0
5
12 +
12 +
8
IBA
10
0
3
0
0
16 +
7 +
3
10
7
2
20
20
12
0
5
15
15 +
3
5
5
8
2
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the table above.
-^Blank spaces represent no data due to contamination.

Table 4-7. I_n vitro somatic embryogenesis from 'Heart' nucellar
callus in response to various plant growth regulators.
Total number of cotyledonary embryos2
uM
0
Kinetin
5 20 80
5
2iP
20
80
5
BA
20
80
0
4
3
0
0
0
0
1
3
y
5
5
0
0
0
10
0
0
5
0
2,4-D
10
0
0
0
0
0
2
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
5
1
3
10
0
15
10+x
0
0
30 +
0
NAA
10
3
20 +
12
5
5
1
10
0
3
5
20
20
0
0
7
0
0
0
0
0
5
0
3
0
15
12 +
10
0
15
0
5
IAA
10
0
10
10
12
20
0
0
10
20
0
5 +
12
0
3
2
0
0
0
5
8
8
5
12 +
0
15
10
0
IBA
10
10
10
10 +
0
10
0
20
0
5
0
20
8
0
5
2
5
0
0
25
0
z
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the table above.
-*Blank spaces represent no data due to contamination.
x+=most embryos over 6 mm in length.

88
Table 4-8. I_n vitro somatic embryogenesis from mango
nucellar callus for cultivars in experiment 1.
Cultivar Embryony Average2
James Saigon
Poly
31.0
Parris
Poly
8.2
Tommy Atkins
Mono
4.9
Heart
Poly
4.2
Simmonds *
Poly
1.1
Florigon *
Poly
0.4
Cambodiana *
Poly
0.3
Irwin *
Mono
0.1
Aroemanis *
Poly
0.05
Gadong *
Poly
0.0
2
Total number of cotyledonary embryos per plate, averaged
^over 130 treatments (200 mg inoculum per 12 ml BN!)
Cultivars may have lost embryogenic potential.
Table 4-9. Ln vitro somatic embryogenesis in response
to various complex organic addenda.
Cultivar2
LCE^
CHX
YEW
LCE&CH
LCE&YE
James Saigon
50
20
5
35
7
Parris
25+V
10 +
0
30 +
0
Tommy Atkins
15 +
12
0
2 +
0
Heart
20
12
0
8
0
Simmonds *
0
0
0
0
0
Florigon *
0
0
0
0
0
Cambodiana *
2
3
3
0
0
Irwin *
20 +
0
0
0
0
Aroemanis *
1
0
0
0
2
Gadong *
0
0
0
0
0
2
200 mg of callus inoculum per 12 ml of BM (callus
initiation medium, Table 4-3) with the addenda listed
in the table above (treatments were not replicated).
yLCE=liquid coconut endosperm 20% v/v.
xCH=casein hydrolysate, 0.015% w/v.
WYE=yeast extract, 0.05% w/v.
v+=Most cotyledonary embryos larger than 6 mm in length.
Cultures may have lost embryogenic competency.

89
could be discerned in 'Parris', the second most embryogenic
cultivar, even though many treatments for this genotupe were
lost due to contamination (Table 4-5). Treatments containing
the auxin NAA generally performed better than treatments
containing the other three auxins. NAA was used in all of
the subsequent embryogenesis experiments with 'Parris'. The
monoembryonic 'Tommy Atkins' (the most important Florida
cultivar) was most responsive on treatments containing IAA
and IBA (Table 4-6). 'Irwin', the other monoembryonic
cultivar tested, exhibited very low levels of somatic
embryogenesis on all treatments except the one treatment
containing only LCE (Tables A-6 and 4-8). The pronounced
genotype and genotype x treatment response found in mango is
quite common in many plant tissue culture systems, e.g.,
alfalfa (Kao and Michaluk, 1980), carrot (Steward et al.,
1975) , and corn (Lu et a_l. , 1982) .
All of the cultures had been maintained in vitro on
solid media for more than one year prior to the initiation
of this experiment. Six of the 10 cultivars: Simmonds,
Florigon, Cambodiana, Irwin, Aroemanis, and Gadong, produced
an average of 0 to 1.1 somatic embryos per inoculation
(Tables A-3 through A-6). These cultures appeared less
responsive than similar cultures initiated by Litz and may
have lost their embryogenic potential (Litz, personal
communication). Therefore, these results may not be
indicative of the embryogenic potential of all nucellar
cultures initiated from these cultivars. The apparent loss

90
of embryogenic potential in over half of the cultivars
perhaps was less significant than the retention of
embryogenicity in the other cultures. An embryogenic culture
of 'James Saigon' has been maintained for 3 years on a low
salt medium with no apparent loss in embryogenic potential.
The use of complex organic addenda did promote somatic
embryogenesis and development in some cultivars but had
little or no effect in others (Table 4-9). In general, LCE
(20%, v/v) was more effective than CH (0.15%, w/v) or YE
(0.5%, w/v) or different combinations of these in promoting
somatic embryogenesis. Casein hydrolysate was more effective
than YE. Liquid coconut endosperm and CH appeared to be
intermediate in effectiveness between LCE and CH alone, with
the notable exception of 'Parris' which performed best with
the LCE and CH. Yeast extract with all but one cultivar
('Aroemanis') was inhibitory. The benefical effects of
complex organic addenda, such as LCE, has been well known
for many years (Steward et al., 1958b). Both LCE and CH are
rich sources of reduced nitrogen and may also contain
natural auxins and cytokinins (Dix and van Staden, 1982) ,
all of which have been implicated in affecting somatic
embryogenesis (Ammirato, 1983). Yeast extract, which
generally had a negative effect on somatic embryogenesis
from mango nucellar callus, may contain inhibitory
substances or may have been used at a concentration (0.5%)
which is inhibitory or lethal.

91
Based on the results of this experiment, the 5 most
responsive treatment combinations were evaluated empirically
as liquid maintenance media formulations with the 5 most
embryogenic genotypes (25 combinations). Many of the
treatment combinations, when used in liquid maintenance
medium, gave poor results. The globular callus often had
large necrotic regions which would spread and eventually
lead to the death of the entire culture. Treatments and
genotypes which produced actively proliferating cultures,
with little to no necrosis, were maintained for further
studies. 'James Saigon', the most embryogenic cultivar of
this study, produced a rapidly dividing, fine callus in
suspension culture completely lacking PGRs. Although these
cultures multiplied very rapidly and showed little necrosis,
they were not embryogenic when transferred to solid media
with a variety of PGR combinations and sucrose regimes.
'Parris', the second most embryogenic cultivar of this
study, produced rapidly dividing cultures with very little
necrosis in liquid modified 1/2 MS medium supplemented with
2,4-D and kinetin. Several of the healthiest appearing
cultures were multiplied and tested for their embryogenic
potential. 'Parris' embryogenic callus was used as a model
in further studies.
In summary, PGRs do not appear to be essential for
somatic embryogenesis in mango nucellar callus. Indeed, in
the highly embryogenic cultures of 'James Saigon', somatic
embryogenesis occurred quite readily in media devoid of

92
PGRs. However PGRs, particularly auxins such as 2,4-D, do
seem to stimulate callus proliferation by suppressing
differention, and in the proper form and concentration PGRs
did improve subsequent embryo production. These findings are
very much in line with the hypothesis set forth by Sharp et
al. , (1980) in which auxins function as cloning agents of
embryogenic predetermined cells and are not directly needed
for somatic embryogenesis in systems where no intermittent
callus is formed.
Results from embryogenic cultures of 'Parris' indicate
that efficient somatic embryogenesis can also be realized by
changing the exogenous auxin and cytokinin sources from
2,4-D and kinetin to NAA and 2iP. Alterations of PGR type
and/or concentration from primary to secondary and tertiary
media are quite common, particularly in the more refined
culture systems, e.g., ginseng (Chang and Hsing, 1980) and
daylily (Krikorian and Kann, 1981).
Experiment 4-2: In Vitro Somatic Embryogenesis from
'Parris' Nucellar Callus in Response to the Callus
Maintenance Medium and Major Salts Formulations
The results are shown in Table 4-10. Embryogenic
'Parris' callus maintained in a liquid modified MS medium
supplemented with 4.5 uM 2,4-D, 4.6 uM kinetin, and 2.05 mM
glutamine (callus maintenance medium B, Table 4-3), upon
transfer to solid embryogenesis medium (Table 4-3) produced
significantly more cotyledonary embryos than callus
maintained on the same BM supplemented with 4.5 uM 2,4-D and

93
Table 4-10. I_n vitro somatic embryogenesis from 'Parris'
nucellar callus in response to the callus
maintenance medium and major salts
formulations.
Somatic
embryogenesis2
Major salt
formulation
Callus
maintenance
medium A^
Callus
maintenance
medium BX
MS
3.2
6.0dw
1/2 MS
4.7
38.1c
WPM
15.8
108.9b
B-5
62.5
135.0a
„ w
Avgerage
21.6b
72.0a
zAverage total number of cotyledonary embryos per
plate, based on 6 replicates.
^Callus grown on 1/2 MS media supplemented with
4.5 uM 2,4-D and 18.6 uM Kinetin (Table 4-3).
xCallus grown on 1/2 MS media supplemented with 4.5 uM
2,4-D, 4.6 uM Kinetin, and 2.05 mM glutamine (Table 4-3).
wMeans separated by Duncan's multiple range test
at the 5% level.

94
18.6 uM kinetin (callus maintenance medium A, Table 4-3).
Morphologically, callus grown on medium with a high kinetin
concentration was more compact and less differentiated than
callus grown on medium with low kinetin and appeared to
contain slightly more necrotic areas. Callus grown with low
concentrations of kinetin and with glutamine supplements
proliferated more slowly (approximately 2/3 the rate of
callus in the other formulation) but produced somatic
embryos with better developed cotyledons.
The separate effects of low kinetin concentrations or
glutamine on the embryogenic potential of mango nucellar
callus cannot be distinguished in this experiment. However,
glutamine supplements have been used effectively in a number
of other embryogenic plant culture systems including carrot
(Kamada and Harada, 1979) and papaya (Litz and Conover,
1982). The 19 uM of kinetin used in formulation A is
significantly higher than the 0.5-5.0 uM of kinetin used in
most plant culture systems (Ammirato, 1983) . This level may
have been inhibitory to somatic embryogenesis.
The modified B-5 major salts formulation (Table 4-1)
used in the callus maintenance medium produced significantly
more somatic cotyledonary embryos than the WPM formulation,
which in turn resulted in significantly more embryos than
either MS or 1/2 MS formulations. Morphologically, the
embryos produced on B-5 medium had well developed, elongated
cotyledons without fasciations, while the embryos produced
on the MS formulations were small, compact, and had small

95
cotyledons (Fig. 4.1). In general, cultures that produced
the highest numbers of somatic embryos also had the most
normal appearing embryos.
The most striking difference between the modified B-5
formulation and the other major salt formulations is the
lack of ammonium (Table 4-1). The importance of the form in
which nitrogen is supplied to the culture medium has been
reviewed in Chapter II. Indeed, one of the major differences
between MS medium and earlier BM formulations is the
inclusion of high levels of nitrogen, especially in the form
of NH4+. Modified B-5 major salts only contain nitrogen in
the nitrate (NO^ ) form. In the modified B-5 medium used in
this study, reduced nitrogen is supplied by glutamine (2
mM), which was added to all medium formulations. Miller
(1986), using a similar modified B-5 medium without ammonium
but supplemented with malt extract, concluded that 'Key'
lime nucellar cultures initiated and grown on this medium
produced more long-term embryogenic calli than cultures
initiated and grown on Murashige and Tucker (1969) medium,
the traditional BM formulation used with Citrus.
The high concentrations of major salts in the MS
formulation appear to be inhibitory to somatic embryogenesis
from mango nucellar callus. Salt concentration cannot be
solely responsible for the low embryogenic response in this
experiment, because the response on WPM, with the lowest
major salt levels, was less than that on modified B-5
formulation.

96
Experiment 4-3: In Vitro Somatic Embryogenesis from 'Parris'
Nucellar Callus in Response to the Callus Maintenance
Medium Formulation and Sucrose Concentration
The results are shown in Table 4-11. Embryogenic
'Parris' callus multiplied in liquid callus maintenance
medium B, (Table 4-3), upon transfer to solid embryogenesis
medium (Table 4-3) containing 4% or more sucrose, produced a
significantly greater number of cotyledonary embryos than
callus multiplied in callus maintenance medium A (Table
4-3). These results are very similar to those in the
previously described experiment (4-2) except that the
overall level of somatic embryogenesis was lower.
Somatic cotyledonary embryo production from callus
grown in maintenance media B was significantly higher on
embryogenesis media containing 5 or 6% sucrose than on lower
concentrations of sucrose. Total callus production
differences between treatments with the exception of the 0%
sucrose did not appear to be significant. Somatic
embryogenesis differencies between treatments were mostly
quantitative, but somatic embryos produced on 3 and 4%
sucrose tended to be less vigorous compared to those
produced on the 5 and 6% sucrose. These results seem to
indicate that callus growth can be supported by lower levels
of sucrose (3-4%), while somatic embryogenesis and growth
require higher levels of sucrose (5-6%). In general, mango
nucellar callus responds well to relatively high sucrose
concentrations, rather than the 2-3% commonly used in other
plant culture systems ( Ozias-Atkins and Vasil, 1985).

97
Table 4-11. Ln vitro somatic embryogenesis from 'Parris'
nucellar callus in response to the callus
maintenance medium formulation and sucrose
concentration.
sucrose
concentration
(%)
Somatic
embryogenesis2
callus
maintenance
medium A^
callus
maintenance
X
medium B
O
•
o
0.0
0.0cw
2.0
0.0
0.0c
3.0
0.0
1.4c
4.0
0.8
5.2b
o
•
in
2.2
8.8a
6.0
3.4
8.0a
Avgeragew
1.1b
3.8a
2
Average total number of cotyledonary embryos per
plate based on 5 replicates.
^Callus grown on 1/2 MS media supplemented with
4.5 uM 2,4-D and 18.6 uM Kinetin (Table 4.3).
X
Callus grown on 1/2 MS media supplemented with 4.5 uM
2,4-D, 4.6 Kinetin, and 2.05 mM glutamine (Table 4-3).
w
Means separated by Duncan s multiple range test
at the 5% level.

98
Experiment 4-4: Callus Production from Nonembryogenic
'James Saigon1 Nucellar Callus in Response to the Major
Salts Formulation of the Callus Maintenance Medium
The results are shown in Table 4-12. One-year-old
nonembryogenic 'James Saigon' callus that was grown in
liquid initiation medium (Table 4-3) without any PGRs did
not produce significantly more callus in response to
different major salts formulations. Although callus
production means ranged from 15.318 g of callus per flask
with the 1/2 MS formulation to 12.834 g with the WPM, they
were not significantly different when compared using a
Duncan's multiple range test. Extrapolations made from this
morphologically and physiologically distinct nonembryogenic
callus to embryogenic callus of other cultivars are perhaps
tenuous; however, the nutrient requirements of this
nonembryogenic callus appear to be less demanding than that
of embryogenic callus.
Experiment 4-5: In Vitro Somatic Embryogenesis from
'James Saigon' Nucellar Callus in Response to the
Solidifying Agent Used in the Embryogenesis Medium
The results are shown in Table 4-13. Two-year-old
embryogenic 'James Saigon' callus multiplied in a liquid
modified B-5 medium supplemented with 4.5 uM 2,4-D and 4.6
uM kinetin (callus maintenance medium C, Table 4-3) upon
transfer to a solid embryogenesis medium solidified with
0.18% Gelrite gellan gum produced an average of 79.4 somatic
cotyledonary embryos per plate. The number of somatic

99
Table 4-12. Callus production from nonembryogenic 'James
Saigon' nucellar callus in response to the
major salts formulation of the callus
maintenance medium.
Major salts
formulation2
MS
1/2 MS
WPM
B-
â–  X
Average callus production^
(X + /- SE)
15.154 + /- 2.65 g
15.318 + /- 0.65 g
12.834 + /- 3.54 g
14.352 + /- 2.25 g
2
For formulations see Table 4-1.
^Average callus production (fresh weight) from 125 ml
^Erlenmeyer flasks, based on 5 replicates.
XModified Gamborg B-5 (Table 4-1).
Table 4-13. Iri vitro somatic embryogenesis from 'James
Saigon' nucellar callus in response to the
gelling agent used in the embryogenesis
medium.
Gelling Average number of Range of
agent somatic embryos2 embryo length
Difco Bacto-agar 48.75 b^ 1-2.9 mm
Gelrite gellan gum 79.4 a 1-3.9 mm
2
Average number of cotyledonary embryos per plate from
300 mg inoculum on 25 ml of embryogenesis medium without
2,4-D (Table 4-3), based on 8 replicates.
^Means separated using a Duncan's mutiple range test at
the 1% level.

100
cotyledonary embryos was significantly greater than on media
solidified with 0.7% Difco Bacto-agar which averaged 48.75
somatic embryos per 100 x 15 mm petri dish.
Osmolarity differencies between the 2 solid media may
be responsible for some of the observed variation. However
it is more likely that organic compounds present in the 2
solidifying products either selectively support or inhibit
the process of somatic embryogenesis from mango nucellar
callus. The use of Gelrite gellan gum resulted in more
normal plantlet production in apple shoot cultures than
other solidifying products tested (Pasqualetto et al.,
1986) .
Experiment 4-6: In Vitro Production of 'James Saigon1
Nucellar Callus in Response to a Liquid or a Solid
Maintenance Medium
The results are shown in Table 4-14. Two-year-old
embryogenic 'James Saigon' callus multiplied in a liquid
modified B-5 medium (callus maintenance medium C, Table 4-3)
produced an average of 4.9 g of callus (fresh weight) from
300 mg inoculum in 50 ml of liquid BM during a 60 day period
with 2 subcultures. This was significantly more callus than
was produced on solid BM of the same composition, regardless
of the solidifying agent during the same period with 2
subcultures. Callus maintenance medium, solidified with
Gelrite gellan gum at 0.2% produced an average of 1.2 g of
callus per 12 ml of BM, while BM solidified with 0.7% Sigma

101
Table 4-14. In vitro production of 'James Saigon'
nucellar callus in response to liquid or
solid callus maintenance medium.
, 2
Type of Callus fresh weight
culture (g per plate or flask)
Solid, stationary cultures
Sigma agar gum (0.7%) 0.9 c^
Gelrite gellan gum (0.2%) 1.2 b
Suspension culturesx 4.9 a
z300 mg of callus inoculated onto 12 ml of solid BM or
into 50 ml of liquid BM (callus maintenance medium C
Table 4-3) 2 subcultures, data collected at 60 days,
average of 10 replicates.
â– ^Means separated using a Duncan's multiple range test
at the 1% level.
x50 ml of liquid BM in 125 ml Erlenmeyer flasks on
a rotary shaker.

102
agar gum produced an average of 0.9 g of callus under the
same conditions.
These results indicate that callus proliferation is
enhanced in suspension cultures in comparison with the use
of solid medium. Suspension cultures produced over 4 times
the amount of callus obtained from solid media during the
same time interval; however, the embryogenic potential of
the callus produced under these different culture systems
was not evaluated in this experiment.
Conclusions
1. Nucellar callus cultures from 10 mango cultivars varied
considerably in their embryogenic response. Some
cultures appear to have lost their embryogenic
competency with time, while other cultures have
remained embryogenic for over 3 years.
2. No single combination of PGRs was optimum for somatic
embryogenesis in all the genotypes, although auxin and
cytokinin concentrations in the range of 0-20 uM
produced the best results for most of the cultivars
tested.
3. A liquid callus maintenance media supplemented with 5
uM 2,4-D, 5 uM kinetin, 2 mM glutamine, and vitamins
(callus maintenance medium B, Table 4-3) resulted in
the production of large quantities of embryogenic
callus in several mango cultivars.

103
4. Four times as much callus (fresh weight) could be
produced using a liquid shaker culture system in
comparison with a solid stationary culture system.
5. A modified Gamborg B-5 medium (embryogenesis medium,
Table 4-3) resulted in efficient somatic embryogenesis
from 'Parris' and 'James Saigon' nucellar callus.
6. Sucrose concentrations of 5 or 6% resulted in a higher
frequency of somatic embryogenesis than lower sucrose
concentrations.
7. Media solidified with Gelrite gellan gum resulted in
significantly more efficient somatic embryogenesis than
media solidified with Sigma agar gum or Difco
Bacto-agar.

104
Figure 4-1
'Parris' somatic embryogenesis in response
to modified B-5, 1/2 MS, WPM, and MS major
salts formulations used in experiment 4-2.

CHAPTER V
SOMATIC EMBRYO MATURATION, GERMINATION,
AND PLANTLET FORMATION
Introduction
The initiation and multiplication of embryogenic mango
nucellar callus cultures were described in Chapter IV.
Somatic embryos produced in vitro according to the protocols
of the previous chapter were utilized for the studies
described in this chapter.
Liquid coconut endosperm (LCE) and CH, prepared
according to Table A-l, were used in many of the embryo
maturation and germination media formulations. Basal medium
(BM) containing LCE was adjusted to a pH of 6.8-8.4
(depending on the pH of the LCE) prior to autoclaving.
Filter-sterilized LCE was added to the cooling media to
bring the final BM pH to approximately 5.75.
Care was taken to minimize the experimental error due
to variability within the populations of somatic embryos
used as inocula for the embryo maturation and germination
experiments. Only large (over 5 mm in length), well
developed somatic embryos were transferred to embryo
maturation or germination media. When possible all the
embryos for a given experiment were derived from a single
105

106
treatment of a somatic embryogenesis experiment. When this
was not possible all the embryos within a single replication
were at least derived from a single source population.
Materials and Methods
Experiment 5-1: In Vitro Somatic Embryo Production and
Maturation from 'James Saigon1 Nucellar Callus in
Response to Culture Regime
Two-year-old embryogenic 'James Saigon1 callus
maintained on solid modified MS consisting of 1/4 MS major
salts, MS
minor
salts,
2.5%
sucrose, 1.0
mM (150
mg l-1)
glutamine,
0.55
mM (100
mg 1
1) inositol,
, 100 uM
(28.7
mg
l-1) FeSO.
7H-0
, and 1.
2 uM
(0.4 mg 1_1)
thiamine
HC1
was
transferred to modified liguid B-5 callus maintenance medium
(callus maintenance medium C, Table 4-3) for multiplication.
Somatic embryogenesis occurred in both liguid and on solid
modified B-5 medium (embryogenesis medium, Table 4-3). Two
gelling agents were used in the solid embryogenesis medium,
Gelrite gellan gum (0.2% w/v) and Sigma agar gum (0.7%).
Five somatic embryo maturation culture regimes were
tested for their effects on embryo development as measured
by fresh weight increases. The 5 regimes were somatic
embryos produced and matured in liguid medium, somatic
embryos produced on Gelrite and matured in liguid media,
somatic embryos produced in liquid and matured on Gelrite
media, embryos produced and matured on Gelrite medium, and
embryos produced and matured on Sigma agar medium. All
embryo maturation media contained modified B-5 major salts,

107
MS minor salts, 20% (v/v) LCE, 1.5% sucrose, 0.025% CH, 2.05
mM (300 mg 1 glutamine, 0.55 mM (100 mg 1 inositol,
53.3 uM (4 mg 1 1) glycine, 3.0 uM (1 mg 1 1) thiamine HC1,
4.1 uM (0.5 mg 1 nicotinic acid, and 2.4 uM (0.5 mg l-1)
pyridoxine HC1.
Somatic embryos (500 mg) produced on either solid or in
liquid embryogenesis medium were transferred to either 50 ml
of liquid sterile BM in 125 ml Erlenmeyer flasks or to 12 ml
of solid sterile BM in 60 x 15 ml plastic petri dishes.
Erlenmeyer flasks were maintained at 100 r.p.m. on rotary
shakers, while petri dishes were stored in plastic boxes.
Cultures were maintained on/in embryogenesis medium for 3
weeks and then transferred to embryo maturation media for 2
1/2 weeks. Cultures were incubated at room temperature
(24-26 C) without supplemental light. The experimental
design was completely randomized with 5 treatments and 6
replicates. Treatments were scored by measuring total fresh
weight per flask 5 1/2 weeks after the experiment was
started.
Experiment 5-2: In Vitro 'Parris' Somatic Embryo Maturation
in Response to Sucrose Concentration and ABA in a Liquid
Embryo Maturation Medium
One-year-old embryogenic 'Parris' callus, initiated and
maintained on a solid modified MS medium (callus initiation
medium, Table 4-3) was transferred to liquid callus
maintenance medium B (Table 4-3) for multiplication and
somatic embryogenesis.

108
Two concentrations of sucrose (5 and 10% w/v) and 1
concentration of ABA (5 uM) were tested for their effect on
embryo maturation. Only the concentration of sucrose and ABA
were altered between treatments. All embryo maturation media
contained 1/2 MS major salts, MS minor salts, 0.025% CH,
2.05 mM (300 mg 1 1) glutamine, 0.55 mM (100 mg 1_1)
inositol, 53.3 uM (4 mg 1 1) glycine, 3.0 uM (1 mg l-1)
thiamine HC1, 4.1 uM (0.5 mg 1 nicotinic acid, and 2.4 uM
(0.5 mg 1 â– *â– ) pyridoxine HC1.
Globular somatic embryos and callus (1.5 g/inoculum)
were transferred to 50 ml of sterile liquid embryo
maturation medium in 125 ml Erlenmeyer flasks. Cultures were
maintained at room temperature (24-26 C) on a rotary shaker
at 100 r.p.m. without supplemental light. Cultures were
subcultured to fresh media after 21 days. The experimental
design was a 2 x 2 factorial with 4 replicates. Treatments
were scored by weighing the total embryo mass (fresh weight)
per flask 30 days after transfer.
Experiment 5-3: In Vitro 'Parris' Somatic Embryo Maturation
in Response to Sucrose Concentration and Supplements to
the Embryo Maturation Medium
One-year-old embryogenic 'Parris' callus, initiated and
maintained on a solid modified MS medium (callus initiation
medium, Table 4-3) was transferred to a liguid callus
maintenance medium (callus maintenance medium B, Table 4-3)
for multiplication. Somatic embryos were harvested 45 days
after the callus was transferred to solid modified B-5

109
embryogenesis medium (embryogenesis medium, Table 4-3)
solidified with 0.75% (w/v) Difco Bacto-agar.
Seven embryo maturation media formulations consisting
of no addenda, 20% (v/v) LCE, 0.025% (w/v) CH, LCE with CH,
LCE with ABA (3 uM), CH with ABA, and LCE with CH and ABA
and 3 sucrose concentrations (3, 6, and 9%) were tested for
their effects on embryo maturation. Only the embryo
maturation medium formulation and the sucrose concentration
were altered between treatments. All embryo maturation media
contained modified B-5 major salts (Table 4-1), MS minor
salts (Table 4-2), 0.75% Difco Bacto-agar, 2.05 mM (300 mg
1 "*") glutamine, 0.55 mM (100 mg 1 ^) inositol, 53.3 uM (4 mg
1 "'") glycine, 3.0 uM (1 mg 1 ^) thiamine HC1, 4.1 uM (0.5 mg
1 'L) nicotinic acid, and 2.4 uM (0.5 mg 1 1) pyridoxine HC1.
Approximately 10 somatic embryos (5-8 mm long) along
with a small amount of
associated
callus
were transferred
to
12 ml of solid sterile
embryo maturation
medium in
60 x
15
mm plastic petri dishes
. Cultures
were
incubated
at room
temperature (24-26 C)
in clear
plastic boxes
with
no
supplemental light. The experimental design was a 7 x 3
factorial with 5 replicates. Treatments were scored 30 days
after transferring by assigning a 0-9 rating (Table 5-1)
based on embryo size and developmental stage.

110
Table 5-1.
In vitro somatic embryo maturation
rating scale used in experiment 5-3z.
0.
all embryos necrotic,
no growth
1.
1/2 of embryos alive,
but no growth
2.
most embryos alive, average 5 mm
3.
embryos average 8 mm
4.
several embryos 10 mm
5.
several embryos 15 mm
6.
most embryos 15 mm
7.
several embryos 20 mm
8.
most embryos 20 mm
9.
most embryos 21-25 mm
2
Cultures were evaluated 30 days after subculturing.
Embryos were approximately 3-5 mm at the time of
transfer.

Ill
Experiment 5-4: In Vitro 'Parris' Somatic Embryo Maturation
in Response to Soilidifying Agent and ABA in the Embryo
Maturation Medium
One-year-old embryogenic 'Parris' callus, initiated and
maintained on solid modified MS medium (callus initiation
medium, Table 4-3) was transferred to liquid callus
maintenance medium B (Table 4-3) for multiplication. Somatic
embryos were formed on solid modified B-5 embryogenesis
medium (Table 4-3) solidified with 0.75% Difco Bacto-agar.
Gelrite gelling gum [0.175% (w/v)] and Difco Bacto-agar
(0.75%), as well as the addition of 3 uM ABA to the medium
were tested for their effect on somatic embryo maturation.
All somatic embryo maturation media contained modified B-5
major salts, MS minor salts, 20% (v/v) LCE, 4.5% sucrose,
0.025% (w/v) CH, 2.05 mM (300 mg 1 ^) glutamine, 0.55 mM
(100 mg 1 1) inositol, 53.3 uM (4 mg 1 ^) glycine, 3.0 uM (1
mg 1 ) thiamine HC1, 4.1 uM (0.5 mg 1 ^) nicotinic acid,
and 2.4 uM (0.5 mg 1 1) pyridoxine HC1.
Approximately 50 somatic embryos together with a small
amount of associated calli were transferred to 25 ml of
sterile embryo maturation medium in 100 X 15 mm plastic
petri dishes. Cultures were incubated at room temperature
(24-28 C) with a 16 hr photoperiod (76 umol s ^ m ^)
provided by cool-white fluorescent lamps. The experimental
design was a 2 X 2 factorial with 7 replicates. Treatments
were scored visually 30 days after the embryos had been
transferred by assigning a 0-9 rating (Table 5-2) based on
embryo size and development.

112
Table 5-2. _In vitro somatic embryo maturation
rating scale used in experiment 5-4z.
0.
1.
2.
3.
4.
5.
6.
7.
8.
9.
all embryos necrotic
1/4 of embryos alive
1/4 of embryos 5 mm,
1/2 of embryos 5 mm,
1/2 of embryos 5 mm,
cotyledons
1/2 of embryos 7 mm,
cotyledons
1/2 of embryos 10 mm
cotyledons
3/4 of embryos 10 mm
cotyledons
3/4 of embryos 15 mm
cotyledons
3/4 of embryos 20 mm
cotyledons
, no growth
but no measurable growth
most with polycotyledony
many with polycotyledony
most with well developed
most with well developed
, most with well developed
, most with well developed
, most with well developed
, most with well developed
z
Cultures were evaluated 30 days after subculturing.
Embryos were approximately 3-5 mm at the time of
transfer.

113
Experiment 5-5: In Vitro 'Parris' Somatic Embryo
Germination and Shoot Formation in Response to the
Embryo Germination Medium Formulation
One-year-old embryogenic 'Parris' callus, initiated and
maintained on solid modified MS medium (callus initiation
medium, Table 4-3) was transferred to liquid callus
maintenance medium B (Table 4-3) for multiplication. Embryos
were formed on modified B-5 embryogenesis medium (Table 4-3)
solidified with 0.75% Difco Bacto-agar.
Four somatic embryo germination medium formulations
consisting of modified B-5, 1/2 modified B-5, 1/2 modified
B-5 & LCE (20%, v/v) & CH (0.025% w/v), and peach
germination medium (Table A-8) were tested for their effect
on somatic embryo germination and shoot development. Only
the embryo germination medium formulation was altered
between treatments
. All
treatments
contained
3%
sucrose
0.59% Difco Bacto-
agar,
2.08 uM (500
mg
r1)
CaNC
>3
4H20
-1
-1
1.37 mM (200 mg
1 )
glutamine,
0.27
mM
(50
mg
1 1
, -i
-1
inositol, 53.3 uM
(4 mg
1 ) glycine
, 3.0
uM
(1
mg
1
thiamine HC1, 4.1 uM (0.5 mg 1 ) nicotinic acid, and 2.4 uM
(0.5 mg 1 â– *â– ) pyridoxine HC1.
Between 3-5 large somatic embryos (10-20 mm long) were
placed on 35 ml of solid sterile embryo germination medium
in 60 x 60 mm glass containers or single somatic embryos
were placed individually on 25 ml of media in 30 x 150 mm
test tubes. Cultures were incubated in growth chambers at a
constant temperature of 25 C, with a 16 hr photoperiod

114
-1 -2
provided by low intensity illumination (76 umol s m )
from cool-white fluorescent lamps. The experimental design
was completely randomized with 4 treatments and 10
replicates. Treatments were scored 2 months after transfer
by recording the total number of germinated embryos, as
determined by the emergence of a root radical and by
subsequent shoot formation. Germinated somatic embryos were
transferred to a commercial potting mixture, watered with
dilute basal salts, and covered with plastic to prevent
dessication. Pots were incubated in a growth chamber with
artifical lighting. The effectiveness of different
treatments on plantlet formation was not measured because of
lack of uniform planting procedures.
Experiment 5-6: In Vitro 'Parris' Somatic Embryo
Germination and Shoot Formation in Response to the
Embryo Germination Medium Formulation
One-year-old embryogenic 'Parris' callus, initiated and
maintained on solid modified MS medium (callus initiation
medium, Table 4-3) was transferred to a liquid callus
maintenance medium B, (Table 4-3) for multiplication.
Somatic embryogenesis occurred on modified B-5 embryogenesis
medium (Table 4-3) solidified with 0.75% (w/v) Difco
Bacto-agar.
Three embryo germination medium formulations, i.e.,
modified B-5, modified B-5 with LCE (20% v/v) and CH (0.025%
w/v), and modified B-5 wtih LCE, CH and GA (3.3 uM) were
tested for their effect on somatic embryo germination and

115
development. All treatments contained modified B-5 major
salts, MS minor salts, 3.0% (w/v) sucrose, 0.19% (w/v)
Gelrite gellan gum, 2.05 mM (300 mg l-1) glutamine, 0.55 mM
(100 mg 1 1) inositol, 53.3 uM (4 mg 1 glycine, 3.0 uM (1
mg 1 ''') thiamine HC1, 4.1 uM (0.5 mg 1 nicotinic acid,
and 2.4 uM (0.5 mg 1 1) pyridoxine HC1.
Seven-12 embryos were transferred to 30 ml of solid
sterile embryo germination medium in 15 x 100 mm plastic
petri dishes. Cultures were incubated in a growth chamber at
a constant temperature of 27 C with a 16 hr photoperiod (76
-1-2
uM s m ) provided by cool-white fluorescent lamps. The
experimental design was completely randomized with 4
treatments and 5 replicates. Treatments were scored 3 weeks
after transfer by recording the percentage of germinated
somatic embryos, as determined by emergence of a root
radical and the formation of a shoot. After recording data
from treatments, germinated somatic embryos were transferred
to a commercial potting mixture watered with dilute basal
salts and covered with plastic to prevent dessication. Pots
were incubated in a growth chamber with artifical lighting.
No data was recorded on plantlet formation because of lack
of uniform planting procedures.

116
Results and Discussion
Experiment 5-1: In Vitro Somatic Embryo Production and
Maturation from 'James Saigon1 Nucellar Callus in
Response to Culture Regime
The results are shown Table 5-3. Embryogenic 'James
Saigon' callus multiplied in liquid callus maintenance
medium C (Table 4-3) produced significantly more and larger
somatic embryos, as measured by fresh weight, in a culture
regime consisting of liquid embryogenesis medium followed by
liquid embryo maturation medium. The second highest
treatment mean was obtained from a culture regime in which
somatic embryogenesis occurred on media solidified with 0.2%
Gelrite gellan gum followed by maturation in liquid medium.
The lowest average fresh weights were obtained from a
culture regime in which somatic embryogenesis and maturation
both occurred on media solidified with Sigma agar gum
(0.7%). No germination studies were conducted to evaluate
the competency of these somatic embryos to produce plants.
Somatic embryos produced in liquid media exhibited more
morphological abnormalities than those produced on solid
media. These included a higher percentage of polycotyledony,
cotyledons with multiple fasciations, and vitrification.
Most of the embryos grown in liquid were quite large (2 cm).
On Gelrite, the embryos were noticably smaller, but 20% did
reach the same size as the liquid-grown embryos. Embryos
grown on Sigma agar were substantially smaller (under 1 cm)
and less vigorous, but there was less vitrification.

117
Table 5-3. In vitro somatic embryo production and
maturation from 'James Saigon' nucellar
callus in response to the culture regime.
• Z V
Culture regime Average fresh weight^
(g per flask or dish)
liquid-liquid
10.8
X
a
Gelrite-liquid
7.8
b
Gelrite-Gelrite
2.5
c
1iquid-Gelrite
2.1
c
Sigma agar-Sigma agar
1.3
c
2
Culture regime includes both embryogenesis and maturation
on either a solid medium (0.2% w/v Gelrite gellan gum or
0.7% Sigma agar gum) or in liquid medium.
^Average of 6 replicates, measurements taken 5 1/2 weeks
after initial transfer to embryogenesis medium.
XMeans separated using a Duncan's multiple range test at
the 5% level.

118
In general, all of the 'James Saigon' somatic embryos
were poorly formed in comparison with 'Parris' embryos to be
described in experiments 5-4 to 5-6. Vitrification, was
extreme with nearly all treatments and replicates being
affected. Although no germination studies were conducted
with these somatic embryos, they did not appear to be
physiologically competent to form plants. No embryos of the
approximately 1000 observed showed any chlorophyll
development in contrast with 'Parris' somatic embryos.
Experiment 5-2: In Vitro 'Parris' Somatic Embryo Maturation
in Response to Sucrose Concentration and ABA in a Liquid
Embryo Maturation Medium
The results are shown in Table 5-4. 'Parris' somatic
embryos were produced in liquid callus maintenance medium B
(Table 4-3) and transferred to modified 1/2 MS liquid embryo
maturation medium supplemented with LCE, CH, glutamine, and
vitamins. Somatic embryos were significantly more numerous
and larger as measured by fresh weight when the sucrose
concentration of the liquid maturation medium was 5% than
when it was 10%. The use of 10% sucrose and the addition of
ABA to the medium suppressed both somatic embryogenesis and
subsequent development. Likewise, somatic embryos grown in
these 2 treatments exhibited slightly more necrosis than
embryos matured in 5% sucrose without ABA.
In general, somatic embryos produced in this
experiment, relative to those produced in the following
experiments, were poorly developed and had aberrant

119
Table 5-4. Ln vitro 'Parris' somatic embryo maturation
in response to sucrose concentration and ABA
in liquid embryo maturation medium.
Treatment means (g per flask)2
ABA Sucrose concentration
5%
10%
0 UM
3.55 ay
3.15 a
5 UM
3.10 b
2.35 c
zAverage fresh weight per flask, 30 days after transferring
callus (1.5 g inoculum per 50 ml BM) to embryo maturation
medium, based on 4 replicates.
yMeans separated using a Duncan's multiple range test at
the 5% level.

120
cotyledons. No germination studies were conducted with these
somatic embryos.
Experiment 5-3: In Vitro 'Parris' Somatic Embryo Maturation
in Response to Sucrose Concentration and Supplements to
the Embryo Maturation Medium
The results are shown in Table 5-5. 'Parris' somatic
embryos produced on solid embryogenesis medium (Table 4-3)
and transferred to solid modified B-5 embryo maturation
medium were significantly larger and better developed as
determined by embryo maturation ratings (Table 5-1) in 9 of
the 21 treatment combinations of medium supplements and
sucrose concentrations. The 9 treatment combinations
receiving the highest ratings were in decending order: LCE
with CH and ABA at 3% sucrose, LCE and CH at 6%, LCE at 6%,
LCE and ABA at 3%, BM without supplements at 6%, LCE and ABA
at 6%, CH at 6%, LCE with CH and ABA at 6%, and LCE and CH
at 3%.
In general, all somatic embryos from this experiment
were very vigorous, healthy, and had few cotyledon
abnormalities. Differences between treatments were primarily
due to embryo size and no treatment combination produced
abnormal embryos in all the replicates. The low amount of
statistical significance between most treatments was in part
due to the heterogeneous nature of the inocula. Low vigor
inocula randomly distributed throughout the experiment
probably resulted in a high experimental error.

121
Table 5-5. _In vitro 'Parris' somatic embryo maturation in
response to sucrose concentration and supplements
to the embryo maturation medium.
Medium
formulation
Treatment means'
Sucrose concentration
3% 6% 9%
B-5
3.6
5.2
4.2
B-5
S.
LCE
3.6
5.8
3.0
B-5
S<
CH
3.2
4.8
3.4
B-5
&
LCE S, CH
4.6
5.8
4.0
B-5
St
LCE S, ABA
5.6
5.0
3.0
B-5
S<
CH St ABA
4.2
3.8
2.8
B-5
St
LCE S< CH S. ABA
6.0
4.8
3.6
zAverage of 5 replicates based on a 0-9 rating (Table 5-1).
A +/- 1.4 was judged nonsignificant using a Duncan's
multiple range test at the 5% level.

122
Abscisic acid did not statistically improve somatic
embryo maturation, even though the highest rating score was
obtained on medium supplemented with ABA, LCE, and CH at 3%
sucrose. Treatments containing ABA were more effective with
3% sucrose than with higher sucrose concentrations. Sucrose
at 6% resulted in better embryo maturation than treatments
containing 3 or 9% sucrose, with the exception mentioned
above. The addition of LCE alone to the BM was more
effective for somatic embryo maturation than CH alone. The
addition of LCE and CH together gave a slightly better
response than LCE alone even though the differences were not
statistically significant.
Experiment 5-4; In Vitro 'Parris' Somatic Embryo Maturation
in Response to Solidifying Agent and ABA in the Embryo
Maturation Medium
The results are shown in Table 5-6. 'Parris' somatic
embryos were produced on solid embryogenesis medium (Table
4-3) and transferred to solid modified B-5 embryo maturation
medium with 4.5% sucrose and supplemented with LCE, CH,
glutamine, and vitamins. Maturation medium produced
significantly larger, well developed embryos when solidified
with Gelrite gellan gum (0.175% w/v) than on medium
solidified with Difco Bacto-agar (0.75%). The addition of
ABA (3 uM) to the maturation medium did not significantly
improve embryo maturation as measured by the maturation
rating scale in Table 5-2.

123
Table 5-6. _In vitro 'Parris' somatic embryo maturation
in response to the solidifying agent and ABA
in the embryo maturation medium.
Treatment means2
Gelrite^
Agarx
With ABAW
6.0 av
1.7 b
Without ABA
5.6 a
2.7 b
Average
5.8
2.2
zAverage of 7 replicates, using a scale of 0-9 (Table 5-2).
-^Gelrite gellan gum (0.175%) from Kelco.
xBacto-agar (0.75%) from Difco.
W3 uM ABA.
vMeans seperated by Duncan's multiple range test, at
the 1% level.

124
Somatic embryos on maturation medium containing Gelrite
were whiter and had larger, more extended cotyledons than
embryos on Difco Bacto-agar (see Fig. 5-1). The Difco
embryos frequently were thin and they were often necrotic at
their bases.
Experiment 5-5: In Vitro 'Parris' Somatic Embryo Germination
and Shoot Formation in Response to the Embryo Germination
Medium Formulation
The results are shown in Table 5-7. 'Parris' somatic
embryos produced on solid embryogenesis medium (Table 4-3)
had a 63% germination rate, as determined by the emergence
of a root radical, on modified B-5 embryo germination medium
solidified with Difco Bacto-agar and supplemented with LCE
and CH. This was a significantly higher germination rate
than on the modified B-5 medium with no supplements, on 1/2
modified B-5 medium with no supplements, or on peach
germination medium (Table A-8). Shoot formation, as
determined by the presence of a swollen distinct shoot bud
or extended shoot stem, was highest on the modified B-5
medium with no supplements.
On germination media somatic embryos typically enlarged
slightly followed by a gradual greening of the entire
embryo. Germination occurred by the emergence of a single
tap root which often grew 2-3 cm before forking into two or
more branch roots. Germination generally occurred 2-3 weeks
after transfer; however sporadic germination was observed up
to 8 weeks. Shoot emergence almost invariably followed 1-3

125
Table 5-7. Irt vitro 'Parris' somatic embryo germination
and shoot formation in response to the embryo
germination medium formulation.
Formulation2
Reps.
Total no.
of embryos
Germ.
(%)
Shoots
(%)
B-5
6
36
35 bY
9 a
1/2 B-5
3
19
27 c
0 c
1/2 B-5+LCE+CHX
6
35
63 a
2 b
Peach germ. med.w
4
21
34 b
0 c
ZA11 media was solidified with Difco Bacto-agar.
V
1 Means separated using a Duncan's multiple range test at
the 5% level.
XLCE 20% (v/v), CH 0.025% (w/v).
w
For formulation see Table A-7.
Table 5-8. Ln vitro 'Parris' somatic embryo germination
and shoot formation in response to the embryo
germination medium formulation.
Formulation2
Reps.
Total no.
of embryos
Germ.
(%)
Shoots
(%)
B-5
5
47
38 aY
4
B-5 + LCE + CH
15
197
38 a
5
B-5+LCE+CH+GA
5
49
15 b
2
ZLCE 20% (v/v), CH 0.025% (w/v), GA 3.3 uM, all media was
solidified with Gelrite gellan gum.
^Means seperated using a Duncan's multiple range test at
the 5% level.

126
weeks after root emergence. Only somatic embryos with good
root systems produced shoots. Several plants with multiple
shoots were observed and these generally had low vigor.
Occassionally plants were observed with well developed root
systems but poorly developed shoots. Data were not presented
on plantlet survival from individual treatments because no
protocol had been devised for the efficient transfer of
germinated somatic embryos to soil.
Experiment 5-6: In Vitro 'Parris' Somatic Embryo
Germination and Shoot Formation in Response the
Embryo Germination Medium Formulation
The results are shown in Table 5-8. 'Parris' somatic
embryos produced on solid embryogenesis medium (Table 4-3),
upon transfer to modified B-5 germination medium with
Gelrite gellan gum, had a 38% germination rate with or
without the addition of LCE and CH. The addition of GA to
LCE- and CH-supplemented media reduced germination
significantly to 15%. Germination medium supplemented with
LCE and CH produced more shoots, although not significantly
more than either of the other two treatments.
Morphologically the embryos in this experiment appeared
to be as normal as embryos from the previous experiment (see
Figures 5-2 and 5-3), although the germination rates for the
best treatment were much lower, i.e., 38 vs. 63%. The major
differences between these 2 treatments were that full
strength modified B-5 salts and Gelrite gellan gum were used
in this experiment whereas 1/2 strength modified B-5 salts

127
and Difco Bacto-agar were used in the previous experiment.
Somatic embryos that produced roots and/or shoots on the
Gelrite media of this experiment had turned green in
contrast to the somatic embryos that did not germinate,
which tended to be cream to pink.
Data were not presented on plantlet survival from
individual treatments because no protocol had been devised
for the efficient transfer of germinated somatic embryos to
soil. Many of the first germinated somatic embryos were lost
to unidentified fungal pathogens. Other plantlets lost vigor
and died when watered with dilute solutions of commerical
plant food. Somatic embryos seemed to grow best when
transferred immediately to soil after root emergence (2-3
cm) rather than after shoots were formed in vitro. Good
plantlet survival was obtained by regularly spraying
germinated somatic embryos with a 1/4 concentration
solutions of B-5 salts, glutamine, and vitamins without
sucrose. Germinated somatic embryos were planted in 4 inch
pots of commercial potting mixture and covered with plastic
to prevent dessication. To avoid fungal contamination,
weekly sprays of Captan were administered. Young plantlets
commonly produced several sets of poorly formed, narrow
leaves, which eventually abscised, prior to the formation of
normal healthy leaves. Plantlets became established slowly
but eventually developed into normal plants (see Fig. 5-4).

Conclusions
Solid medium was superior to liquid medium for somatic
embryogenesis and maturation, as determined by a
morphological rating system based on embryo size and
cotyledon development.
Somatic
embryo
maturation
was most
successful
on
modified
solid
B-5
medium
supplemented with
LCE
(20% v/v)
o
ac
o
.025%
w/v) ,
ABA (3 uM),
and 3%
(w/v)
sucrose; LCE, CH, and 6% sucrose; or LCE and 6% sucrose
in comparison with the other formulations tested.
Somatic embryos with fewer developmental abnormalities
occurred on modified B-5 medium supplemented with LCE,
CH, glutamine, vitamins, and 4.5% sucrose, and
solidified with Gelrite gellar gum (0.175% w/v) than
on the same medium solidified with Difco Bacto-agar
(0.75%). The addition of ABA (3 uM) to the embryo
maturation medium did not improve embryo development.
Sixty three percent of 'Parris' somatic embryos
successfully germinated in vitro, as determined by the
emergence of a root radical on modified 1/2 strength
B-5 germination medium supplemented with LCE, CH,
glutamine, vitamins, 3% sucrose, and 0.59% Difco
Bacto-agar. The highest percentage of somatic embryos
which developed shoot buds was obtained on modified
full strength B-5 germination medium without
supplements. Good germination (38%) was also achieved
on modified B-5 medium solidified with Gelrite gellan

129
gum with or without the LCE and CH supplements in
comparison with the other formulations tested.

Figure 5-1 'Parris' somatic embryo maturation in response
to Difco Bacto-agar (top), Gelrite gellan gum
(bottom), and ABA (right) in experiment 5-4.
130

Figure 5-2 A culture of germinating 'Parris' somatic embryos
on a modified B-5 germination medium solidifyed
with Gelrite gellan gum and supplemented with LCE
and CH (experiment 5-6).
131

Figure 5-3 Germinated 'Parris' somatic embryos from Figure 5-2
ready to be transferred to the soil.
132

Figure 5-4 Young 'Parris' plants regenerated via _in vitro
somatic embryogenesis, established in the soil.
133

CHAPTER VI
SUMMARY AND CONCLUSIONS
Histological Investigations
Histological investigations with polyembryonic 'James
Saigon' and 'Parris' ovules confirmed the nucellar origin of
adventive embryos. Adventive nucellar embryos arose in the
micropylar half of the ovule prior to the cellularization of
the endosperm. The adventive embryos appeared to pass
through developmental stages similar to those of young
zygotic embryos of other plant species.
Embryogenic calli could be induced from both micropylar
and chalzal portions of excised nucelli in vitro, and were
therefore not restricted to those regions directy associated
with adventive embryony, i.e., the micropyle. Nucellar
callus initiated on solid medium and multiplied in
suspension culture produces a cream-dark brown, compact,
nonfriable, globular callus. At low magnification this
callus can be seen to be composed of 5-15 mm diameter
clusters of globular to early cotyledonary stage somatic
embryos.
Cross sections through this globular callus idicated
that each cluster is composed of a central core, consisting
of a degenerating somatic embryo. From the periphery of this
134

135
degenerating somatic embryo newly formed somatic embryos
radiate outward. I_n vitro somatic embryogenesis in mango
appears to be very similar to that described from Citrus
nucellar callus. In both systems embryogenesis usually
occurs directly from the epidermal layer of preformed
somatic embryos and no subculturable, undifferentiated
callus is produced.
In mango nucellar callus, the addition of 2,4-D and
kinetin to the suspension culture medium appears to suppress
both vascularization and meristematic differentiation in
developing somatic embryos and a wide range of developmental
abnormalities are seen. These abnormalities include somatic
embryos with poorly formed epidermis and noncompact
morphology, polycotyledony, and secondary budding. Somatic
embryogenesis occurs from the epidermal cells of preformed
somatic embryos. The young proembryos proliferate for a
short time, with a small percentage developing into globular
and heart-shaped embryos. These somatic embryos in the
presence of 2,4-D and kinetin are generally developmentally
arrested prior to the differentiation of embryonic organs
and secondary epidermal budding is initiated.
Observations of the epidermal layer of globular callus
during somatic embryogenesis reveal that the epidermis loses
its continuity as highly cytoplasmic cells with prominent,
densely staining nuclei become distinguishable. These cells
eventually form colonies of proembryonic cells. This pattern
of somatic embryogenesis is in conflict with the hypothesis

136
of Sharp et al. (1980), in which 2,4-D is envisioned as a
cloning agent for embryogenic predetermined cells with no
intermittent de- or re-differentiation event occurring. It
also appears to be different from histological work done by
Konar et a_l. (1972) on somatic embryogenesis in Ranunculus,
in which morphologically distinguishable cells are present
in the explant (stem epidermis) prior to somatic
embryogenesis.
Histology performed on somatic embryos grown on solid
medium reveals a closed vascular system with bipolar
meristems. This work provides conclusive evidence that
regeneration from mango nucellar callus occurs via somatic
embryogenesis and corroborates the reports of Litz et al.
(1982) .
Tissue Culture Studies
A great deal of genotypic variability is observed in
the embryogenic response of nucellar callus to various PGRs.
Although the proper combination and concentrations of PGRs
improved the efficiency of somatic embryogenesis in vitro,
there does not seem to be an absolute requirement for
exogenous PGRs in the highly embryogenic cultures. The use
of complex organic addenda, such as LCE and CH, did improve
somatic embryogenesis and subsequent embryo development in
some cultivars. Polyembryonic 'Parris' and 'James Saigon'
nucellar calli were particularly embryogenic and were used
for all of the regeneration studies.

137
The following is a summary of the protocol developed
with 'Parris' nucellar cultures to regenerate plants in
vitro via somatic embryogenesis. All media contain modified
B-5 major salts [lacking (NH4)2 SO^] (Table 4-1), MS minor
salts (Table 4-2), 2 mM (300 mg *) glutamine, 0.5 mM (100
mg *) myo-inositol, 50 uM (4 mg *) glycine, 4 uM (0.5 mg-*)
nicotinic acid, 3 uM (2 mg *) thiamine HC1, and 2 uM (0.5
mg *) pyridoxin HC1. Media are solidified with 0.2% (w/v)
Gelrite gellan gum.
1. Embryogenic nucellar callus is initiated in the dark
from excised nucelli or longitudinally bisected ovules
on basal medium containing 5% (w/v) sucrose, 5 uM (1
mg *) 2,4-D, and 5 uM (1 mg *) kinetin.
2. Proliferating embryogenic callus is transferred to
liquid initiation medium for multiplication and
maintained on a rotary shaker at 100 r.p.m. without
supplemental light.
3. Nucellar callus is transferred from suspension culture
to solid medium containing 5% sucrose, 10 uM (2 mg *)
NAA, and 5 uM (1 mg *) 2iP for somatic embryogenesis.
4. Five mm length somatic embryos are transferred to solid
basal medium containing 4.5% sucrose, 0.025% (w/v)
casein hydrolysate (CH), and 20% (v/v) filter-
sterilized liquid coconut endosperm (LCE). The pH of
the basal medium is adjusted prior to autoclaving so
that the addition of the LCE will bring the final

138
medium pH to 5.7. Embryos are matured under
supplemental light.
5. Ten to 20 mm length somatic embryos are germinated on
1/2 strength basal medium containing 3% sucrose under
supplemental light.
6. Germinated somatic embryos (root radicle 2 cm length)
are transferred to commercial potting mixture and
watered with 1/5 strength basal salts supplemented with
1 mM (250 mg”1) Ca(NC>3)2 4H20, 75 uM (20 mg”1) FeS04
7H20, and 75 uM (30 mg *) Na2 EDTA. The pots are
covered with plastic bags to prevent desiccation and
placed under supplemental light. Germinated embryos and
plantlets are watered weekly with fungicide [1% (w/v)
Captan]. After plants have formed several sets of
leaves the plastic is gradually removed.
Conclusions
The application of this protocol to all mango
cultivars and cultures has not been demonstrated. It is
not unlikely that modifications will be needed in the
PGRs used to facilitate regeneration with other
cultivars, e.g., 'Tommy Atkins' responded well to IAA
and IBA. Experiments 5-5 and 5-6 with germination media
indicate that the use of Difco Bacto-agar may be more
useful than Gelrite gellan gum for the germination of
somatic embryos. Similarly, the usefulness of the
complex organic addenda (LCE and CH) in the modified

139
B-5 medium is somewhat suspect based on the results of
experiment 5-2, 5-5, and 5-6. It may be worthwhile to
test these supplements in other controlled experiments.

APPENDIX

141
Table A-l. Plant growth regulators (PGRs) used with iri vitro culture of mango.
Abreviation
Chemical Name
Soluble in
MW
1 -1
1 mg
(uM)
2,4-D
(2,4-Dichlorophenoxy)acetic acid
ethanol
221.04
4.5
NAA
2-Naphthaleneacetic acid
NaOH or KOH
186.2
5.4
IAA
Indole-3-acetic acid
ethanol
175.18
5.7
IBA
Indole-3-butyric acid
ethanol
203.0
4.9
BA
6-benzylaminopurine(N^ Benzyladenine)
HC1
225.2
4.4
2 iP
(2-Isopentenyl)adenine
HC1
203.3
4.9
KIN
Kinetin
HC1
215.2
4.6
ABA
Abscisic acid (filter-sterilized)
ethanol
264.3
3.8
GA
Gibberellic acid (filter-sterilized)
ethanol
346.4
2.9
LCE Liguid coconut endosperm (coconut water), prepared from immature coconuts,
filter-sterilized and stored frozen, refilter-sterilized and added to
cool, sterile media with pH adjusted to allow for the addition of the
addition of the acidic filtrate.
CH Casein hydrolysate, added directly to the media prior to adjusting pH and
autoclaving.
YE Yeast extract, added directly to the media prior to adjusting pH and
autoclaving.

142
Table A-2. List of mango cultivars with
successful j_n vitro nucellar
callus initiation.
Cultivar2 Seed^
Aroemanis
Poly
Brander
Poly
Brooks
Mono
Cambodiana
Poly
Everbearing
Mono
Florigon
Poly
Gadong
Poly
Golek
Poly
Heart
Poly
Hone Cambodia
Poly
Irwin
Mono
James Saigon
Poly
Keitt
Mono
Kensington
Poly
Kur
Poly
Madoe
Poly
Manzano
Poly
Mikongensis
Poly
Mulgoba
Poly
Mun
Poly
Nam Donk Mai
Poly
Ono
Poly
Parris
Poly
Peach
Poly
Rockdale Saigon
Poly
Sabre
Poly
Simmonds
Poly
Stringless Peach
Poly
Tommy Atkins
Mono
Tuehau
Poly
Turpentine
Poly
z
Trees were located in germplasm collections
at the University of Florida Tropical
Research and Education Center, Homestead,
and the U.S.D.A. Subtropical Horticultural
Research Unit, Miami, Florida.
-^Type of seed: monoembryonic or polyembryonic.

Table A-
-3. In vitro somatic embryogenesis from 'Simmonds' nucellar
callus in response to various plant growth regulators.
uM
Total number of co
Kinetin
0 5 20 80
tyledonary
2iP
5 20
embryc
80
>sz
5
BA
20
80
0
0
0
0
0
0
8
0
0
y
0
5
0
0
0
0
0
0
0
0
0
2,4-D 10
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
NAA 10
0
0
0
0
0
0
0
0
0
2
20
0
0
0
0
0
0
0
15
0
0
5
0
0
0
0
0
0
0
0
0
0
IAA 10
2
0
0
0
1
0
1
0
0
0
20
0
0
0
0
0
3
0
0
0
0
5
0
0
0
0
0
30
0
3
8
IBA 10
0
0
0
0
0
0
0
20
23
0
20
0
0
0
0
0
0
0
0
20
0
2
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the above table.
yBlank spaces represent no data due to contamination.
143

Table A-4. In vitro somatic embryogenesis
callus in response to various
from
plant
'Florigon' nucellar
growth regulators.
uM
0
Total number of
Kinetin
5 20 80
cotyledonary embryos2
2iP
5 20 80 5
BA
20
80
0
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
2,4-D
10
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
5
0
0
5
0
0
0
0
8
0
0
NAA
10
0
4
0
0
y
0
0
0
0
17
20
3
0
0
0
0
0
0
0
10
0
5
0
0
0
0
0
0
0
0
0
0
IAA
10
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
IBA
10
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
2
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the table above.
^Blank spaces represent no data due to contamination.
144

Table A-5.
In
vitro
somatic
embryogenessis
from
'Cambodinana'
' nucellar
callus in
response to
various plant
growth
regulators.
Total number of
cotyledonary embryos2
Kinetin
2iP
BA
uM
0
5
20
80
5
20
80
5
20
80
0
0
0
0
0
0
0
0
0
y
0
5
0
0
0
0
0
0
0
0
0
0
2,4-D 10
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
NAA 10
0
1
0
0
0
0
0
0
0
0
20
0
0
15
0
0
0
0
0
0
0
5
0
0
6
0
0
0
0
0
0
0
IAA 10
0
0
0
0
0
0
0
0
0
0
20
0
13
0
0
0
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
IBA 10
0
0
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
0
0
z
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the table above.
yBlank spaces represent no data due to contamination.
145

Table A-6.
In vitro somatic embryogenesis from 'Irwin' nucellar
callus in response to various plant growth regulators.
uM
0
5
2,4-D 10
20
5
NAA 10
20
5
IAA 10
20
5
IBA 10
20
0
0
0
0
0
0
2
0
0
2
3
0
0
0
Total number of
Kinetin
5 20 80
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 3 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
cotyledonary
2iP
5 20
0 0
0 0
0 0
0 0
0 0
0 0
0 0
0 2
0 0
1 0
0 0
0 0
0 0
embryos2
80
0
0
0
1
0
0
0
1
0
0
0
0
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
BA
20
y
0
0
0
0
0
0
0
0
0
0
0
0
80
0
200 mg of callus inoculated onto 12 ml of callus initiation medium
(Table 4-3) supplemented with PGRs according to the table above.
yBlank spaces represent no data due to contamination.
OOO OOO MOO ooo

147
Table A-7. Peach embryo germination medium.
Salt2
Concentration
(mg/1)
Ca(N03)2 4H20
1140.00
MgSO 7H 0
370.00
KN03
190.00
Kh2P°4
170.00
Myo-inositol
100.00
Na EDTA
37.20
FeS04 7H20
27.90
MnSO^ 4H20
22.30
ZnSO^ 7H20
8.60
H B04
6.20
Glycine
2.00
KI
0.83
Nicotinic Acid
0.50
Pyridoxine HC1
0.50
Thiamine HC1
0.50
Na MoO. 2H20
0.25
CoCl2 5H20^
0.03
CuS04 5H20
0.03
2
Modified Knop's Solution, Jose Chaparro,
M.S. Thesis, 1986 University of Florida,
Gainesville.

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BIOGRAPHICAL SKETCH
Stephen Gregory DeWald was born on June 3, 1950, in
Fort Wayne, Indiana, where he attended both primary and
secondary school. Stephen started college as a business
major in 1969 at Indiana University in Bloomington but left
school for a career in horticulture and landscaping. After
several years of working as a free-lance landscaper and
nurseryman, he reentered school to pursue his interests in
plant breeding and genetics. He obtained a Bachelor of Arts
degree in plant science from Indiana University in 1979.
The following year he entered Graduate School in the
Department of Botany and Plant Pathology at Purdue
University in West Lafayette, Indiana. His master's thesis
concerned the greenhouse screening of young apple hybrid
seedlings for resistance to powdery mildew. He received his
Master of Science degree there specializing in fruit tree
pathology and plant breeding and genetics.
In 1983 Stephen entered the Fruit Crops Department at
the University of Florida, Gainesville, to obtain a Doctor
of Philosophy degree. He specialized in tissue culture and
plant cultivar development and improvement.
162

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.
Richard E. Litz, Chairman
Associate Professor of
Horticultural 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.
Ct • VVvo~ Gloria A. Moore
Associate Professor of
Horticultural 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.
Wayn¿ B.
Professor
of
• K) —
Sherman
of Horticultural
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.
&u\. MIL
Robert JJ^/Knight, Jr
Professor of Horticultural 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.
O â– 
Vy Prem S. Chourey ^
Professor of Plant Pathology

This dissertation was submitted to the Graduate Faculty
the College of Agriculture and to the Graduate School
was accepted as partial fulfillment of the requirements
the degree of Doctor of Philosophy.
May, 1987
Dean, Col/Lege of Agriculture
y^y
of
and
for
Dean, Graduate School

UNIVERSITY OF FLORIDA



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