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Tissue culture and electrophoretic studies of pineapple (Ananas comosus) and related species

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Title:
Tissue culture and electrophoretic studies of pineapple (Ananas comosus) and related species
Added title page title:
Ananas comosus
Creator:
DeWald, Maria Grazia, 1948-
Publication Date:
Language:
English
Physical Description:
xiv, 141 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Pineapple -- Propagation -- In vitro ( lcsh )
Pineapples ( jstor )
Cayenne ( jstor )
Dehydrogenases ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references (leaves 130-140).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Maria Grazia DeWald.

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University of Florida
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University of Florida
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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.
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030389584 ( ALEPH )
AER2773 ( NOTIS )
16928890 ( OCLC )

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Full Text












TISSUE CULTURE AND ELECTROPHORETIC STUDIES OF
PINEAPPLE (Ananas comosus) AND RELATED SPECIES









By

MARIA GRAZIA 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 husband, Steve,

for his constant support, understanding, love, and help

through these last years while we were students and parents.


















ACKNOWLEDGMENTS


I express gratitude to Dr. Wayne Sherman for his

tireless encouragement, thoughtful guidance, and friendship

during all these years. Special thanks are extended to Dr.

Gloria Moore for allowing the use of laboratory, continuous

support, and suggestions for this dissertation. Appreciation

is extended to Dr. Richard Litz for allowing use of

laboratory and equipment and advice during the tissue

culture studies. Sincere appreciation is given to Dr. Paul

Lyrene and Dr. Walter Judd for their help in reviewing this

dissertation and for the knowledge I gained when attending

their courses. The help of Anne Harper with laboratory work

is appreciated.

Financial support provided by CDCH and Universidad

Central de Venezuela is also acknowledged.

Many thanks go to my parents, Francesco and Antonina

Antoni, for their love and support over the years.

Finally, I extend my appreciation to the graduate

students, the service staff, and the faculty of the

University of Florida Fruit Crops Department, Gainesville,

for the many ways in which they have helped.




















TABLE OF CONTENTS


Page

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

LIST OF TABLES ................. ................................vi

LIST OF FIGURES... ............................ .................viii

KEY TO ABBREVIATIONS.................................. .... x

ABSTRACT.................... .......... ............. ..... xii

CHAPTER


INTRODUCTION... ......... .......... ............. 1

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

Ananas Taxonomy.................................3
Pineapple Propagation......................... 4
Pineapple Tissue Culture...................... 6
Somaclonal Variation............................8
Application of Isozyme Analysis in
Horticultural Plant Systematics.............12

PRODUCTION OF PINEAPPLE PLANTS IN VITRO....... 17

Introduction................................17
Materials and Methods.........................18
Genotypes Cultured ............................18
Sterilization and Initiation of Cultures....18
Culture Multiplication......................19
Establishment in Soil........................20
Results and Discussion.........................21

ELECTROPHORETIC STUDIES IN ANANAS..............35

Introduction........................................ 35
Materials and Methods.........................36
Genotypes Used for Electrophoretic Studies..36
Starch Gel Preparation......................38


I

II


III









IV









CHAPTER Page

Protein Extraction and Gel Loading..........40
Electrophoretic Buffers and Staining
Systems...................................41
Electrophoresis............................44
Enzyme Activity Staining.....................45
Analysis of Banding Patterns................45
Results and Discussion........................46
Selection of Electrophoretic Buffers
and Enzyme Systems Useful for Studying
Isozymes in the Genus Ananas...............46
Monomorphic Enzymes........................ 48
Enzyme Systems with Variable Banding
Patterns................................... 49
Linkage Analysis for Enzyme Loci............55
Conclusions...................................56

V IDENTIFICATION OF ANANAS GENOTYPES
BY ISOZYME PHENOTYPES.........................77

Introduction...................... ...........77
Materials and Methods........................78
Results and Discussion.......................78
Cultivar Identification..................... 79
Species Identification...................... 83
Feral Type Identification....................86
Conclusions ..................... ....... ..... 88

VI PHENETIC RELATIONSHIPS IN ANANAS BASED
ON ISOZYME ELECTROPHORETIC ANALYSIS..........101

Introduction................................ 101
Material and Methods........................103
Results and Discussion.......................104

VII CHARACTERIZATION OF REGENERATED
PINEAPPLE PLANTS ........................... 116

Introduction................................116
Materials and Methods.......................117
Results and Discussion..................... 118
Electrophoretic Characterization..........118
Morphological Characterization.............119

VIII SUMMARY AND CONCLUSIONS ...................... 124

LITERATURE CITED.........................................130

BIOGRAPHICAL SKETCH............................ ..........141


















LIST OF TABLES


Table Page

3.1 Murashige and Skoog salts formulation..............23

3.2 In vitro pineapple plantlet production for
each initiated bud................................24

3.3 In vitro pineapple plantlet production for
each initiated bud..................................25

3.4 In vitro pineapple plantlet production in
response to the presence or absence of plant
growth regulators in the culture medium.............26

4.1 Ananas genotypes surveyed for isozyme banding
patterns ............... .......... ... .. ........ 58

4.2 Electrophoretic buffer systems used for isozyme
detection in Ananas...................... ...... 59

4.3 Enzyme activity stains and buffer systems tested
for isozyme detection in Ananas....................60

4.4 Well resolved enzyme activity staining systems
and electroporetic parameters used in Ananas.......61

4.5 Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived from
an open-pollinated 'Smooth Cayenne' fruit..........62

4.6 Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived from
an open-pollinated 'Cambray' fruit.................63

4.7 Genotypic ratios and chi-square goodness of
fit values for 6 isozyme loci in pineapple..........64

4.8 Segregation ratios and chi-square goodness of
fit values for independent inheritance of
peroxidase and phosphoglucomutase loci in
'Smooth Cayenne'-derived seedlings..................65









Table Page


4.9 Segregating ratios and chi-square goodness
of fit values for independent inheritance
of peroxidase and phosphoglucomutase loci
in 'Cambray'-derived seedlings.....................66

5.1 Peroxidase and phosphoglucomutase isozyme
banding patterns observed for pineapple
cultivars .......................................... 91

5.2 Pineapple cultivar classification based on
peroxidase banding patterns........................92

5.3 Pineapple cultivar classification based on
phosphoglucomutase banding patterns.................93

5.4 Pineapple cultivar identification based on
peroxidase (Per) and phosphoglucomutase (Pgm)
isozyme banding patterns ..........................94

5.5 Peroxidase and phosphoglucomutase isozyme
banding patterns for Ananas species.................95

5.6 Phosphoglucomutase isozyme banding patterns
observed for various Ananas genotypes...............96

6.1 Designation of genotypes analyzed
electrophoretically... .......................... 109

6.2 Presence-absence data matrix for electrophoretic
analysis of Ananas genotypes......................111

7.1 Regenerate pineapple plants evaluated for
electrophoretic and morphological variation.......122



















LIST OF FIGURES


Figure Page

3.1 Initiation of axillary bud culture..................27

3.2 Proliferation of culture 4 months after
inoculation........................................28

3.3 Actively dividing culture ready for subculture.....29

3.4 Regenerated plantlets established in individual
pots or in flats.............. ....................30

3.5 Comparison of multiplication cultures with
and without plant growth regulators................ 31

3.6 Ananas bracteatus var. tricolor.....................33

4.1 Non-variable isozyme banding patterns observed
for Ananas genotypes..............................67

4.2 Schematic representation of peroxidase isozymes
in 2 pineapple segregating populations.............70

4.3 Peroxidase isozyme patterns observed in pineapple..71

4.4 Schematic representation of phosphoglucomutase
isozyme in Ananas.................................. 72

4.5 Schematic representation of phosphoglucomutase
isozyme in 2 pineapple segregating populations.....73

4.6 Phosphoglucomutase banding pattern in pineapple....74

4.7 Malate dehydrogenase patterns observed in
pineapple.........................................75

5.1 Schematic representation of peroxidase (Per)
and phosphoglucomutase (Pgm) isozymes in
pineapple cultivars................................97


viii









Figure Page

5.2 Schematic representation of peroxidase (Per)
and phosphoglucomutase (Pgm) isozymes in
Ananas species......................................98

5.3 Variation in phosphoglucomutase isozyme in
Ananas genotypes leaf extracts.....................100

6.1 Cluster analysis dendogram of 27 pineapple
cultivar isozyme phenotypes........................113

6.2 Cluster analysis dendogram of isozyme phenotypes
of Ananas species and feral types.................114

6.3 Cluster analysis dendogram of the isozyme
phenotypes of 62 Ananas genotypes................115

7.1 Abnormal pineapple regenerated plants............123



















KEY TO ABBREVIATIONS


ATP:

BA:

CW:

H:

IAA:

IBA:

K:

KIN:

LBTC:

MS:

MTT:



NAA:

NAD:

NADP:

Na2EDTA

NBT:

PMS:

Rb:
Rf:


adenosine 5' triphosphate disodium salt

6-benzylaminopurine (N6 benzyladenine)

coconut water

histidine buffer

indole-3-acetic acid

indole-3-butyric acid

K buffer

kinetin

lithium borate/tris citrate buffer

Murashige and Skoog basal medium formulation

3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-

tetrazolium bromide

2-naphthaleneacetic acid

beta-nicotinamide adenine dinucleotide

beta-nicotinamide adenine dinucleotide phosphate

disodium ethylenediaminetetracetic acid

nitro blue tetrazolium

phenazine methosulfate

band migration relative to band sample

band migration relative to borate front









TB: tris borate buffer

TC: tris citrate buffer

Tris: tris-(hydroxymethyl) amino methane

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


















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



TISSUE CULTURE AND ELECTROPHORETIC STUDIES OF
PINEAPPLE (Ananas comosus) AND RELATED SPECIES

By

MARIA GRAZIA DEWALD

May, 1987

Chairman: Wayne B. Sherman
Cochairman: Gloria A. Moore
Major Department: Horticultural Science (Fruit Crops)

Tissue culture studies were conducted with pineapple

(Ananas comosus) and A. bracteatus var. tricolor in order to

evaluate the feasibility of in vitro techniques as a method

for the rapid multiplication of desirable genotypes. A

system utilizing shake cultures derived from axillary buds

of crowns, slips, and stems was devised. This system

resulted in the efficient production of large numbers of

pineapple plantlets that were easily established in soil

under greenhouse conditions. The regenerated plants were

characterized morphologically and on the basis of isozyme

banding patterns. High levels of phenotypic variability,

especially for leaf morphology, were observed shortly after

the small plants were transferred to the soil. After one









year only 4 variant phenotypes persisted in the 2000

regenerated plants. No isozyme variants were identified.

Electrophoretic characterization of Ananas with

emphasis on economically important pineapple cultivars was

also conducted to help establish phenetic relationships. A

method was developed whereby leaf sap could be quickly

extracted and analyzed for isozyme polymorphisms using

starch gel electrophoresis. One hundred and ninety

combinations consisting of 38 enzyme activity stains and 5

electrophoretic buffer systems were evaluated for isozyme

banding resolution. From this survey, 8 enzyme staining

systems giving good banding resolution were identified.

Three of these, peroxidase, phosphoglucomutase, and malate

dehydrogenase, were polymorphic. Two segregating seedling

populations were utilized to establish a genetic model for

the 3 polymorphic enzymes. The genetic analysis revealed

that both peroxidase and phosphoglucomutase were controlled

.by 3 independent, monomeric loci.

A taxonomic survey of 67 Ananas genotypes was conducted

utilizing the peroxidase and phosphoglucomutase staining

systems. Fifteen of the 27 pineapple cultivars and 5 of the

7 species analyzed could be uniquely distinguished using the

2 enzyme systems. Ananas comosus, one of the species that

could not be unambiguously distinguished, was highly

polymorphic due to the large number of cultivars included in

the species. Ananas monstrosus, the other species without a

unique isozyme phenotype, was confirmed to be a simple crown


xiii









mutation of A. comosus that occurs in many cultivars and

therefore is not considered a valid species. Most of the 33

feral types analyzed could be included under species with

similar banding patterns. Phenetic relationships of the

genotypes based on cluster analysis dendograms were

established.



















CHAPTER I
INTRODUCTION


Pineapple is one of the most important fruit crops in

the world. Nearly 12 million metric tons are produced

annually in the tropical and subtropical regions of the

world (FAO Production Yearbook, 1984). It is particularly

well suited to arid and semiarid tropical locations where

few other crops do well.

One reason for the popularity of pineapple is that it

has proved very amenable to large-scale commercial

production and has good canning qualities. Most of the world

production can be attributed to 'Smooth Cayenne' and its

sports. 'Smooth Cayenne' is characterized by its relatively

smooth leaves (spiny tip) and large cylindrical fruit with

good canning qualities. However, it is susceptible to

several economically important diseases and does not produce

slips, which are needed for propagation. Numerous high

quality spiny cultivars also exist that are more adapted to

tropical regions, where 'Smooth Cayenne' does not develop

good fruit qualities, or are better suited for the fresh

market. The presence of spines and the scarcity of planting

material on these cultivars severely restricts their









suitability to large scale planting. Pineapple breeding

efforts have been directed mainly at the improvement of

'Smooth Cayenne', because most of the breeding programs have

been conducted by large commercial companies whose

production is dependent on the specific canning

characteristics of this cultivar.

The genus Ananas, to which pineapple belongs, is poorly

characterized taxonomically. One explanation for this may be

that the center of origin and diversity of this genus is

located in the isolated regions of the upper Amazonian River

basin. This lack of characterization limits the ability of

plant breeders to utilize the variability present in both A.

comosus and related species.

The objectives of this research were 1) to develop an

efficient in vitro propagation system for pineapple; 2) to

morphologically and electrophoretically evaluate the genetic

fidelity of the regenerated plants; 3) to identify

techniques that are useful for visualizing isozymes in

Ananas; 4) to survey the available Ananas germplasm to

determine if variability in isozyme banding patterns exists;

5) to study the possible genetic basis for variability in

isozyme banding patterns; 6) to uniquely identify Ananas

species, including pineapple cultivars, and to categorize

feral types into species using isozyme polymorphism; and 7)

to establish phenetic relationships among Ananas genotypes

based on isozyme banding patterns.


















CHAPTER II
LITERATURE REVIEW


Ananas Taxonomy


Pineapples [Ananas comosus (L.) Merr.] have been

studied and characterized by naturalists and botanists for

hundreds of years; however, there has been much variation in

their taxonomic treatment. Some botanists believe Ananas is

a monotypic genus, i.e., a single species, comprised of many

varieties. Others have considered the genus to include many

species, which are often separated on the basis of a few,

slight character variations (Collins, 1960).

The latest classification of the genus Ananas was by

Smith and Downs (1979). Smith was the botanist who also

classified most of the Bromeliaceae (the Ananas family). The

species considered valid in this latest treatment are A.

ananassoides, A. bracteatus, A. comosus, A. fritzmuelleri,

A. lucidus, A. nanus, and A. parguazensis. Classification is

based upon the presence or absence of a crown (coma

foliaceous), syncarp size, presence and direction of spines,

and floral bract characteristics. This is the most

comprehensive survey of the pineapple genus to date,

providing botanists for the first time with a taxonomic key.









A chronological review of pineapple taxonomy was later

made by Antoni (1983), indicating that the present

classification of the genus Ananas is highly confused and

questioning the validity of some species, e.g., A.

monstrosus. The same author stressed the importance of using

techniques such as tissue culture and electrophoretic

separation of isozymes to differentiate species.



Pineapple Propagation

Pineapple is a self-incompatible perennial crop that is

vegetatively propagated. The crop is usually cultivated in

high density fields. The spineless cultivar 'Smooth Cayenne'

is planted in Hawaii at 60,000 to 80,000 plants per ha. (Py

and Tisseau, 1965). In other regions where spiny cultivars

are used, only 15,000 to 35,000 plants are planted per ha.

(Py and Tisseau, 1965). At such high planting densities,

propagules are a major limiting factor in the establishment

of new fields. The problem can be even more extreme in the

case of newly developed cultivars. The number of propagules

per pineapple plant varies with the cultivar. However, a

typical plant produces between 6-10 propagules for crop

cycle (18-22 months). These propagules include the crown

(produced on the fruit apex), slips (produced on the fruit

peduncle), and suckers (produced on the plant stem). A

solitary sucker is usually left in place to produce the

subsequent ratoon fruit. Culturally, slips are the most

desired propagule. They are more favorable than crowns









because they are uniform, root faster, and can be planted

deeper, giving rise to a more uniform stock of plants.

The most economically important cultivar, 'Smooth

Cayenne', has low slip production. Slip production is

further reduced in this cultivar when it is planted at high

densities to produce a fruit size acceptable for efficient

processing (Ravcof and Yamane, 1970). Crowns are sold with

the fruit in fresh market situations, thereby further

reducing the amount of propagules.

Several methods have been developed over the past 40

years to increase the amount of propagules (Py, 1979). One

method, developed by Macluskie (1939), is to section the

mature stems. The individual pieces are placed in sand boxes

and incubated in a warm moist place. After several weeks the

axillary buds grow and form adventitious roots. This

technique was later tried by several researchers using

sections of crowns and slips (Tkatchenko, 1947; Collins,

1960; Py and Estanove, 1964). A second method consists of

propagating a single leaf with intact axillary bud dissected

from the crown or slip (Seow and Wee, 1970). This method is

laborious and produces a limited amount of plantlets per

crown.

The most recent advance in pineapple multiplication

employs plant morphactins (Sanford and Abdul, 1971). Plant

morphactins are a diverse group of synthetic chemicals which

dramatically alter the morphological development of the

plant. Morphactins in pineapple are used in combination with









flower-inducing ethylene compounds, e.g., ethrel. Mature

plants (12-16 month old) are first induced to flower. The

flowering process is then converted into a multiplicant

process by the application of a morphactin, e.g., Maintain

CF-125. Using this method, up to 12 "sliplets" per plant can

be produced (Ravcof and Yamane, 1970). This is an economical

and rapid (2 years) method that allows the production of

over half a million vigorous propagules similar to slips per

ha. Planting material for most large-scale commercial

plantings of 'Smooth Cayenne' types is now produced by this

method of multiplication (Krezdorn, personal communication,

1985).

All of these methods require large fields to establish

plants. Rapid and more efficient pineapple propagation

systems still limit the establishment of new commercial

plantings. Tissue culture appears to be a very attractive

alternative method of multiplication and has particular

utility in situations where clonal material is scarce.



Pineapple Tissue Culture

Pineapples have been regenerated in vitro from the

culture of shoot tips, axillary buds, and immature syncarps.

Direct plant production from excised axillary buds was first

reported by Aghion and Beauchesne (1960). A single plantlet

was obtained from each cultured axillary bud on solid, half

strength Knop's medium supplemented with 15% (v/v) coconut

water (CW). Sita et al-. (1974) also regenerated single










plants from individual slip shoot tips on a solid medium of

Knudson major salts and Nitsch minor salts supplemented with

5.4 uM NAA.

Mapes (1974) was the first to describe a method for

multiple shoot production directly from axillary buds

present in cultured shoot. The shoot tips were placed in

liquid Murashige and Skoog (MS) medium supplemented with

0.22 mM adenine to initiate proliferation. Subsequently,

they were transferred to solid MS medium supplemented with

0.15 mM adenine to allow shoot development. The

proliferating mass of shoots was termed a protocorm-like

structure. Similar results were obtained by Teo (1974) and

Pannetier and Lanaud (1976). Wakasa et al. (1978) reported

the initiation of shoots from protocorm-like calli derived

from immature syncarps, axillary buds of suckers and slips,

immature crowns, and immature slips cultured on solid MS

media containing 5.4-54.0 mM NAA and 4.4-44.0 uM BA. The

best results were obtained on medium supplemented with 10.8

uM NAA and 8.8 uM BA. Wakasa et al. (1978) described 2 types

of protocorm-like calli. Calli derived from axillary buds

were described as nodule-like in appearance, while the calli

derived from axillary buds were described as globule-like.

Mathews et al. (1976) demonstrated that liquid culture

medium appeared to be most suitable for shoot proliferation

from axillary bud cultures. They used an MS medium

containing 9.7 uM NAA, 9.8 uM IBA, and 9.2 uM KIN. Shoots

were initiated on solid medium supplemented with 4.4 uM BA









and 4.9 uM IBA (Mathews and Rangan, 1979). Later studies by

these authors indicated that callus could also be initiated

from axillary bud cultures on MS medium supplemented with

54.0 uM NAA, 15% (v/v) CW, and 0.04% (w/v) casein

hydrolysate. Shoot proliferation occurred on MS medium

supplemented with 9.7 uM NAA, 9.8 uM IBA, and 9.2 uM KIN.

Root formation occurred on White's medium containing 0.27 uM

NAA and 0.196 uM IBA. Zepeda and Sagawa (1981) reported

limited shoot proliferation from axillary buds cultured in

liquid MS medium supplemented with 25% (v/v) CW.

Shoot proliferation from hybrid embryo-derived callus

was reported by Srinivasa Rao et al. (1981) on MS medium

with 10.8 uM NAA, 9.8 uM IBA, and 11.0 uM BA. Immature leaf

explants have also been used for shoot proliferation on MS

solid medium containing 9.7 uM NAA, 9.8 uM IBA, and 48.4 uM

BA (Mathews and Rangan, 1979).

Drew (1980) published a report on pineapple tissue

culture theorizing the possibility of obtaining 100,000

plants from a single axillary bud in 12 months. There is no

mention in the literature in regard to the number of

regenerated plants established in soil, with the exception

of Wakasa et al. (1978) who reported growing 400 regenerated

plants.



Somaclonal Variation

The term somaclonal variation was coined by Larkin and

Scowcroft (1981) to describe the phenotypic variation









observed in plants regenerated from somatic tissue in vitro.

Somaclonal variation appears to be common to all in vitro

propagation systems. However, the quantity and quality of

the variation differs greatly with the genotype and

propagation protocol.

A number of reviews have been written on this subject

(Bayliss, 1980; Larkin and Scowcroft, 1981; Orton, 1983a;

1983b; 1984; Evans et al. 1984). Agromonically useful

somaclonal variation has been reported in many crop species,

e.g., alfalfa (Johnson et al., 1980), maize (Edallo et al.,

1981), oats (McCoy et al., 1982), potato (Shepard et al.,

1980), sugarcane (Heinz and Mee, 1969), tobacco (Chaleff and

Keil, 1981), tomato (Evans et al., 1984), and wheat (Larkin

et al., 1984).

The variation observed in tissue culture-produced

somaclones may be stable and heritable or it may be

transient, nonheritable, and epigenetic in nature. It is

particularly critical for the scientist to distinguish

between types of phenotypic variation. Epigenetic variation,

because it is nonheritable, has limited usefulness to

cultivar improvement.

A wide range of chromosomal alterations are possible in

tissue culture-regenerated plants. Single base pair

alterations or deletions (Edallo et al., 1981) represent one

end of the spectrum, while major karyotypic alterations,

including the doubling or deletion of complete sets of









chromosomes (Janick et al., 1977), represent the other

extreme.

Stable, heritable variation implies genetic

variability. This genetic variability may be due to

pre-existing heterogeneity present in the cells of the

explant and/or de novo genetic alterations occurring during

the culturing process (Evans et al., 1984). Unfortunately,

scientists have not been able to determine the relative

amount of variation attributable to these 2 sources (Larkin

and Scowcroft, 1981).

The full range of genetic variability that can be

generated and expressed in tissue culture-regenerated plants

has not yet been established. It does appear, however, that

the full range of genetic variability present in cultured

cells is not present in the regenerated plants (Edallo et

al., 1981). The regeneration process may be serving as a

selective screen for those cells that are genetically

competent to form plants.

Perhaps the best documented source of variation is the

explant. Barbier and Dulieu (1980) observed higher levels of

somaclonal variation in tobacco plants that were regenerated

from protoplasts and newly initiated callus cultures in

comparison with plants regenerated from well established

callus cultures. Wakasa (1979) also reported significant

differences in the amount of somaclonal variation observed

for vegetative morphology in 'Smooth Cayenne' pineapple

plants regenerated from 3 explant sources. He found that









plants regenerated from syncarp (immature fruit) tissues

exhibited 100% variability, while plants derived from

axillary buds of crowns and slips were 34% variable. The

lowest levels of variation (7%) were observed in plants

regenerated from small crowns. Varibilities in leaf wax,

color, spines, and foliage density were observed in the

plants derived from the syncarp, while only spine

variability was observed in the plants from slips and

axillary buds. It should be noted that this study was done

on up to 2-year-old regenerated plants that were grown in

the greenhouse, and no long-term study on the stability of

this variation was conducted.

The amount of time in culture can also influence the

variability of regenerated plants. Karyotypic changes have

been correlated with increasing time in culture (Bayliss,

1980). Increasing time in culture has been shown to increase

somaclonal variation in tobacco (Syono and Furuya, 1972),

Chrysanthemum (Bush et al., 1976; Sutter and Langhans,

1981), and oats (McCoy, 1980 cited by Reisch, 1983). In

contrast, long-term cultures of corn were reported to be

stable (McCoy, 1980 cited by Reisch, 1983).

The phytohormones used in the culture medium are

another possible factor affecting somaclonal variation. High

concentrations (18 uM) of the auxin 2,4-D were reported to

produce unique variation in regenerated barley plants that

was not observed in plants regenerated using lower

concentrations (Deambrogio and Dale, 1980). Similarly, a









medium supplemented with NAA (27 uM) and KIN (0.5 uM) was

shown to produce significantly more karyotypic alterations

in regenerated Haworthia plants than a medium supplemented

with 0.6 uM IAA (Ogihara, 1981).

The genotype has also been shown to influence

somaclonal variation. Liu and Chen (1976) evaluated 8

sugarcane cultivars and found that regenerated plants from

the cultivars ranged from 1.8% variable to 34% variable.

Skirvin and Janick (1976) found somaclonal variation

cultivar differences in Pelargonium sp. DeWald and Moore

(1987) indicated in their review that certain crops, e.g.,

polyploids and asexually propagated crops in which sexual

fertility is not important, appear to tolerate higher levels

of chromosomal variation produced via somaclonal variation

than other crops.



Applications of Isozyme Analysis in Horticultural Plant

Systematics

Isozymes are multiple molecular forms of an enzyme

having the same substrate specificity (Markert and Moller,

1959). Hunter and Markert (1957), using the starch gel

electrophoresis technique developed by Smithies (1955),

demonstrated that isozymes can be visualized on starch gels

stained with specific histochemical stains. Bands appearing

on a starch gel correspond to different alleles and/or loci

coding for a specific enzyme.









Isozymes have been used extensively as genetic markers

in plants. They have a number of advantages over

conventional markers. Isozymes are generally not affected by

the morphological or physiological status of the plant. They

interact codominantly at a single locus, rarely exhibit

epistatic interactions, and can be detected in a variety of

tissues (Tanksley and Rick, 1980; Tanksley et al., 1982;

Tanksley, 1983).

The use of electrophoretic analysis to characterize

isozyme variation as an aid for plant systematics is now

widely applied and has been the subject of several reviews

(Gottlieb, 1977; Crawford, 1983). The electrophoretic

phenotype can be directly equatable with genotype, unlike

many morphological characters, which may demonstrate a great

deal of environmental or developmental plasticity (Crawford,

1983; Gottlieb, 1977).

In plant systematics, isozyme electrophoresis has been

successfully utilized for both cultivar and species

identification by providing a way to quantitatively assess

the genetic variation among conspecific populations and the

genetic divergence among congeneric species. Other important

applications include the resolution of polyploid taxa and

insight into their origins, the investigation of the origin

of cultivated crops, and the testing of broad systematic or

evolutionary hypotheses.

Reliable identification of cultivars using classical

methods based on morphological and physiological characters









has become increasily difficult, partially due to the large

number of lines or varieties now available. Isozymes as

bio-chemical markers for cultivar identification have proven

reliable, consistent, and essentially unaffected by

environmental conditions (Bailey, 1983). They can be used as

"fingerprints" to aid in the visual identification of

cultivars (Pierce and Brewbaker, 1973). The utility of this

technique has already been demonstrated in a number of

species, e.g., Anthurium (Kobayashi et al., 1987), alfalfa

(Quiros, 1980), apple (Menendez et al., 1986; Weeden and

Lamb, 1985), avocado (Torres et al., 1978a), barley

(Bassiri, 1976), bean (Bassiri and Rouhani, 1977; Weeden,

1984), Camellia (Wendel and Parks, 1983), Citrus (Torres et

al., 1978b), grape (Schwennesen et al., 1982), grasses

(Wilkinson and Beard, 1972), maize (Cardy and Kannenberg,

1982), Musa (Jarret and Litz, 1986b), peach (Carter and

Brock, 1980), pear (Santamour and Demuth, 1980), pecan

(Mielke and Wolfe, 1982), poinsettia (Werner and Sink,

1977), rice (Sarkar and Bose, 1984), strawberry (Bringhurst

et al., 1981), and wheat (Menke et al., 1973).

The use of isozymes in the investigation of plant

taxonomy, particularly species circumscription is relatively

recent. Zymogram techniques have been used most extensively

in Nicotiana. Characteristic enzyme patterns for most

Nicotiana species have been observed using proteins (Hart

and Bathia, 1967), peroxidase alone (Sheen, 1970), and in

combination with esterases (Smith et al., 1970). Species









identifications using isozymes have also been reported in

Datura (Conklin and Smith, 1971), Citrus (Button et al.,

1976), Musa (Jarret and Litz, 1986c), and Pistacia (Loukas

and Pontikis, 1979).

Specific isozymes or proteins have been used to

identify particular genomes in polyploids. In bananas,

Jarret and Litz (1986a) confirmed the bispecific origin of

the triploid 'Chato' from Musa acuminata and Musa balbisiana

using leaf isozyme polymorphisms. An allopolyploid origin

for the apple has been proposed based on inheritance data of

7 pollen enzymes (Chevreau et al., 1985). Isozyme analysis

of Gossypium hirsutum, a natural allopolyploid, has shown

that esterases exhibit greater intergenomic variability than

intraspecific or intravarietal variability (Cherry et al.,

1970). In a later study, Cherry et al. (1971) showed that

evolutionary changes had occurred in the species that were

reflected in isozyme differences.

One of the most common uses of isozymes has been to

assess the validity of proposed parentages of cultivars and

progeny. This has been done in apple (Chyi and Weeden, 1984;

Weeden and Lamb, 1985), avocado (Golching et al., 1985;

Torres and Bergh, 1980; Torres et al., 1978a), Citrus

(Torres et al., 1978b), and date palm (Torres and Tisserat,

1980).

Protein and isozyme analyses have proved particularly

helpful in deducing systematic relationships between groups

where morphological and cytological data have been






16


inconclusive. Electrophoretic studies have been used

successfully to establish phylogenetic relationships in

rice, wheat, barley, soybean, and cotton (Ladizinsky and

Hymowitz, 1979). More recently, similar studies have been

conducted with Amaranthus (Hauptli and Jain, 1984) and

Capsicum (Panda et al., 1986).


















CHAPTER III
PRODUCTION OF PINEAPPLE PLANTS IN VITRO


Introduction


Pineapple is routinely propagated vegetatively by means

of lateral shoots, basal suckers, or crowns. Plant material

is often limited, especially for the 80,000 plants/ha needed

in the establishment of new plantations, and for the

propagation of improved cultivars or newly discovered

sports.

In vitro propagation of pineapple was first achieved in

1960 by Aghion and Beauchesne (1960), but it was not until

1974, with the simultaneous reports of Laksmi Sita et al.

(1974) in India, Teo (1974) in Malaya, and Mapes (1974) in

Hawaii that in vitro production of pineapple was considered

as a commercial alternative. Most recent reports (Pannetier

and Lanaud, 1976; Wakasa et al., 1978; Mathews and Rangan,

1979; Drew, 1980; Zepeda and Sagawa, 1981) deal only with

the factors involved in the establishment of axillary buds

in culture. They do not consider the number of plants

regenerated or the efficiency of the system for reduced

labor cost. Furthermore, high levels of variability were

observed in regenerated 'Smooth Cayenne' plants by Wakasa









(1979). Such variability would compromise the commercial use

of in vitro propagation of pineapple, since regenerated

plants could not be assured to be clonal in nature.

This study was conducted using several species and

cultivars to improve the technique of tissue culture of

axillary buds for rapid pineapple propagation and to

investigate the possible variation occurring in the

regenerated plants.



Materials and Methods


Genotypes Cultured

Two cultivars of the common pineapple Ananas comosus

(L.) Merr. were used: 'PR-1-67', a spiny commercial cultivar

grown for fresh fruit and canning in Puerto Rico and

'Perolera', a smooth-leaved cultivar grown in the Northen

Andes of South America and used for fresh fruit. The third

genotype was Ananas bracteatus var. tricolor Smith, a

small-fruited ornamental characterized by showy colored

bracts and tricolored leaves (Fig. 3.6A). Two plants of

PR-1-67 and 1 plant of the other 2 genotypes were used in

order to determine intracultivar and intercultivar

variability in response to tissue culture.



Sterilization and Initiation of Cultures

Crowns and stems were rinsed in water, defoliated and

surface-sterilized by agitating in a 20% sodium hypochlorite

solution with 2-3 drops of a surfactant (Tween 20 or Tween









80) for 20 min, followed by 3 rinses of 10 min each in

sterile deionized water. Terminal and axillary buds were

then excised aseptically and again surface-sterilized in a

2% sodium hypochlorite solution for 10 min followed by 3

more 10 min rinses in sterile deionized water (Fig. 3.1A).

Buds were inoculated onto a modified Murashige and Skoog

(1962) medium (Table 3.1), supplemented with 3% sucrose,

0.8% Difco Bacto-agar, 0.57 mM inositol, 1.2 uM thiamine

HC1, 10.8 uM NAA and 8.8 uM BA. Growth regulators were added

and the medium pH was adjusted to 5.7 prior to autoclaving

at 121 C, and 1.1 kg cm for 20 min. The medium was poured

into 60 X 15 mm sterile Petri dishes for initial bud

explants or into test tubes for subculture (Fig.3.1B).

Cultures were incubated at room temperature (24-27 OC)
-1 -2
with a 16 hr photoperiod of 76 umol s m provided by

cool-white fluorescent lamps. Explants were subcultured onto

fresh medium in test tubes at 6 week intervals until

adequate growth had occurred for transfer to liquid medium

(Fig. 3.2).

In the experiment described, 3 buds from each of the 2

plants of 'PR-1-67' and 1 plant of 'Perolera' and Ananas

bracteatus var. tricolor were established in this manner.



Culture Multiplication

Proliferating explants were multiplied in liquid

cultures consisting of 50 ml of the medium described above

in a 125 ml Erlenmeyer flask, maintained at 100 rpm on









gyrorotary shakers under 16 hr photoperiod (76 umol s- m 2)

provided by fluorescent lamps. Liquid shake cultures were

subcultured at approximately 4 week intervals, when flasks

had become tightly packed with plantlets (Fig. 3.3). In this

way, numerous flasks of callus and regenerating plants were

obtained from one original bud.

An experiment was conducted to determine the necessity

of transferring the callus and plantlets to a medium without

hormones to induce root formation and thus plant survival,

and to evaluate the influence of plant growth regulators on

the number and size of plants produced. Ten grams of an

actively dividing 'Perolera' culture were inoculated into

flasks containing 50 ml of basal medium with or without

hormones (10.8 uM NAA and 8.8 uM BA). The 2 treatments were

replicated 9 times.



Establishment in Soil

After each subculture plantlets 2.5 cm and larger were

harvested. They were transferred to individual pots or to

flats containing a commercial soil mixture and enclosed in

plastic bags, creating a greenhouse effect (Fig. 3.4). The

plants were incubated in a growth chamber at 28 C under

fluorescent lamps and gradually hardened off by removing the

plastic covers. Acclimated plants were transplanted to

larger pots and placed in a greenhouse under a black cloth

screen to prevent scorching. The screen was removed 3 weeks

later and surviving plants were counted. Fertilization was









initially done biweekly with an acid-forming plant

fertilizer and later on with a solution of 1/10 MS salts as

apparent nutrient deficiencies developed. Phenotypic

observations of the plants were made periodically.



Results and Discussion


Production of plantlets from axillary bud cultures

initiated on solid medium and multiplied in liquid shaker

cultures started 9 months after explanting. The total number

of harvestable plantlets doubled with each monthly

subculture after the 11th month (Table 3.2). From 300 to 350

plantlets could be obtained by the 13th month from a single

bud culture (Table 3.3). Approximately 25 plantlets could be

harvested from each 125 ml flask with each additional

subculture. In all genotypes, plantlets larger than 3.0 cm

had a survival rate of nearly 100% when transferred to soil.

A 2-fold increase in the total number of harvestable

plantlets was obtained in 6 weeks when plant growth

regulators were removed from the multiplication medium

(Table 3.4; Fig. 3.5). However, after 2 subcultures (14

weeks), all plantlets had been harvested and no further

callus production or multiplication occurred. Further,

transferring to a medium without plant growth regulators to

induce root formation was not necessary for plantlet

survival. Plantlets without roots produced in multiplication

medium with plant growth regulators also had a high survival

rate and, while initially smaller than plantlets produced in









hormone-free medium, attained the same level of growth after

2 months in soil. The cultures periodically transferred into

multiplication medium with plant growth regulators appear to

be capable of indefinite multiplication. Phenotypic

variation using leaf morphology as a marker was assessed in

approximately 2000 regenerated plants and will be discussed

in detail in Chapter VII.

Tissue culture of the species A. bracteatus var.

tricolor produced regenerated plants that were either

tricolor, green or albino (Fig. 3.6). This indicates the

chimeral origin of this genotype. It is clear that these

variegated plants should not be given formal varietal rank.























Table 3.1. Murashige and Skoog salts formulation.


Salts mg 1-1


Major Salts
NH NO3 1650.00
KNO 1900.00
MgS 4.7H20 370.00
CaCl 440.00
KH2 P4 170.00

Minor Salts
Na EDTA 37.30
FeSO4.7H O 27.80
MnSO4.4H O 22.30
ZnSO4.7H O 8.60
H BO 6.20
K 10.83
NaMoO ..2H 0 0.25
Co0S4.6H20 0.025
CuSO4.5H20 0.025























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ON, A



























0i

















Figure 3.1. Initiation of axillary bud culture. A. Buds
aseptically removed; B. Bud expansion 8 days
after inoculation.













































Figure 3.2. Proliferating culture 4 months after
inoculation and ready to be transferred
to liquid multiplication medium.


"I

















































Figure 3.3. Actively dividing culture ready for
subculture. Plantlets larger than
2.5 cm will be transferred to soil.




























ro

a.



co






U) U)
"-H 4-4








.QU




cno
(c
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Figure 3.5. A. Comparison of multiplication cultures
with (right) and without (left) plant
growth regulators.







L A


Figure 3.5.


continued. B. Plantlets produced in medium
with plant growth regulators; C. Plantlets
produced in medium without plant growth
regulators.


Y






















































Figure 3.6. A. Ananas bracteatus var. tricolor.












B





















C





















Figure 3.6. continued. B. In vitro multiplication showing
resolution of chimera; C. Regenerated plants.
Albino plants are incapable of survival in
soil.



















CHAPTER IV
ELECTROPHORETIC STUDIES IN ANANAS


Introduction


Electrophoretic characterization of plants based on

differential patterns of protein migration is a powerful

technique to separate and classify genotypes. Isozyme

analysis has aided taxonomists, breeders, and physiologists

in understanding the underlying causes of phenotypic

variation. The plant breeder is particularly interested in

classification and characterization studies in order to

better utilize existing germplasm resources. The purpose of

this study was to determine what enzyme and buffer systems

work well in Ananas and which enzymes show polymorphism. The

analysis was conducted with taxonomy, breeding, and cultivar

improvement in mind.

No previous reports of isozyme studies have been

published for pineapple. For this reason, an initial study

was undertaken using a small number of Ananas genotypes to

determine which enzyme staining systems would give well

resolved banding patterns. Horizontal starch gel

electrophoresis was used for isozyme characterization

because it is non-toxic and yields multiple slices from a









single gel, thus allowing the rapid screening of a large

number of enzyme staining systems. The procedures followed

were similar to those currently being used with Citrus by

Moore (personal communication, 1985). The enzyme staining

systems that resulted in well resolved banding patterns were

used to survey a larger number of Ananas genotypes to

identify and characterize isozyme polymorphism. Genetic

studies were performed using seedling populations to

determine the basis of variability observed among genotypes.

Information from these studies was used to formulate a

system by which cultivars, feral types, and species could be

identified and distinguished, and their phenetic affinities

could be established. These systems will be described in

Chapters V and VI. Selected enzyme staining systems were

used to determine phenotypic stability of tissue

culture-regenerated plants and will be discussed in Chapter

VII.



Materials and Methods


Genotypes Used for Electrophoretic Studies

A list of the genotypes used in this study is given in

Table 4.1. The population included all of the species

presently considered valid in the genus Ananas (Smith and

Downs, 1979; Antoni, 1983) with the exception of A.

fritzmuelleri, which was not available in any of the major

world collections. The A. comosus cultivars surveyed

represent the most important cultivars in use world-wide.









The Ananas genotypes indicated by numbers are introductions

made by the author and originally established at the

Facultad de Agronomia, Universidad Central de Venezuela, or

material from the collection at the USDA Subtropical

Horticultural Research Unit, Miami, Florida. These feral

types were collected in the regions of southern Venezuela

and northern Brazil, an area that has been suggested as the

center of origin for the genus Ananas (Leal and Antoni,

1980). The suspected taxonomic classification of the feral

types follows the introduction number. The identification

was based on the botanical characters considered valid for

each Ananas species by Smith and Downs (1979). High levels

of phenotypic variation were present for several characters

among the collected types, including presence or absence of

spines, leaf color, and fruit shape, size, and color.

Genetic studies were based on isozyme analysis from

both the Ananas germplasm survey and 2 seedling populations.

The seedling populations were obtained from a commercial

planting 'of 'Smooth Cayenne' and 'Cambray' growing in

adjacent fields in northwestern Ecuador. Since pineapple has

only been reported as self-incompatible within a cultivar

and cross-compatible between cultivars (Brewbaker and

Gorrez, 1967), these seedling populations were assumed to be

2 segregating populations representing reciprocal crosses

between 'Smooth Cayenne' and 'Cambray'. The seeds were taken

from mature fruit, rinsed in water, and air dried. Seeds

were scarified by a treatment of 30-60 sec in concentrated









sulphuric acid followed by a rinse in sterile water and were

planted in covered petri dishes containing a sterilized soil

mixture moistened with sterile water. Periodic sprays with a

1% Captan solution were made to avoid fungal contamination.

Germinated seeds were transferred to pots containing a

commercial soil mix composed of sphagnum peat moss,

horticultural grade vermiculite and perlite, composted pine

bark, and washed granite sand. The pots were covered with

polyethylene bags to prevent dessication. Transplants were

initially held in a growth chamber at 27 oC with 16 hr

-1 -2
fluorescent light (76 umol s m ), and were taken to the

greenhouse after acclimation.

All plants of the collection were kept at Gainesville,

Florida, under greenhouse conditions during the cold months

(Nov.-Apr.) and outside the rest of the year. Plants were

kept in 25 cm pots with the same soil mixture as the

seedlings and were periodically fertilized with a standard

water-soluble 20-20-20 fertilizer.



Starch Gel Preparation

A 10% (w/v) starch gel consisting of a 2:1 (w/w) mixture

of Sigma and Connaught hydrolyzed potato starch was used

throughout this study. All the starch was from the same lot

number. Thirty grams of the starch mixture along with 300 ml

of a gel buffer solution (Table 4.2) were poured into a 1000

ml side arm flask. The mixture was swirled vigorously by

hand and placed on a magnetic stir pad at medium speed for









10 min to assure a well-mixed suspension and to avoid the

formation of lumps in the starch. The flask was then placed

on a magnetic hot plate at high heat and stirred at medium

speed. The solution turned translucent as it reached maximum

viscosity. At this point, the flask was again swirled by

hand and the cofactor NADP (10 mg dissolved in 1 ml water)

was added for those staining systems requiring it. The flask

was heated again until large air bubbles appeared on the

surface of the solution.

The solution was degassed immediately by vacuum before

it started to cool, to avoid air bubbles forming in the gel

that could interfere with the migration of proteins. The

vacuum source was provided by attaching an aspirator to a

sink faucet and to the flask side arm. The solution was

ready to be poured when no small bubbles were present.

The molds used had inside dimensions of 19 x 19 x 0.5 cm

and were made from plexigas sealed with methylene chloride.

Any bubbles that formed while pouring the starch solution

into the molds were removed with a spatula before the gel

started to harden. The gel was allowed to cool for

approximately 30 min at room temperature, and then further

cooled by refrigeration (4 C) for 1 hr prior to loading the

samples. Gels could be kept for 12-16 hr at room temperature

before being placed in the refrigerator and they were

covered with polyethylene film to prevent dessication.









Protein Extraction and Gel Loading

Leaf sap was used as the main protein source throughout

this study because it was easily prepared, readily

available, and gave good results. All leaf samples were

taken from the fourth leaf at the center of the plant,

counting from the first visible leaf, and were used

immediately or stored in plastic bags at 4 C for later use.

Samples were used within 7 days of collection, although no

decrease in enzyme activity could be detected for up to 3

weeks in storage. Leaf sap was expressed from the proximal

portion of the adaxial surface of the lamina, using a pestle

and a filter paper wick (Whatman No. 3, 4 x 8 mm). The white

waxy epidermis and underlying layer of fibers were scraped

from the leaf surface to aid in leaf sap absorption into the

wick. The saturated wick was then inserted into a slit 5 cm

from the top (cathodal side) of the chilled gel for anodal

migrating enzymes and at 8 cm from the top for cathodal

migrating enzymes, e.g. peroxidase. Once all samples had

been inserted, glass rods were placed at the top and bottom

of the gel to ensure good contact between wicks and gel

during electrophoresis.

Pollen was collected from flowering plants just prior

to anthesis, dried at room temperature for 24 hr, and frozen

at -4 C until used in electrophoretic assays. Proteins were

extracted from the pollen u-sing the method outlined by

Weeden and Gottlied (1980). A small amount of frozen pollen









was placed in 0.1 ml of extraction buffer for 4 hr. The

supernatant was then absorbed into the wick.



Electrophoretic Buffers and Staining Systems

Five electrophoretic buffer systems were tested:

histidine citrate (H), lithium borate/tris citrate (LBTC),

tris borate (TB), tris citrate (TC), and K buffer (K) [Table

4.2], as were 38 enzyme activity stains. Tested combinations

are listed in Table 4.3.

The formulations used for the enzyme staining solutions

were derived from several authors. Endopeptidase (E.C.

3.4.22.?) was according to Melville and Scandalios (1972).

Formate dehydrogenase (E.C. 1.2.1.2) was from Wendel and

Parks (1982). Creatine kinase (E.C. 2.7.3.2) and hexokinase

(E.C. 2.7.1.1) were as described by Shaw and Prasad (1970).

Malate dehydrogenase (E.C. 1.1.1.37) was from Cardy et al.

(1981). The following were according to Vallejos (1983):

adenylate kinase (E.C. 2.7.4.3), alcohol dehydrogenase (E.C.

1.1.1.1), aldolase (E.C. 4.1.2.13), alpha-amylase (E.C.

3.2.1.1), ascorbate oxidase (E.C. 1.10.3.3), catalase (E.C.

1.11.1.6), diaphorase (E.C. 1.6.4.3), fumarase (E.C.

4.2.1.1), galactose dehydrogenase (E.C. 1.1.1.48), beta-D-

galactosidase (E.C. 3.2.1.23), using alpha-napthyl-D-

galactopyranosidase as a substrate, glucose-6-phosphate

dehydrogenase (E.C. 1.1.1.49), beta-D-glucosidase (E.C.

3.2.1.21), glutathione reductase (E.C. 1.6.4.2), isocitrate

dehydrogenase (E.C. 1.1.1.42), laccase (E.C. 1.10.3.2),





42


lactate dehydrogenase (E.C. 1.1.1.27), 6-phosphogluconate


aenyarogenase (E.C. .i.i.'i),


pyruvate


&.iiilbe (iC;.


2.7.1.40), shikimate dehydrogenase (E.C. 1.1.1.25), triose

phosphate isomerase (E.C. 5.3.1.1), urease (E.C. 3.5.1.5),

and xanthine dehydrogenase (E.C. 1.2.1.37). In addition to

these, the following staining systems were modified from

Vallejos (1983) as follows:

Acid phosphatase (E.C. 3.1.3.2)
100 mg alpha-napthyl acid phosphatase, Na salt
50 mg Fast Black salt
100 mg 0.1 M sodium acetate, pH adjusted to 4.7
with glacial acetic acid


Alkaline
100
100
100

Esterase
100
42
40
18
3


phosphatase (E.C. 3.1.3.1)
mg beta-naphthylacid phosphatase, Na salt
mg Fast Blue RR salt
mg 0.1 M tris HC1, pH 8.5

(E.C. 3.1.1.2)
mg Fast Blue RR salt
ml H 0
ml 0.2 M NaH2PO
ml 0.2 M Na2 HP
ml of the following solution: 1 g alpha-
napthyl acetate and 1 g beta-napthyl acetate
dissolved in acetone and brought to 100 ml
with H20


Glutamate oxaloacetate transaminase (Aspartate amino-
transferase, E.C. 2.6.1.1)
500 mg L-aspartic acid
70 mg alpha-keto-glutaric acid
50 mg pyridoxal-5'-phosphate
200 mg Fast Blue BB salt
100 ml 0.1 M tris HC1, pH 8.0

Leucine aminopeptidase (E.C. 3.4.11.1)
40 mg L-leucine-beta-napthylamide HC1
100 mg Black K salt
50 ml 0,2 M tris + 0.2 M maleic anhydride
20 ml 0.2 N NaOH
30 ml H20









Phosphoglucomutase (E.C. 2.7.5.1)
500 mg glucose-1-phosphate, Na2 salt
20 mg NADP
30 mg MTT
4 mg PMS
100 mg MgC1
100 ml 0.1 M tris HC1, pH 8.0
40 units glucose-6-phosphate dehydrogenase

Phosphohexose isomerase (Glucophosphate isomerase,
E.C. 5.3.1.9)
100 mg fructose-6-phosphate, Na2 salt
10 mg NADP
20 mg MTT
4 mg PMS
100 mg MgC1
100 ml 0.1 M tris HC1, pH 8.0
10 units glucose-6-phosphate dehydrogenase

Malic enzyme was modified from Cardy et al. (1981);

peroxidase and superoxide dismutase were modified from Moore

(unpublished) and Moore and Collins (1983), as follows:

Malic enzyme (E.C. 1.1.4.0)
30 mg NADP
30 mg NBT
4 mg PMS
100 mg MgCI
20 ml 0.1 A malic acid, pH adjusted to 7.0
with NaOH
1 ml 0.2 M NaH2PO4
48 ml 0.2 M Na2HPO4
30 ml H20

Peroxidase (E.C. 1.11.1.7)
250 mg para-phenylenediamine
50 mg MnSO4
5 ml 1.0 M sodium acetate, pH adjusted to 4.7
with glacial acetic acid
30 ml 95% ethanol
65 ml H 0
0.5 ml 30% H 02 added after above solution is
poured onto gel









Superoxide dismutase (E.C. 1.15.1.1)
2 mg Riboflavin
75 mg Na EDTA
10 mg NBE
100 ml 0.1 M tris HC1, pH 8.0


Electrophoresis

The gel with sample wicks in place was set on top of 2

plexiglass buffer tanks. The electrode consisted of a 19 cm

length of platinum wire. Five hundred milliliter of buffer

solution was added to each electrode buffer tank, covering

the electrode. The bridges between the electrode buffer and

the gel consisted of 4-ply chromatography paper (Whatman No.

3). A glass plate was placed on top of the gel to assure

good contact between the gel and electrode bridges.

Electrophoresis was carried out under constant voltage

in a refrigerator at 4 0C. Power settings and running time

varied with each buffer and enzyme staining system. Gels

were typically run for the initial survey of buffers at 100

to 180 volts and approximately 20 milliamps for 16 to 18 hr

or at 200 to 250 volts and 25 to 50 milliamps for 4 to 6 hr.

Protein migration was monitored using several techniques. In

the gels containing borate buffers, a light brown line was

visible that migrated anodally during electrophoresis. In

gels using K buffer, a sample wick saturated with

bromophenol blue was used to follow the progress of

electrophoresis. Protein migration was monitored in gels

using histidine buffers by noting a pink color in the anode

electrode buffer solution. Protein migration could also be










monitored by a decrease in current readings when using a

constant voltage source during electrophoresis.



Enzyme Activity Staining

Gels were sliced horizontally into 4 slices (2 mm

thick), the top slice was discarded. The gel slices were

placed in plastic boxes and refrigerated until staining

(usually less than 2 hr).

Staining solutions were prepared fresh, poured over the

gel slice and swirled lightly to eliminate air bubbles

trapped underneath. Gels were placed in the dark and

observed every 15 to 30 min to follow the progress of

staining. When optimum resolution was observed, the gels

were rinsed in deionized water and examined. Gels were fixed

for long-term storage using a (7:7:1 v/v/v) solution of

methanol, deionized water, and glacial acetic acid. The gels

were then wrapped in polyethylene film and refrigerated.



Analysis of Banding Patterns

The banding patterns (zymograms) were recorded by

measuring the migration rate of each band. An internal

standard was created in each gel system to calculate

relative migration rates. The borate front produced in the

TB and LBTC buffer systems and the bromophenol blue band in

the K buffer systems served as an internal standard for

these systems. Buffer systems that did not produce a front

or a bromophenol blue band (H and TC) were evaluated by









placing a sample with a unique banding pattern in the gel,

one band of which served as the internal standard. The

migration of the band relative to the front (Rf value) or to

the unique band (Rb value) was calculated by dividing the

band migration distance by the migration distance of the

internal standard.

A genetic mechanism to explain variability was

hypothesized when zymogram polymorphism was observed among

the genotypes. The 2 segregating populations available were

used to test the genetic mechanism by comparing the observed

ratios with the expected ratios using chi-square analysis.

The possibility of genetic linkage was investigated by

analyzing the segregation ratios at more than 1 locus.

Linkage between loci could be suspected if chi-square

analysis revealed a significant deviation from ratios

expected with independent segregation.



Results and Discussion


Selection of Electrophoretic Buffers and Enzyme Systems
Useful for Studying Isozymes in the Genus Ananas

The results of the survey testing 38 enzyme staining

systems with 5 buffers can be seen in Table 4.3.

Well-resolved banding patterns were obtained with 8 enzyme

activity stains: isocitrate dehydrogenase, malate dehydro-

genase, peroxidase, phosphoglucomutase, 6-phosphogluconate

dehydrogenase, phosphohexose isomerase, superoxide dismu-

tase, and triose phosphate isomerase. Table 4.4 lists these










enzyme staining systems along with important electrophoretic

parameters. These systems were selected for further study

and are detailed below.

Twelve additional enzyme systems showed limited

resolution or enzyme staining activity and were grouped into

3 categories: those with activity but no discrete banding,

those with poorly resolved banding and no apparent

variability, and those with poorly resolved banding but some

apparent variability. Enzyme staining systems giving no

discrete banding included beta-D-galactosidase, format

dehydrogenase, and urease. Systems giving poorly resolved

banding with similar activity among genotypes were esterase

and glutathione reductase. Staining systems giving poorly

resolved banding patterns with some variable activity among

genotypes included acid phosphatase, glucose-6-phosphate

dehydrogenase, glutamate oxaloacetate transaminase, leucine

aminopeptidase, malic enzyme, 6-phosphogluconate dehy-

drogenase, and shikimate dehydrogenase. Although 2

additional protein extraction buffers were tried with these

enzyme staining systems, no improved resolution was

observed.

No activity was detected for the following 18 enzymes:

adenylate kinase, alcohol dehydrogenase, aldolase, alkaline

phosphatase, alpha-amylase, ascorbic oxidase, catalase,

creatine kinase, diaphorase, endopeptidase, fumarase,

galactose dehydrogenase, beta-D-glucosidase, hexokinase,









laccase, lactate dehydrogenase, pyruvate kinase, and

xanthine dehydrogenase.

The 8 enzyme activity stains that gave well-resolved

banding patterns were used to survey the 62 genotypes listed

in Table 4.1. In 5 of the staining systems, monomorphic

banding patterns were obtained. The other 3 staining systems

gave polymorphic banding patterns.



Monomorphic Enzymes

These enzymes included isocitrate dehydrogenase,

phosphohexose isomerase, superoxide dismutase and triose

phosphate isomerase. Banding phenotypes observed for these

monomorphic systems are illustrated in Fig. 4.1. and are

described below:

Isocitrate dehydrogenase. Two anodally migrating bands

were observed with this stain, 1 at 2.0 and the other at 2.5

cm from the origin (Fig. 4.1A). The second band appeared

less intense and was not always detected. No variability was

observed among the genotypes studied.

6-Phosphogluconate dehydrogenase. The banding pattern

obtained is shown in Fig. 4.1B. Two anodally migrating

bands, 1 major and 1 minor, were observed for all genotypes

investigated. The major band exhibited a mobility of 2.3 cm

from the origin. The fainter minor band migrated 1.1 cm from

the origin. Fainter poorly resolved bands were also present.

Phosphohexose isomerase. The banding pattern for this

enzyme consisted of 3 bands (Fig. 4.1C). The first and









second bands stained more intensely and migrated at 2.3 and

2.6 cm, respectively, from the origin. The third band, which

migrated at 3.0 cm, usually stained less intensely and was

not always detected. Another unresolved zone of activity was

present.

Superoxide dismutase. The banding pattern is

illustrated in Fig. 4.2D. A monomorphic pattern consisting

of 2 bands was observed for all the genotypes in this enzyme

staining system. The acromatic bands migrated 3.7 and 4.7 cm

from the origin.

Triose phosphate isomerase. Three well resolved bands

were observed for this enzyme with relative migrations of

2.0, 2.5, and 2.8 cm from the origin (Fig. 4.2E). No

variability was detected among the genotypes surveyed.



Enzyme Systems with Variable Banding Patterns

Variable banding patterns were observed for 3 enzyme

staining systems: malate dehydrogenase, peroxidase, and

phosphoglucomutase. Hypotheses to explain the observed

variability in these enzyme systems were based on a genetic

analysis of 2 segregating populations of plants from

putative reciprocal crosses between 'Smooth Cayenne' and

'Cambray'. Isozyme patterns for peroxidase and

phosphoglucomutase observed for other genotypes surveyed

will be discussed in detail in Chapter V.

Peroxidase. Three regions, 2 cathodal (Per-1 and Per-2)

and 1 anodal (Per-3), of well resolved variable banding










patterns were observed for all the genotypes tested, and are

illustrated in Figs. 4.2 and 4.3. All 3 variable regions

exhibited the same general banding patterns, consisting of

either a single fast-migrating band (FF), a single

slow-migrating band (SS), or a combination of the 2 (FS).

The slow band for Per-i was at Rf -0.26, while the fast band

was at Rf -0.28. The slow band for Per-2 was at Rf -0.04 and

the fast band was at Rf -0.11. The slow band for the

anodally migrating Per-3 was at Rf 0.04 and the fast band

at Rf 0.07. Zones of activity with no defined bands were

observed between Rf 0.11 to 0.25 and 0.32 to 0.38.

The variability detected for peroxidase may be

explained by a model of a monomeric enzyme system with 2

alleles (F and S) at each of 3 loci (Per-1, Per-2, and

Per-3). The distal band (F) and proximal band (S) are

produced by different alleles. Heterozygous genotypes would

produce both a F and S band, while homozygous genotypes

would produce either an FF or SS band. Progeny produced from

the cross with 'Smooth Cayenne' (SS) as the seed parent gave

all nonparental isozyme phenotypes (FS) for Per-1, which

would result if parents homozygous (SS, FF) for different

alleles at a locus were crossed. 'Cambray', the putative

pollen parent of the seedlings, had a Per-i phenotype of FF.

Progeny showed a 1:1 ratio of FS and FF phenotypes for both

Per-2 and Per-3, which would be expected if parents of the

seedlings were homozygous (FF) and heterozygous (FS)

respectively for the alleles at the loci. This was the case









with 'Smooth Cayenne' (FF) and 'Cambray' (FS) [Tables 4.5

and 4.7; Figs. 4.2 and 4.3A].

Seedlings obtained from open-pollinated crosses with

'Cambray' as the seed parent and 'Smooth Cayenne' as the

putative pollen parent were expected to show a similar type

of segregation, since this would be the reciprocal cross of

the one described above and only the 2 cultivars were

growing in the region. Instead, progeny from the 'Cambray'

female parent exhibited only the 'Cambray' (FF) banding

pattern for Per-1, as would be expected from a cross between

2 plants that were homozygous at this locus (Table 4.6 and

Fig. 4.2). Chi-square values (Table 4.7) showed a very close

fit to a 1:2:1 (FF:FS:SS) ratio for both Per-2 and Per-3,

which indicates a cross between plants heterozygous at these

2 loci (Fig. 4.2). The banding patterns obtained from this

progeny suggested that 'Smooth Cayenne' did not participate

in the cross and that the seedlings are most likely the

result of selfing.

Phosphoglucomutase. Three anodal regions (Pgm-1, Pgm-2,

and Pgm-3) of well resolved variable banding patterns were

observed in all the genotypes tested and are illustrated in

Fig. 4.4. The banding patterns for the Pgm-1 region were

unique in comparison to Pgm-2 and Pgm-3, and consisted of 5

total bands in the wide population of genotypes surveyed; F,

S, M, L, or P were present in various combinations of 2 of

these bands. Both Pgm-2 and Pgm-3 exhibited the same

variable banding pattern consisting of either a single









fast-migrating band (FF), a single slow-migrating band (SS),

or combination of the 2 (FS). Values for Rb were calculated

using P.I. 115, a feral genotype exhibiting a distinctive

banding pattern (Figs. 4.4 and 4.6) which served as an

internal standard. The 5 bands for Pgm-1 were at Rb 0.43

(L), 0.63 (S), 0.73 (M), 0.83(F), and 1.0 (P). The slow band

(S) for Pgm-2 was at Rb 0.84 and the fast band (F) was at Rb

0.98. The slow band (S) for Pgm-3 was at Rb 1.00 and the

fast band (F) was at Rb 1.19.

The majority of plant species reported in the

literature have contained either 1 or 2 loci governing the

expression of phosphoglucomutase, with the exception of

Camellia japonica, in which a third locus was reported by

Wendel and Parks (1982). In the present study, it was found

that the banding pattern observed in Ananas also may be

explained by hypothesizing a monomeric enzyme with 3 loci

(Pgm-1, Pgm-2, and Pgm-3). Locus Pgm-1 would be composed of

5 alleles, while Pgm-2 and Pgm-3 would each have 2 allelic

forms.

The 2 seedling populations represent only 3 (F, M, and

S) of the 5 possible Pgm-1 allelic forms. Heterozygous

genotypes would show combinations of these 3 bands, i.e.,

FM, FS, or MS, while homozygous genotypes would produce

either a FF, MM or SS band. Likewise, according to the

model, the other 2 loci, Pgm-2 and Pgm-3, produce a distal

band (F) and proximal band (S) as the result of different

alleles. Heterozygous genotypes would produce both a F and S









band, while homozygous genotypes would produce either a FF

or SS band (Tables 4.5, 4.6, and 4.7).

The 'Smooth Cayenne'-derived population showed a 1:1

ratio of phenotypes for Pgm-1 (FS:MS), Pgm-2 (FF:FS) and

Pgm-3 (FS:SS), which would be expected if the parents were

heterozygous and homozygous for each locus, i.e., Pgm-1: FM

x SS, Pgm-2: FF x FS, and Pgm-3: SS x FS (Table 4.7).

'Smooth Cayenne' is heterozygous for Pgm-1 (FM) and

homozygous for Pgm-2 (FF) and Pgm-3 (SS). Conversely,

'Cambray' is homozygous for Pgm-1 (SS) and heterozygous for

Pgm-2 and Pgm-3 (FS). The segregation pattern for the 3 loci

indicate that the seedlings are the results of a cross

between 'Smooth Cayenne' x 'Cambray' (Figs. 4.5 and 4.6A).

The 'Cambray'-derived seedlings again exhibited only

the 'Cambray' banding pattern for Pgm-1, as would be

expected for progeny from parents homozygous at this locus

(Table 4.6). Segregation was observed for Pgm-2 and Pgm-3,

and chi-square values (Table 4.7) showed a very close fit to

a 1:2:1 (FF:FS:FS) ratio., indicating a cross between 2

heterozygous types (FS). As discussed above, 'Smooth

Cayenne' is heterozygous for Pgm-1 (FS) and homozygous for

Pgm-2 (FF) and Pgm-3 (SS), so that it could not have been

the pollen parent in this cross. Thus, the

phosphoglucomutase banding patterns obtained for this

population exhibited segregation ratios which corroborate

the hypothesis that the seedlings are indeed the result of

selfing and not outcrossing (Figs. 4.5 and 4.6B).










Self-incompatibility in pineapple is due to inhibition

of pollen-tube growth in the upper third of the style. It is

gametophytically controlled by a single S locus with

multiple alleles (Brewbaker and Gorrez, 1967). In general,

pineapples set no seed when self-pollinated, but can set

seed when cultivars are crossed (Collins, 1960). Collins

(1960) reports isolated self-fertile 'Smooth Cayenne'

plants. Seed production was variable and germinated seed

gave rise to seedy offspring. These plants exhibited very

low vigor and continued selfing was not possible.

Pareja (1968) hypothesized that the frequent occurrence

of seeds in 'Cambray' may be due to self-compatibility with

pollination being accomplished by mites. The isozyme banding

patterns displayed by the seedling population obtained from

'Cambray' comfirms the self-compatibility of this cultivar.



Malate dehydrogenase. One large anodal region

consisting of many closely adjacent bands was observed for

all genotypes tested and is illustrated in Fig. 4.5. Six of

the bands were well resolved and migrated to 3.5, 3.8, 4.1,

4.5, 5.0, and 5.5 cm from the origin. The 2 fastest

migrating bands, 5.0 and 5.5, were the only ones to show

clear segregation. Banding patterns for the 'Smooth

Cayenne'-derived seedlings exhibited segregation for these 2

bands (Fig. 4.7A) while no segregation (all seedlings had 2

bands) was observed for the 'Cambray'-derived seedlings

(Fig. 4.7C). When the power setting during electrophoresis









was changed from 9 to 16, more separation of the bands was

observed and segregation for other bands seemed to appear

(Fig. 4.7B).

Malate dehydrogenase has been reported as a dimeric

enzyme in numerous diverse plant species, e.g., avocado

(Torres and Bergh, 1980), celery (Orton, 1983c), citrus

(Torres et al., 1982), eucalyptus (Moran and Bell, 1983),

and maize (Goodman and Stuber, 1983). Pollen zymograms from

different cultivars, including 'Smooth Cayenne' and

'Cambray', were compared with their respective leaf

zymograms to determine if intra-allelic heterodimer

formation was present, as would be expected in a polymeric

enzyme. Two bands were absent in pollen zymograms (Fig.

4.7D), implicating intra-allelic heterodimer formation. The

presence of these interaction bands suggests that malate

dehydrogenase functions as a polymeric enzyme in Ananas.

However, more segregating populations must be analyzed

before the genetics of this enzyme system can be clarified.



Linkage Analysis for Enzyme Loci

Linkage analysis was carried out between the

peroxidase and the phosphoglucomutase loci. The results are

given in Tables 4.8 and 4.9. Chi-square analysis by

contingency tables showed that the observed ratios fit

ratios expected for independent segregation.










Conclusions


1. Eight enzyme staining systems of the 38 investigated

were found to give well-resolved banding patterns.

2. Well-resolved non-variable isozyme banding patterns

were observed for isocitrate dehydrogenase, 6-phospho-

gluconate dehydrogenase, phosphohexose isomerase,

superoxide dismutase, and triose phosphate isomerase.

3. Well-resolved variable isozyme banding patterns were

observed for malate dehydrogenase, peroxidase, and

phosphoglucomutase.

4. A genetic model was hypothesized to account for the

variability observed for peroxidase and phosphoglu-

comutase staining systems in the Ananas germplasm

surveyed and in segregating seedling populations from

'Smooth Cayenne' and 'Cambray' pineapple cultivars. The

model hypothesizes that both peroxidase and phospho-

glucomutase function as monomeric enzymes coded for

by 3 loci each: Per-1, Per-2, Per-3, Pgm-1, Pgm-2,

and Pgm-3. Each locus contains 2 allelic forms, F

and S, with the exception of Pgm-1, which exhibits

5 allelic forms: F, S, M, L, and P.

5. Independent segregation was found between all of the

peroxidase and phosphoglucomutase loci.

6. The isozyme studies confirmed that the 2 seedling

populations were the products of crosses between

'Smooth Cayenne' x 'Cambray' and 'Cambray' x self.






57


7. The existence of the 'Cambray' x self population calls

into question belief that domestic pineapple cultivars

are obligated self-incompatible.












Table 4.1. Ananas genotypes surveyed for isozyme banding
patterns.


Species
A. ananassoides
A. bracteatus
A. comosus
A. lucidus
A. nanus
A. monstrosus
A. parguazensis

A. comosus cultivars
Abaka
Black Jamaica
Brecheche
Bumanguesa
Cabeza de Mono
Cabezona
Cacho de Venado
Caicara
Cambray
Cumanesa
Dupuis Smooth
Esmeralda
Injerta
Maipure
Masmerah
Monte Oscuro
Morada
Nacional
Negrita
Panare
Perola
Perolera
Pina de Brazil
PR-1-67
Queen
Red Spanish
Rondon
Smooth Cayenne
Valera Amarilla


Feral TypesZ
046 A. ananassoides
064 A. nanus
072-1 A. comosus
079 A. ananassoides
083 A. ananassoides
085 A. comosus
086 A. comosus
092-1 A. comosus
092-2 A. comosus
093 A. comosus
094 A. comosus
095 A. comosus
097 A. parguazensis
108 A. parguazensis
110 A. comosus
115 ?
116 ?
117 ?
126 A. comosus
188 ?
189 ?
192 A. comosus
193 ?
196 A. comosus
25291 A. comosus
487439 A. comosus
487440 A. comosus
487442 A. comosus
487443 A. comosus
487444 A. comosus


ZNumbers correspond to introductions followed by the
suspected taxonomical classification. "?" indicates that
the botanical characteristics of the plant do not fit any
of the presently described species.














Table 4.2. Electrophoretic buffer systems used for isozyme
detection in Ananas.


Buffer Electrode Gel
system buffery buffery


H 0.065 M histidine 0.009 M histidine
0.02 M citric acid 0.003 M citric acid
pH adjusted to 5.7 pH adjusted to 5.7
with citric acid with citric acid

K 0.18 M trizma base 0.045 M trizma base
0.1 M boric acid 0.025 M boric acid
0.004 M Na2EDTA 0.001 M Na2EDTA
pH 8.6 pH 8.6

LBTC 0.016 M LiOH 0.0016 M LiOH
0.192 M boric acid 0.019 M boric acid
pH 7.2 0.007 M citric acid
0.046 M trizma base
pH 7.7

TB 0.038 M trizma base 0.03 M boric acid
0.002 M citric acid pH 8.5
pH 8.6

TC 0.05 M trizma base 0.017 M trizma base
0.016 M citric acid 0.005 M citric acid
pH adjusted to 7.0 pH adjusted to 7.0
with citric acid with citric acid

ZH = Histidine citrate (Cardy et al., 1981); K = K buffer
(Loukas and Pontikis, 1979); LBTC = Lithium borate/Tris
citrate (modified Scandalios, 1969); TB = Tris borate;
TC = Tris citrate (Moore, unpublished).

Y10 mg NADP was added to buffer in cathodal tank and to gel
buffer when staining for enzyme system that required NADP
as a cofactor.






60




Table 4.3. Enzyme activity stains and buffer systems tested
for isozyme detection in Ananas.



Buffer system

Enzyme activity stain LBTC H TC TB K


Acid phosphatase
Adenylate kinase
Alcohol dehydrogenase
Aldolase
Alkaline phosphatase
alpha-Amylase
Ascorbate oxidase
Catalase
Creatine kinase
Diaphorase
Endopeptidase
Esterase
Formate dehydrogenase
Fumarase
Galactose dehydrogenase
beta-D-Galactosidase
Glucose-6-phosphate dehydrogenase
beta-D-Glucosidase
Glutamate oxalloacetate transaminase
Glutathione reductase
Hexokinase
Isocitrate dehydrogenase
Laccase
Lactate dehydrogenase
Leucine aminopeptidase
Malate dehydrogenase
Malic enzyme
Peroxidase
Phosphoglucomutase
6-phosphogluconate dehydrogenase
Phosphohexose isomerase
Piruvate kinase
Shikimate dehydrogenase
Superoxide dismutase
Superoxide dismutase + KCN
Triose phosphate isomerase
Urease
Xanthine dehydrogenase


+



















+





+++




++

++

+++Y
+++
+++


+
*
*
*
*
*
*
*
*
*
*
+
*
*
*
*
++
*
++
*
*
++
*
*
*
++
*

*
++
++
*
++

*


- *


- = no activity; + = poor migration or resolution;

++ = fair migration or resolution; +++ = well resolved
bands; = assay not performed.

YModified to 0.065 M, pH 6.3.



















U






0
-H
a)






'o0
C










E
a)
4-J
U)
>1



EH


Cu
*4-



CO
>
a)







0 )






0 C
C- o


OJC
C

E





r-f



E-40
S)





S 0.







6-<


E-







El Q.
ma)
0





E








N
U)








C =
4-(
1-4





0





a) )
4-4 4-)
4-4 W





























co
a)




>1


N

rz


X
~ e~~ G~


n o0 0 n 0 0 0 0








n omN c4 N m 'o.
-4 -4 r-1 l







Ln 0 Ln In 0 In q In







o o o 0 0 0 0 0
0o o In o 0 VIn I
mm c m cN


>1 >1
a a tc; o


ul



0
k0
0 )


-4



u c
W C












a) 0
u e4
O(






?0 a
U
O








>4J 0




S 4-4
3 44


0 41
a)


00




4 4-







co (a
CE 4)






U) -H-






s-I r
40 ( 4
rCiC C0









E- 0














Table 4.5. Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived from
an open-pollinated 'Smooth Cayenne' fruit.


Isozyme genotypes

Seedlings Per-i Per-2 Per-3 Pgm-1 Pgm-2 Pgm-3





















Table 4.6. Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived
from an open-pollinated 'Cambray' fruit.


Isozyme genotypes

Seedlings Per-1 Per-2 Per-3 Pgm-1 Pgm-2 Pgm-3


1 FF FS FS SS FS FS
2 FF FS FF SS FS FF
3 FF FS FS SS FF FF
4 FF FS SS SS SS FS
5 FF FF SS SS FF FS
6 FF FS FF SS SS SS
7 FF FS SS SS FS FS
8 FF SS FS SS FS FS
9 FF FF SS SS FS FS
10 FF FS SS SS SS FS
11 FF FS FSS S SS SS
12 FF SS FF SS FF FS
13 FF F F FS SS FF SS
14 FF FF FS SS FS FS
15 FF FS FF SS SS SS
16 FF SS FS SS FS FS
17 FF FF FS SS FF SS
18 FF SS FS SS FF FS
19 FF FS FS SS FS FF
20 FF SS FS SS FS FF
















0o


I I
LU'-4


*Ml

0
,-4

0)
c
>1
N
0
U)




-4

4-I







OU




0)




CH
4-4










4O


0
U)
U)


ro
0
0
0







U)
a





c
0
1)










-4,








OC
UO
0












*E
H (U
^ -P



I-


(N (n (n

0- CN (N
0 C4 C
rlNN
*** V


I I l r rl r
I ** ** ** ** **
r] l rol





I I I I I





III LOi
r-





T--
0o o o-I m co C0





I CM IOr-
r-Il r- rV-


XXX







0) 0) (1
I II

U)U)U


eU) n W

XXX





r-it CP
I<& iI


o Ln
o r-
0*
* I







00
0 0


00


I *.N C
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LALA










00
00


00


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(N




I I I I I I





1 0 i I -
- -l


0 LAn c
(N















U) CnU) )




V --- V
0)
U) r-I NM C
(I II

> 0) (U U)




(0
u


I ow O























r-4 N en


On m t7
P4 P 0
EEE
0 tr CD


LA)CN

-i


-Q

0*
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X












E
(0



U
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-o









241
(0

UO
Cn















'4-I

-U)
0 0



E

0 0







0 -H



u 0


4-)Q)
- 41
U- (a
U u

Va)
CN4








0
4C4

























)-H







-H +







65






( IlIIIIIII
O > LnooLnminMmmaLn
Qc) a4






0fa O.- w CN
E X
CO

U0 0 0
3r-I r-4 r- r-i r-4 r-4 r-4 r-l r-A -4


-I 4 r- r- r-4 -
S.. .. .. .. .. .. .. .. ..
H U ( r- r-I v.-4 .-4
44 W



4-1 tQ E-i
(0
m4 4J

0 .
E 0 -- -oo o- -- o-




0 0 w 1,m r- m %
0 0-) rJW W C W W b W CQ M M
00 4OC2www




mC -O
r--







4 -4
0- -





o Iz I I I I I I IQI I
0 Q 0
I (0 44 ~ U 5C/ rZz 44 rjj r4 M W .4
OH r

u w r.
(0 -






(0 0 U)r rM4M m N
*H *04 ao wU) ) -H
-Ik CC UrJ Cf l
(0 44 -H (I

V 0- r. U)







S0 w0l0C0(7
C U(D ) HNCDI -O ,-0 v-4 V
*-r U) (0 -- )
-Pu N U) U)4 nMU)U (nCA)C CncU) W
(z '- P U ) Q ) I w x w m x ww 0



U) -H V -W

S4 WE E EEEEEEE H
* w tp tp tr tU) 0 tr '0'00 tp0 V



0 4 W 4 W 4 W W E E E I
(0 0 U w ) ) )U U w U t'p p T
E-4 4- a4 P404 04 134 0i ai P4 (i a4~ A4a













a)
0 0 L LA LA L 0 0
0) (N 0 (N N H
P > (0 .
S-H > I I I I
S0 0 0 o Ln
W a 4 Ln in in N CN
0 I
04- a)
(U > 0
0 ( o o o oo
c o N o > o
r > m m m in o

OU X
m -l
0 C

00 *X

(0 U i -4l r-l W( r- ri l 1 r r-1 i l -l 1-4
> o ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** **
> 0 .. .. .. .. .. .. .. .. 5* .. .. 0. *. Co *.
4-0 L .. ** ** ** ** ** ** ** ** ** ** ** ...
-HO) W) -1 .-i .- .-4l --4 r- .-4 .- I r-4- r-4 V-i -l 4 -
4-4O E-

41 4-

C0 3

U) 0 Un z-1ti) MCI) fciZ.04 LUn y) 6CN
rX4 rT4 rL4a d rZ.4 r 4 rL| r24 CL| PLa


a) 0 0 -- -.- 1- -


O0. 0 (N ( (O (N O
o0 0 U)( U(n U) E U) ( UI (no U)





04 rici) FZd j) IT4 U) rZ4 W EI U)
00 0
44 U) rNL mm r34 -I r m c
0 U) 441 LIC w C(n W W 4 I2 CrO2 W CJ
1 3 r4 ) WzUi4 UM1s) r4 M44 M(0
W EM I II I ) XC4 nCa II an Ia CI U tO



O 0
0 x



0 a) -H -1-
00 n C14 U O0 (nMX MA M W MM MM
(U 44 (U ra a)




0 0 4I I mI
-HC ) --- --- -- --- (1)






O)sIN ON(N (N mmO i
4J (U ) U N raM U M U) W fi) (n W m w aw wm 44 (n U) r2 V) U)M M)
(-I -J H I I I I 6 W U)



41 1 N) 1 )
(W r--lN n r-4N (e NM m
*HI I I II I I I I I u
4 4 W E E EE E EEE H
< A 04 04 04 04 04 a4 94 P4 Pc PL C
W C ( N C% (N ce m m t AI

SU4 4 W w W 4 4 E E 'I
(13 0 () Q) () (1) a) (U Q) (U) 0>0l t
E-4 P4 04 PL4 a4 a4 14 a4 A A4ia4 0 a 4
















A


















B


















Figure 4.1. Non-variable isozyme banding patterns
observed for Ananas genotypes. A. Isocitrate
dehydrogenase; B. 6-Phosphogluconate
dehydrogenase. Sample origin is at the bottom
of each photograph and band migration is
anodal.





































Figure 4.1. continued. C. Phosphohexose isomerase;
D. Superoxide dismutase.


I ~~~HUIlr I I I






















Imsp't*3g "~9ffl


Figure 4.1. continued. E. Triose phosphate isomerase.


























+

m-mmmm -m mmm

Per-3

--a --- -

Pe r-2
um- mmm- mmm -


m mmmm


m Per-i


1 2 3 4 5 6 7 8 9 10 11 12 13


Figure 4.2. Schematic representation of peroxidase isozymes
in 2 pineapple segregating populations.
1. 'Smooth Cayenne'; 2. 'Cambray'; 3-6. Hybrids
of 'Smooth Cayenne' x 'Cambray' cross;
7-13. Hybrids of 'Cambray' x self cross.


0.0


.1


M


III II























*1"



~ I


I'Y ?



r~I


I e


I


Figure 4.3. Peroxidase banding patterns observed in
pineapple. A. From right to left, 'Smooth
Cayenne' ,'Cambray', and progeny from the
putative cross 'Smooth Cayenne' x 'Cambray';
B. 'Smooth Cayenne', 'Cambray', and progeny
from the putative cross 'Cambray' x self.
Sample origin is at the middle of each
photograph and band migration is both
anodal and cathodal.

























Pgm-3


Pgm-2


m -m


Pgm-I


m
mmmm


1 2 3 4 5 6 7 8 9


Figure 4.4. Schematic representation of phosphoglucomutase
isozyme in Ananas. 1. P.I. 115; 2. 'Maipure';
3. 'Smooth Cayenne'; 4. P.I. 072; 5. P.I. 085;
6. 'Black Jamaica'; 7. P.I. 095; 8. P.I. 27281;
9. 'Brecheche'. Black bands = Pgm-1; cross-
hatched bands = Pgm-2; dotted bands = Pgm-3.


~S~i~B ~ g~ ~3~ ~Sg~two
























1.2-
1.1 -
1.0


........ ........


Pgm-3


Pg m-2


m m m


I m


m m


Pgm-1


m M


1 2 3 4 5 6 7 8 9





Figure 4.5. Schematic representation of phosphoglucomutase
isozyme in 2 pineapple segregating
populations. 1. 'Smooth Cayenne';
2. 'Cambray'; 3-6. Hybrids of 'Smooth
Cayenne' x 'Cambray' cross; 7-9. Hybrids
of 'Cambray' x self cross.


Am= As= am ~~8W


M =- ill 8 i I ME=H~s

























*4* *fb* | ,


Figure 4.6.


Phosphoglucomutase banding pattern in
pineapple. A. From right to left, P.I. 115,
'Smooth Cayenne', 'Cambray', and progeny
from the putative cross 'Smooth Cayenne' x
'Cambray'; B. 'Smooth Cayenne', 'Cambray',
and progeny from the putative cross
'Cambray' x self. Sample origin is at the
bottom of each photograph and band migration
is anodal.


-

























































Figure 4.7.


A





































Malate dehydrogenase patterns observed in
pineapple. A. and B. Gels run at different
power settings (9 and 16), from right to left,
'Smooth Cayenne', 'Cambray', and seedlings
from the putative cross 'Smooth Cayenne'
x 'Cambray'. Sample origin is at the bottom
of each photograph and band migration is
anodal.


I













C













;,,ii









Figure 4.7. continued. C. Progeny from the putative
cross 'Cambray' x self; D. Pollen and leaf,
from right to left, 'Smooth Cayenne',
'Brecheche', and 'Cambray'.


















CHAPTER V
IDENTIFICATION OF ANANAS GENOTYPES
BY ISOZYME PHENOTYPES


Introduction


The utilization of isozyme techniques for cultivar

identification requires high levels of clearly recognizable

inter-cultivar isozyme polymorphism, preferably with

monomorphic cultivar phenotypes. The phenotypes should be

stable over a variety of environmental and developmental

conditions. The techniques used to characterize the

phenotypes should be simple and efficient.

Ananas comosus appears to be an ideal candidate for

electrophoretic characterization using isozyme polymor-

phisms. The self-incompatible and outcrossing nature of the

species would be expected to generate high levels of

inter-specific genetic variation, while intra-clonal

variability would be expected to be relatively low due to

the apomictic nature of pineapple.

The taxonomy and origin of the genus Ananas has been

the subject of much debate and speculation. Species are

generally distinguished by the presence or absence and

direction of leaf spines, petal morphology, and fruit size.









The purpose of this study was to electrophoretically

characterize the genus Ananas using isozyme polymorphisms

with emphasis on gaining information on the origin of the

commercially important pineapple cultivars of the world.



Materials and Methods


Starch gels were prepared and electrophoresis was

conducted as described in Chapter IV. Two buffer systems

were used for electrophoresis, a histidine system at pH 6.3

and a K buffer at pH 8.6 (Table 4.2). The histidine buffer

was used to assay for phosphoglucomutase activity and the K

buffer for peroxidase activity. At least 2 plants of each

genotype (Table 4.1) were examined electrophoretically for

peroxidase and phosphoglucomutase polymorphisms, except that

no peroxidase determination was made for the feral types.

Assays were repeated until consistent results were obtained.

The banding patterns were recorded using both schematic

drawings and photographs. Band migration distances were

based on Rf and Rb values as calculated in Chapter IV.



Results and Discussion


Considerable isozyme polymorphism was observed among

the genotypes examined for both peroxidase and

phosphoglucomutase. In both systems, bands were

characterized for each locus according to inheritance

studies discussed in Chapter IV. Peroxidase showed either a









single or a double banded pattern at 2 migrating regions,

corresponding to the Per-2 and Per-3 loci. No clear bands

were present for Per-1. Phosphoglucomutase exhibited the

most useful isozyme polymorphisms. Bands corresponding to

the Pgm-1 locus gave consistent and clear resolution but

overlapping bands corresponding to Pgm-2 and Pgm-3 loci

complicated the characterization of certain genotypes.



Cultivar Identification

Peroxidase. Limited isozyme polymorphism was detected

for peroxidase. A total of 7 different banding patterns was

observed and designated A1 through A7 (Table 5.1; Figs. 5.1

and 5.2). Patterns A3, A4, and A7 were unique to 'Negrita',

'Pina de Brazil', and 'Maipure' respectively. 'Panare' and

'Queen' shared pattern A6, while the other cultivars showed

patterns A1, A2 or A5 (Table 5.2).



Phosphoglucomutase. Eleven different phosphoglucomutase

banding patterns were identified and designated B1 through

B11 (Tables 5.1 and 5.2; Figs. 5.1 and 5.2). Unique banding

patterns were observed for 'Black Jamaica', 'Rondon',

'Valera Amarilla', 'Cacho de Venado', and 'Cabezona'. These

patterns were B3, B4, B7, B and B10 respectively. The rest

of the cultivars were distributed over the patterns B1, B2,

B5, B6, B9, and B11 (Table 5.3).

The polymorphic enzyme systems of peroxidase and

phosphoglucomutase were very useful for cultivar









classification. Using only the peroxidase or the

phosphoglucomutase isozyme system, 8 cultivars could be

distinguished by their unique banding patterns (Tables 5.2

and 5.3). However, using information combined from both the

peroxidase and phosphoglucomutase isozyme systems, 20 unique

classes of banding patterns could be distinguished. These

classes permitted the identification of 15 cultivars out of

the total 27 examined (Table 5.4).

The origin and exact relationship of many of the

pineapple cultivars studied remains unclear. A wide variety

of clones and cultivars exists because of the prevalence of

inter-clonal fertility and apomixis accompanied by

self-incompatibility. Certain pineapple cultivars and clones

exhibit high rates of mutation and many unique sports exist;

e.g., 'Smooth Cayenne', the most economically important

cultivar, is really a group of clones (Collins, 1960). These

factors considerably complicate characterization and

classification schemes.

Based on the peroxidase and phosphoglucomutase isozyme

banding patterns, it appears that there is a very close

relationship among many of the cultivars tested. 'PR-1-67',

a reputed hybrid widely grown in Puerto Rico, was obtained

from an open-pollinationed seed of 'Red Spanish' growing

adjacent to a field of 'Smooth Cayenne' (Ramirez et al.,

1970 and 1972). The parental relationship of these 2

cultivars to 'PR-1-67' is corroborated by their isozyme

banding patterns (Table 5.1). 'Esmeralda', a cultivar grown









in Mexico (Samuels, 1970), and 'Dupuis Smooth' are presumed

to be sports of 'Smooth Cayenne'. The 3 cultivars exhibited

the same banding pattern, indicating a common origin.

'Cumanesa' is very similar morphologically to 'Red Spanish'

and is believed to be a sport of the latter (Leal and

Antoni, 1980b). The identical banding pattern showed by the

2 cultivars suggests that 'Cumanesa' could indeed have

originated as a sport of 'Red Spanish'.

'Bumanguesa' and 'Perolera' are the 2 cultivars

commonly grown in the Andean regions of Venezuela and

Colombia. Both cultivars have similar morphological

characteristics of plant growth and differ only in mature

fruit color. 'Bumanguesa' produces a bright red fruit, while

'Perolera' produces a yellow fruit. Because of the

similarities of these 2 cultivars, Leal et al. (1979)

hypothesized that 'Bumanguesa' was a sport of 'Perolera'.

However, the peroxidase and phosphoglucomutase banding

pattern observed for these 2 cultivars do not indicate a

close relationship, unless the mutation involved the Per-2,

Per-3, and Pgm-3 loci (Table 5.1).

The cultivars 'Queen' and 'Panare', which represent the

"Queen" group of pineapples, exhibited identical isozyme

banding patterns for both peroxidase and phosphoglucomutase.

They also are very similar morphologically. This is

remarkable since cultivars belonging to this horticultural

group are grown commercially only in South Africa and

Australia (Samuels, 1970). 'Panare' was described by Leal et









al. (1979) from a clone being cultivated in southern

Venezuela (Bolivar State). Local people reported that the

original plants were "wild" types found growing nearby.

Since the area including southern Venezuela and northern

Brazil has been suggested as the center of origin for the

Ananas genus (Leal and Antoni, 1980a) it seems likely that

the "Queen" group probably was derived from 'Panare'-like

wild types. This finding confirms the presence of "Queen"

types growing in the Western Hemisphere.

There seems to be some correlation among the Pgm-1

alleles and the horticultural groups of pineapple (Table

5.1). All cultivars within the "Cayenne", "Queen", and

"Maipure" groups exhibit identical banding patterns, i.e.,

FM. Phylogenetic relationships between these groups will be

discussed in Chapter VI. Most of the cultivars in the

"Spanish" group had a common banding pattern for Pgm-1,

i.e., SS. However, the 4 cultivars in the "Abacaki" group,

showed different allelic combinations for Pgm-1, i.e., FM,

MS, SS.

Pineapple cultivars are classified into horticultural

groups without regard to their origin or genetic

relationships, e.g., 'PR-1-67' is included in the "Spanish"

group even though it is a known hybrid (Samuels, 1970; Leal

and Soule, 1977). Similarly, other cultivars could be of

hybrid origin, which would explain the isozyme banding

variability observed between individuals within a group.

'Caicara' is from the same region as 'Panare' and









'Brecheche' and it may have been derived from these. 'Black

Jamaica' may be a hybrid between 'Smooth Cayenne' x 'Red

Spanish', and 'Valera Amarilla' may be a hybrid of

'Perolera' x 'Red Spanish', since the parents and hybrids

are all cultivated in the same regions. 'Cacho de Venado',

which has spiny leaves and conical-shape fruit, has been

included in the "Abacaki" group created by Py and Tisseau

(1965). Based on isozyme analysis of the Pgm-1 locus, it

appears to be more related to the "Spanish" group.

Furthermore, 'Cacho de Venado' is from the same region as

'Monte Oscuro' and the feral type 086 (Monagas State,

Venezuela). The banding pattern of these cultivars also

suggests a hybrid origin for 'Cacho de Venado'.



Species Identification

Peroxidase and phosphoglucomutase banding patterns for

7 Ananas species are shown in Table 5.5, Figs. 5.2 and 5.3.

Based on the banding patterns of peroxidase and

phosphoglucomutase, 5 of the 7 species can be distinguished.

The 2 exceptions are A. comosus, which includes all of the

commercial pineapple cultivars, and A. monstrosus. Ananas

comosus, as indicated in the previous section, exhibited

high levels of isozyme polymorphism, particularly for the

Pgm-1 locus. Ananas monstrosus also exhibited isozyme

polymorphism in the 2 individuals tested.

The amount of polymorphism observed within A. comosus

may be attributed to the present very broad circumscription









of this species. Smith (1961) separates A. comosus from the

other Ananas species by the length of the fruit, i.e.,

greater than 15 cm. Thus, in this classification system,

this species includes all the edible pineapples regardless

of other morphological differences such as leaf color,

presence or absence of spines, and fruit morphology. The

same flexibility is not allowed in the classification of the

other species and only individuals that exactly fit the

description, which includes leaf, flower, and fruit

morphology and growth habit, are included.

Ananas monstrosus was originally described by Carriere

(1870) and included in the later classification by Smith

(1961). The description was based on a single individual

plant found. It differs from A. comosus (pineapple) only in

that the fruit lacks a crown. The validity of this species

has been questioned by Antoni (1983), who reported finding a

mutation of the cultivar 'Negrita' that mimics the A.

montrosus characteristic, i.e., lack of crown. Based on

growth habit and leaf and fruit morphology, the 2 plants

representing A. monstrosus used in this study appear to be

crown mutations of 'Perola' and 'Red Spanish' respectively.

The peroxidase and phosphoglucomutase banding patterns

observed for these plants are also typical of 'Perola' and

'Red Spanish'. The taxon thus seems to be of polyphyletic

origin. The questionable validity of A. monstrosus as a

species is further corroborated by the fact that crown









mutations have commonly been seen in other pineapple

cultivars by the author.

The lack of intra-specific variability observed in the

isozyme banding patterns of A. ananassoides, A. bracteatus

var. tricolor, A. lucidus, A. nanus, and A. parguazensis may

have been due to the low sample number (2) used to

characterize each species. However, when the electrophoretic

data from all 5 of these species are considered together, it

is quite surprising that low inter-specific variability was

detected as well. It may be that this lack of variability

reflects the narrow circumscription of these species. Many

of the feral types used in this study closely resembled

individuals fitting the definition of species recognized by

Smith (1961), but they were not included in any of the

prsently recognized species because they differed in 1 or 2

characters from the species descriptions given by Smith and

Downs (1979). These genotypes may belong within the limits

of some of the presently delimited species, thus giving this

taxa greater intra-specific variability, as in the case of

the different clones of pineapple (A. comosus). Perhaps the

most valid explanation for the high degree of intra-specific

homology observed in all the 'Ananas' species, except for A.

comosus, is that they are self-pollinated. In contrast, A.

comosus is self-incompatible, with obligate out-crossing

favoring a high degree of isozyme and allelic heterogenity.










Feral Type Identification

The phosphoglucomutase banding patterns for the 29

feral types characterized electrophoretically are shown in

Table 5.6, Fig. 5.3. The full range of isozymic polymorphism

at each of the 3 phosphoglucomutase loci was represented in

this diverse collection of genotypes. The Pgm-1 locus was

particularly polymorphic, with all of the 5 alleles

represented, i.e., L, S, M, F, and P. Two of these alleles,

L and P, were unique to this collection and had not been

observed in any of the species or pineapple cultivars

previously characterized. Nine different banding patterns

(SL, ML, SS, MS, MM, FM, FS, FF, and PS) were observed at

the Pgm-1 locus. Five of these (SL, ML, MM, FF, and PS) were

new allelic combinations not previously observed.

The FF Pgm-1 genotype was observed in P.I. 095, typical

pineapple plants that produce large, conical 3 kg fruit. The

2 sampled plants of this introduction were collected from a

small plot of plants being cultivated by the Piaroas Indian

River community of El Gavilan. The region is an isolated

portion of dense tropical forest located in the north

central portion of the Amazonian Territory in southern

Venezuela. The cultivated plants were reportedly collected

from the wild by the local Indians.

The Pgm-1 genotype MM was observed in the 2 plants of

the introduction P.I. 487443. These plants were collected

from the tropical rain forest region of San Carlos de Rio

Negro. This region is located in the southernmost portion of




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TISSUE CULTURE AND ELECTROPHORETIC STUDIES OF
PINEAPPLE (Ananas comosus) AND RELATED SPECIES
By
MARIA GRAZIA 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 husband, Steve,
for his constant support, understanding, love, and help
through these last years while we were students and parents.

ACKNOWLEDGMENTS
I express gratitude to Dr. Wayne Sherman for his
tireless encouragment, thoughtful guidance, and friendship
during all these years. Special thanks are extended to Dr.
Gloria Moore for allowing the use of laboratory, continuous
support, and suggestions for this dissertation. Appreciation
is extended to Dr. Richard Litz for allowing use of
laboratory and equipment and advice during the tissue
culture studies. Sincere appreciation is given to Dr. Paul
Lyrene and Dr. Walter Judd for their help in reviewing this
dissertation and for the knowledge I gained when attending
their courses. The help of Anne Harper with laboratory work
is appreciated.
Financial support provided by CDCH and Universidad
Central de Venezuela is also acknowledged.
Many thanks go to my parents, Francesco and Antonina
Antoni, for their love and support over the years.
Finally, I extend my appreciation to the graduate
students, the service staff, and the faculty of the
University of Florida Fruit Crops Department, Gainesville,
for the many ways in which they have helped.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES VÍ
LIST OF FIGURES viii
KEY TO ABBREVIATIONS x
ABSTRACT xii
CHAPTER
I INTRODUCTION 1
II LITERATURE REVIEW 3
Ananas Taxonomy 3
Pineapple Propagation 4
Pineapple Tissue Culture 6
Somaclonal Variation 8
Application of Isozyme Analysis in
Horticultural Plant Systematics 12
III PRODUCTION OF PINEAPPLE PLANTS IN VITRO 17
Introduction 17
Materials and Methods 18
Genotypes Cultured 18
Sterilization and Initiation of Cultures.... 18
Culture Multiplication 19
Establishment in Soil 20
Results and Discussion 21
IV ELECTROPHORETIC STUDIES IN ANANAS 35
Introduction 35
Materials and Methods 36
Genotypes Used for Electrophoretic Studies..36
Starch Gel Preparation 38
iv

CHAPTER Page
Protein Extraction and Gel Loading 40
Electrophoretic Buffers and Staining
Systems 41
Electrophoresis 44
Enzyme Activity Staining 45
Analysis of Banding Patterns 45
Results and Discussion 46
Selection of Electrophoretic Buffers
and Enzyme Systems Useful for Studying
Isozymes in the Genus Ananas 46
Monomorphic Enzymes 48
Enzyme Systems with Variable Banding
Patterns 49
Linkage Analysis for Enzyme Loci 55
Conclusions 56
V IDENTIFICATION OF ANANAS GENOTYPES
BY ISOZYME PHENOTYPES 7 7
Introduction 77
Materials and Methods 78
Results and Discussion 78
Cultivar Identification 79
Species Identification 83
Feral Type Identification 86
Conclusions 88
VI PHENETIC RELATIONSHIPS IN ANANAS BASED
ON ISOZYME ELECTROPHORETIC ANALYSIS 101
Introduction 101
Material and Methods 103
Results and Discussion 104
VII CHARACTERIZATION OF REGENERATED
PINEAPPLE PLANTS 116
Introduction 116
Materials and Methods 117
Results and Discussion 118
Electrophoretic Characterization 118
Morphological Characterization 119
VIII SUMMARY AND CONCLUSIONS 124
LITERATURE CITED 130
BIOGRAPHICAL SKETCH 141
v

LIST OF TABLES
Table Page
3.1 Murashige and Skoog salts formulation 23
3.2 In vitro pineapple plantlet production for
each initiated bud 24
3.3 In vitro pineapple plantlet production for
each initiated bud 25
3.4 In vitro pineapple plantlet production in
response to the presence or absence of plant
growth regulators in the culture medium 26
4.1 Ananas genotypes surveyed for isozyme banding
patterns 58
4.2 Electrophoretic buffer systems used for isozyme
detection in Ananas 59
4.3Enzyme activity stains and buffer systems tested
for isozyme detection in Ananas 60
4.4Well resolved enzyme activity staining systems
and electroporetic parameters used in Ananas 61
4.5 Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived from
an open-pollinated 'Smooth Cayenne' fruit 62
4.6 Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived from
an open-pollinated ' Cambray ' fruit 63
4.7 Genotypic ratios and chi-square goodness of
fit values for 6 isozyme loci in pineapple 64
4.8 Segregation ratios and chi-square goodness of
fit values for independent inheritance of
peroxidase and phosphoglucomutase loci in
'Smooth Cayenne '-derived seedlings 65
vi

Table Page
4.9 Segregating ratios and chi-square goodness
of fit values for independent inheritance
of peroxidase and phosphoglucomutase loci
in 'Cambray '-derived seedlings 66
5.1 Peroxidase and phosphoglucomutase isozyme
banding patterns observed for pineapple
cultivars 91
5.2 Pineapple cultivar classification based on
peroxidase banding patterns 92
5.3 Pineapple cultivar classification based on
phosphoglucomutase banding patterns 93
5.4 Pineapple cultivar identification based on
peroxidase (Per) and phosphoglucomutase (Pgm)
isozyme banding patterns 94
5.5 Peroxidase and phosphoglucomutase isozyme
banding patterns for Ananas species 95
5.6Phosphoglucomutase isozyme banding patterns
observed for various Ananas genotypes 96
6.1 Designation of genotypes analyzed
electrophoretical ly 109
6.2 Presence-absence data matrix for electrophoretic
analysis of Ananas genotypes Ill
7.1 Regenerate pineapple plants evaluated for
electrophoretic and morphological variation 122
vii

LIST OF FIGURES
Figure Page
3.1 Initiation of axillary bud culture 27
3.2 Proliferation of culture 4 months after
inoculation 28
3.3 Actively dividing culture ready for subculture 29
3.4 Regenerated plantlets established in individual
pots or in flats 30
3.5 Comparison of multiplication cultures with
and without plant growth regulators 31
3.6 Ananas bracteatus var. tricolor 33
4.1 Non-variable isozyme banding patterns observed
for Ananas genotypes 6 7
4.2 Schematic representation of peroxidase isozymes
in 2 pineapple segregating populations 70
4.3 Peroxidase isozyme patterns observed in pineapple..71
4.4 Schematic representation of phosphoglucomutase
isozyme in Ananas 72
4.5 Schematic representation of phosphoglucomutase
isozyme in 2 pineapple segregating populations 73
4.6 Phosphoglucomutase banding pattern in pineapple.... 74
4.7 Malate dehydrogenase patterns observed in
pineapple 75
5.1 Schematic representation of peroxidase (Per)
and phosphoglucomutase (Pgm) isozymes in
pineapple cultivars... 97
viii

Figure Page
5.2 Schematic representation of peroxidase (Per)
and phosphoglucomutase (Pgm) isozymes in
Ananas species 98
5.3 Variation in phosphoglucomutase isozyme in
Ananas genotypes leaf extracts 100
6.1 Cluster analysis dendograrn of 27 pineapple
cultivar isozyme phenotypes 113
6.2 Cluster analysis dendograrn of isozyme phenotypes
of Ananas species and feral types 114
6.3 Cluster analysis dendograrn of the isozyme
phenotypes of 62 Ananas genotypes 115
7.1 Abnormal pineapple regenerated plants 123
ix

KEY TO ABBREVIATIONS
ATP:
adenosine 5' triphosphate disodium salt
BA:
6-benzylaminopurine (N^ benzyladenine)
CW:
coconut water
H:
histidine buffer
IAA:
indole-3-acetic acid
IBA:
indole-3-butyric acid
K:
K buffer
KIN:
kinetin
LBTC:
lithium borate/tris citrate buffer
MS:
Murashige and Skoog basal medium formulation
MTT:
3-(4,5-dimethy1thiazol-2-yl)-2,5 diphenyl-
tetrazolium bromide
NAA:
2-naphthaleneacetic acid
NAD:
beta-nicotinamide adenine dinucleotide
NADP:
beta-nicotinamide adenine dinucleotide phosphate
Na2EDTA
disodium ethylenediaminetetracetic acid
NBT:
nitro blue tetrazolium
PMS:
phenazine methosulfate
V
band migration relative to band sample
Rf:
band migration relative to borate front
x

TB:
TC:
Tris:
2,4-D:
tris borate buffer
tris citrate buffer
tris-(hydroxymethyl) amino methane
(2,4-dichlorophenoxy)acetic acid
xi

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
TISSUE CULTURE AND ELECTROPHORETIC STUDIES OF
PINEAPPLE (Ananas comosus) AND RELATED SPECIES
By
MARIA GRAZ IA DEWALD
May, 1987
Chairman: Wayne B. Sherman
Cochairman: Gloria A. Moore
Major Department: Horticultural Science (Fruit Crops)
Tissue culture studies were conducted with pineapple
(Ananas comosus) and A. bracteatus var. tricolor in order to
evaluate the feasibility of in vitro techniques as a method
for the rapid multiplication of desirable genotypes. A
system utilizing shake cultures derived from axillary buds
of crowns, slips, and stems was devised. This system
resulted in the efficient production of large numbers of
pineapple plantlets that were easily established in soil
under greenhouse conditions. The regenerated plants were
characterized morphologically and on the basis of isozyme
banding patterns. High levels of phenotypic variability,
especially for leaf morphology, were observed shortly after
the small plants were transferred to the soil. After one
xii

year only 4 variant phenotypes persisted in the 2000
regenerated plants. No isozyme variants were identified.
Electrophoretic characterization of Ananas with
emphasis on economically important pineapple cultivars was
also conducted to help establish phenetic relationships. A
method was developed whereby leaf sap could be quickly
extracted and analyzed for isozyme polymorphisms using
starch gel electrophoresis. One hundred and ninety
combinations consisting of 38 enzyme activity stains and 5
electrophoretic buffer systems were evaluated for isozyme
banding resolution. From this survey, 8 enzyme staining
systems giving good banding resolution were identified.
Three of these, peroxidase, phosphoglucomutase, and malate
dehydrogenase, were polymorphic. Two segregating seedling
populations were utilized to establish a genetic model for
the 3 polymorphic enzymes. The genetic analysis revealed
that both peroxidase and phosphoglucomutase were controlled
by 3 independent, monomeric loci.
A taxonomic survey of 67 Ananas genotypes was conducted
utilizing the peroxidase and phosphoglucomutase staining
systems. Fifteen of the 27 pineapple cultivars and 5 of the
7 species analyzed could be uniquely distinguished using the
2 enzyme systems. Ananas comosus, one of the species that
could not be unambiguously distinguished, was highly
polymorphic due to the large number of cultivars included in
the species. Ananas monstrosus, the other species without a
unique isozyme phenotype, was confirmed to be a simple crown
xiii

mutation of A. comosus that occurs in many cultivars and
therefore is not considered a valid species. Most of the 33
feral types analyzed could be included under species with
similar banding patterns. Phenetic relationships of the
genotypes based on cluster analysis dendograms were
established.
xiv

CHAPTER I
INTRODUCTION
Pineapple is one of the most important fruit crops in
the world. Nearly 12 million metric tons are produced
annually in the tropical and subtropical regions of the
world (FAO Production Yearbook, 1984). It is particularly
well suited to arid and semiarid tropical locations where
few other crops do well.
One reason for the popularity of pineapple is that it
has proved very amenable to large-scale commercial
production and has good canning qualities. Most of the world
production can be attributed to 'Smooth Cayenne' and its
sports. 'Smooth Cayenne' is characterized by its relatively
smooth leaves (spiny tip) and large cylindrical fruit with
good canning qualities. However, it is susceptible to
several economically important diseases and does not produce
slips, which are needed for propagation. Numerous high
quality spiny cultivars also exist that are more adapted to
tropical regions, where 'Smooth Cayenne' does not develop
good fruit qualities, or are better suited for the fresh
market. The presence of spines and the scarcity of planting
material on these cultivars severely restricts their
1

2
suitability to large scale planting. Pineapple breeding
efforts have been directed mainly at the improvement of
'Smooth Cayenne', because most of the breeding programs have
been conducted by large commercial companies whose
production is dependent on the specific canning
characteristics of this cultivar.
The genus Ananas, to which pineapple belongs, is poorly
characterized taxonomically. One explanation for this may be
that the center of origin and diversity of this genus is
located in the isolated regions of the upper Amazonian River
basin. This lack of characterization limits the ability of
plant breeders to utilize the variability present in both A.
comosus and related species.
The objectives of this research were 1) to develop an
efficient in vitro propagation system for pineapple; 2) to
morphologically and electrophoretically evaluate the genetic
fidelity of the regenerated plants; 3) to identify
techniques that are useful for visualizing isozymes in
Ananas; 4) to survey the available Ananas germplasm to
determine if variability in isozyme banding patterns exists;
5) to study the possible genetic basis for variability in
isozyme banding patterns; 6) to uniquely identify Ananas
species, including pineapple cultivars, and to categorize
feral types into species using isozyme polymorphism; and 7)
to establish phenetic relationships among Ananas genotypes
based on isozyme banding patterns.

CHAPTER II
LITERATURE REVIEW
Ananas Taxonomy
Pineapples [Ananas comosus (L.) Merr.] have been
studied and characterized by naturalists and botanists for
hundreds of years; however, there has been much variation in
their taxonomic treatment. Some botanists believe Ananas is
a monotypic genus, i.e., a single species, comprised of many
varieties. Others have considered the genus to include many
species, which are often separated on the basis of a few,
slight character variations (Collins, 1960).
The latest classification of the genus Ananas was by
Smith and Downs (1979). Smith was the botanist who also
classified most of the Bromeliaceae (the Ananas family). The
species considered valid in this latest treatment are A.
ananassoides, A. bracteatus, A. comosus, A. fritzmuelleri,
A. lucidus, A. nanus, and A. parguazensis. Classification is
based upon the presence or absence of a crown (coma
foliaceous), syncarp size, presence and direction of spines,
and floral bract characteristics. This is the most
comprehensive survey of the pineapple genus to date,
providing botanists for the first time with a taxonomic key.
3

4
A chronological review of pineapple taxonomy was later
made by Antoni (1983), indicating that the present
classification of the genus Ananas is highly confused and
questioning the validity of some species, e.g., A.
monstrosus. The same author stressed the importance of using
techniques such as tissue culture and electrophoretic
separation of isozymes to differentiate species.
Pineapple Propagation
Pineapple is a self-incompatible perennial crop that is
vegetatively propagated. The crop is usually cultivated in
high density fields. The spineless cultivar 'Smooth Cayenne'
is planted in Hawaii at 60,000 to 80,000 plants per ha. (Py
and Tisseau, 1965). In other regions where spiny cultivars
are used, only 15,000 to 35,000 plants are planted per ha.
(Py and Tisseau, 1965). At such high planting densities,
propagules are a major limiting factor in the establishment
of new fields. The problem can be even more extreme in the
case of newly developed cultivars. The number of propagules
per pineapple plant varies with the cultivar. However, a
typical plant produces between 6-10 propagules for crop
cycle (18-22 months). These propagules include the crown
(produced on the fruit apex), slips (produced on the fruit
peduncle), and suckers (produced on the plant stem). A
solitary sucker is usually left in place to produce the
subsequent ratoon fruit. Culturally, slips are the most
desired propagule. They are more favorable than crowns

5
because they are uniform, root faster, and can be planted
deeper, giving rise to a more uniform stock of plants.
The most economically important cultivar, 'Smooth
Cayenne', has low slip production. Slip production is
further reduced in this cultivar when it is planted at high
densities to produce a fruit size acceptable for efficient
processing (Ravcof and Yamane, 1970). Crowns are sold with
the fruit in fresh market situations, thereby further
reducing the amount of propagules.
Several methods have been developed over the past 40
years to increase the amount of propagules (Py, 1979). One
method, developed by Macluskie (1939), is to section the
mature stems. The individual pieces are placed in sand boxes
and incubated in a warm moist place. After several weeks the
axillary buds grow and form adventitious roots. This
technique was later tried by several researchers using
sections of crowns and slips (Tkatchenko, 1947; Collins,
1960; Py and Estanove, 1964). A second method consists of
propagating a single leaf with intact axillary bud dissected
from the crown or slip (Seow and Wee, 1970) . This method is
laborious and produces a limited amount of plantlets per
crown.
The most recent advance in pineapple multiplication
employs plant morphactins (Sanford and Abdul, 1971). Plant
morphactins are a diverse group of synthetic chemicals which
dramatically alter the morphological development of the
plant. Morphactins in pineapple are used in combination with

6
flower-inducing ethylene compounds, e.g., ethrel. Mature
plants (12-16 month old) are first induced to flower. The
flowering process is then converted into a multiplicant
process by the application of a morphactin, e.g., Maintain
CF-125. Using this method, up to 12 "sliplets" per plant can
be produced (Ravcof and Yamane, 1970). This is an economical
and rapid (2 years) method that allows the production of
over half a million vigorous propagules similar to slips per
ha. Planting material for most large-scale commercial
plantings of 'Smooth Cayenne' types is now produced by this
method of multiplication (Krezdorn, personal communication,
1985).
All of these methods require large fields to establish
plants. Rapid and more efficient pineapple propagation
systems still limit the
establishment
of
new
commercial
plantings. Tissue culture
appears to be
a
very
attractive
alternative method of multiplication
and
has
particular
utility in situations where clonal material is scarce.
Pineapple Tissue Culture
Pineapples have been regenerated in vitro from the
culture of shoot tips, axillary buds, and immature syncarps.
Direct plant production from excised axillary buds was first
reported by Aghion and Beauchesne (1960). A single plantlet
was obtained from each cultured axillary bud on solid, half
strength Knop's medium supplemented with 15% (v/v) coconut
water (CW). Sita et al. (1974) also regenerated single

7
plants from individual slip shoot tips on a solid medium of
Knudson major salts and Nitsch minor salts supplemented with
5.4 uM NAA.
Mapes (1974) was the first to describe a method for
multiple shoot production directly from axillary buds
present in cultured shoot. The shoot tips were placed in
liquid Murashige and Skoog (MS) medium supplemented with
0.22 mM adenine to initiate proliferation. Subsequently,
they were transferred to solid MS medium supplemented with
0.15 mM adenine to allow shoot development. The
proliferating mass of shoots was termed a protocorm-like
structure. Similar results were obtained by Teo (1974) and
Pannetier and Lanaud (1976). Wakasa et al. (1978) reported
the initiation of shoots from protocorm-like calli derived
from immature syncarps, axillary buds of suckers and slips,
immature crowns, and immature slips cultured on solid MS
media containing 5.4-54.0 mM NAA and 4.4-44.0 uM BA. The
best results were obtained on medium supplemented with 10.8
uM NAA and 8.8 uM BA. Wakasa et al. (1978) described 2 types
of protocorm-like calli. Calli derived from axillary buds
were described as nodule-like in appearance, while the calli
derived from axillary buds were described as globule-like.
Mathews et al. (1976) demostrated that liquid culture
medium appeared to be most suitable for shoot proliferation
from axillary bud cultures. They used an MS medium
containing 9.7 uM NAA, 9.8 uM IBA, and 9.2 uM KIN. Shoots
were initiated on solid medium supplemented with 4.4 uM BA

8
and 4.9 uM IBA (Mathews and Rangan, 1979). Later studies by
these authors indicated that callus could also be initiated
from axillary bud cultures on MS medium supplemented with
54.0 uM NAA, 15% (v/v) CW, and 0.04% (w/v) casein
hydrolysate. Shoot proliferation occurred on MS medium
supplemented with 9.7 uM NAA, 9.8 uM IBA, and 9.2 uM KIN.
Root formation occurred on White's medium containing 0.27 uM
NAA and 0.196 uM IBA. Zepeda and Sagawa (1981) reported
limited shoot proliferation from axillary buds cultured in
liquid MS medium supplemented with 25% (v/v) CW.
Shoot proliferation from hybrid embryo-derived callus
was reported by Srinivasa Rao et al. (1981) on MS medium
with 10.8 uM NAA, 9.8 uM IBA, and 11.0 uM BA. Immature leaf
explants have also been used for shoot proliferation on MS
solid medium containing 9.7 uM NAA, 9.8 uM IBA, and 48.4 uM
BA (Mathews and Rangan, 1979).
Drew (1980) published a report on pineapple tissue
culture theorizing the possibility of obtaining 100,000
plants from a single axillary bud in 12 months. There is no
mention in the literature in regard to the number of
regenerated plants established in soil, with the exception
of Wakasa et al. (1978) who reported growing 400 regenerated
plants.
Somaclonal Variation
The term somaclonal variation was coined by Larkin and
Scowcroft (1981) to describe the phenotypic variation

9
observed in plants regenerated from somatic tissue in vitro.
Somaclonal variation appears to be common to all in vitro
propagation systems. However, the quantity and quality of
the variation differs greatly with the genotype and
propagation protocol.
A number of reviews have been written on this subject
(Bayliss, 1980; Larkin and Scowcroft, 1981; Orton, 1983a;
1983b; 1984; Evans et al. 1984). Agromonically useful
somaclonal variation has been reported in many crop species,
e.g., alfalfa (Johnson et al., 1980), maize (Edallo et al.,
1981), oats (McCoy et al., 1982), potato (Shepard et al.,
1980), sugarcane (Heinz and Mee, 1969), tobacco (Chaleff and
Keil, 1981), tomato (Evans et al., 1984), and wheat (Larkin
et al., 1984).
The variation observed in tissue culture-produced
somaclones may be stable and heritable or it may be
transient, nonheritable, and epigenetic in nature. It is
particularly critical for the scientist to distinguish
between types of phenotypic variation. Epigenetic variation,
because it is nonheritable, has limited usefulness to
cultivar improvement.
A wide range of chromosomal alterations are possible in
tissue culture-regenerated plants. Single base pair
alterations or deletions (Edallo et al., 1981) represent one
end of the spectrum, while major karyotypic alterations,
including the doubling or deletion of complete sets of

10
chromosomes (Janick et al., 1977), represent the other
extreme.
Stable, heritable variation implies genetic
variability. This genetic variability may be due to
pre-existing heterogeneity present in the cells of the
explant and/or de novo genetic alterations occurring during
the culturing process (Evans et al., 1984). Unfortunately,
scientists have not been able to determine the relative
amount of variation attributable to these 2 sources (Larkin
and Scowcroft, 1981).
The full range of genetic variability that can be
generated and expressed in tissue culture-regenerated plants
has not yet been established. It does appear, however, that
the full range of genetic variability present in cultured
cells is not present in the regenerated plants (Edallo et
al., 1981). The regeneration process may be serving as a
selective screen for those cells that are genetically
competent to form plants.
Perhaps the best documented source of variation is the
explant. Barbier and Dulieu (1980) observed higher levels of
somaclonal variation in tobacco plants that were regenerated
from protoplasts and newly initiated callus cultures in
comparison with plants regenerated from well established
callus cultures. Wakasa (1979) also reported significant
differences in the amount of somaclonal variation observed
for vegetative morphology in 'Smooth Cayenne' pineapple
plants regenerated from 3 explant sources. He found that

11
plants regenerated from syncarp (immature fruit) tissues
exhibited 100% variability, while plants derived from
axillary buds of crowns and slips were 34% variable. The
lowest levels of variation (7%) were observed in plants
regenerated from small crowns. Varibilities in leaf wax,
color, spines, and foliage density were observed in the
plants derived from the syncarp, while only spine
variability was observed in the plants from slips and
axillary buds. It should be noted that this study was done
on up to 2-year-old regenerated plants that were grown in
the greenhouse, and no long-term study on the stability of
this variation was conducted.
The amount of time in culture can also influence the
variability of regenerated plants. Karyotypic changes have
been correlated with increasing time in culture (Bayliss,
1980) . Increasing time in culture has been shown to increase
somaclonal variation in tobacco (Syono and Furuya, 1972),
Chrysanthemum (Bush et al., 1976; Sutter and Langhans,
1981) , and oats (McCoy, 1980 cited by Reisch, 1983) . In
contrast, long-term cultures of corn were reported to be
stable (McCoy, 1980 cited by Reisch, 1983) .
The phytohormones used in the culture medium are
another possible factor affecting somaclonal variation. High
concentrations (18 uM) of the auxin 2,4-D were reported to
produce unique variation in regenerated barley plants that
was not observed in plants regenerated using lower
concentrations (Deambrogio and Dale,
1980) . Similarly, a

12
medium supplemented with NAA (27 uM) and KIN (0.5 uM) was
shown to produce significantly more karyotypic alterations
in regenerated Haworthia plants than a medium supplemented
with 0.6 uM IAA (Ogihara, 1981).
The genotype has also been shown to influence
somaclonal variation. Liu and Chen (1976) evaluated 8
sugarcane cultivars and found that regenerated plants from
the cultivars ranged from 1.8% variable to 34% variable.
Skirvin and Janick (1976) found somaclonal variation
cultivar differencies in Pelargonium sp. DeWald and Moore
(1987) indicated in their review that certain crops, e.g.,
polyploids and asexually propagated crops in which sexual
fertility is not important, appear to tolerate higher levels
of chromosomal variation produced via somaclonal variation
than other crops.
Applications of Isozyme Analysis in Horticultural Plant
Systematics
Isozymes are multiple molecular forms of an enzyme
having the same substrate specificity (Markert and Moller,
1959). Hunter and Markert (1957) , using the starch gel
electrophoresis technique developed by Smithies (1955),
demonstrated that isozymes can be visualized on starch gels
stained with specific histochemical stains. Bands appearing
on a starch gel correspond to different alleles and/or loci
coding for a specific enzyme.

13
Isozymes have been used extensively as genetic markers
in plants. They have a number of advantages over
conventional markers. Isozymes are generally not affected by
the morphological or physiological status of the plant. They
interact codominantly at a single locus, rarely exhibit
epistatic interactions, and can be detected in a variety of
tissues (Tanksley and Rick, 1980; Tanksley et al., 1982;
Tanksley, 1983).
The use of electrophoretic analysis to characterize
isozyme variation as an aid for plant systematics is now
widely applied and has been the subject of several reviews
(Gottlieb, 1977; Crawford, 1983). The electrophoretic
phenotype can be directly equatable with genotype, unlike
many morphological characters, which may demonstrate a great
deal of enviromental or developmental plasticity (Crawford,
1983; Gottlieb, 1977).
In plant systematics, isozyme electrophoresis has been
successfully utilized for both cultivar and species
identification by providing a way to quantitatively assess
the genetic variation among conspecific populations and the
genetic divergence among congeneric species. Other important
applications include the resolution of polyploid taxa and
insight into their origins, the investigation of the origin
of cultivated crops, and the testing of broad systematic or
evolutionary hypotheses.
Reliable identification of cultivars using classical
methods based on morphological and physiological characters

14
has become increasily difficult, partially due to the large
number of lines or varieties now available. Isozymes as
bio-chemical markers for cultivar identification have proven
reliable, consistent, and essentially unaffected by
enviromental conditions (Bailey, 1983). They can be used as
"fingerprints" to aid in the visual identification of
cultivars (Pierce and Brewbaker, 1973). The utility of this
technique has already been demonstrated in a number of
species, e.g., Anthurium (Kobayashi et al., 1987), alfalfa
(Quiros, 1980), apple (Menendez et al., 1986; Weeden and
Lamb, 1985), avocado (Torres et al., 1978a), barley
(Bassiri, 1976), bean (Bassiri and Rouhani, 1977; Weeden,
1984), Camellia (Wendel and Parks, 1983), Citrus (Torres et
al., 1978b), grape (Schwennesen et al., 1982), grasses
(Wilkinson and Beard, 1972), maize (Cardy and Kannenberg,
1982), Musa (Jarret and Litz, 1986b), peach (Carter and
Brock, 1980), pear (Santamour and Demuth, 1980), pecan
(Mielke and Wolfe, 1982), poinsettia (Werner and Sink,
1977), rice (Sarkar and Bose, 1984), strawberry (Bringhurst
et al., 1981), and wheat (Menke et al., 1973).
The use of isozymes in the investigation of plant
taxonomy, particularly species circumscription is relatively
recent. Zymogram techniques have been used most extensively
in Nicotiana. Characteristic enzyme patterns for most
Nicotiana species have been observed using proteins (Hart
and Bathia, 1967), peroxidase alone (Sheen, 1970), and in
combination with esterases (Smith et al.,
1970). Species

15
identifications using isozymes have also been reported in
Datura (Conklin and Smith, 1971), Citrus (Button et al.,
1976), Musa (Jarret and Litz, 1986c), and Pistacia (Loukas
and Pontikis, 1979).
Specific isozymes or proteins have been used to
identify particular genomes in polyploids. In bananas,
Jarret and Litz (1986a) confirmed the bispecific origin of
the triploid 'Chato' from Musa acuminata and Musa balbisiana
using leaf isozyme polymorphisms. An allopolyploid origin
for the apple has been proposed based on inheritance data of
7 pollen enzymes (Chevreau et al., 1985). Isozyme analysis
of Gossypium hirsutum, a natural allopolyploid, has shown
that esterases exhibit greater intergenomic variability than
intraspecific or intravarietal variability (Cherry et al.,
1970). In a later study, Cherry et al. (1971) showed that
evolutionary changes had occurred in the species that were
reflected in isozyme differences.
One of the most common uses of isozymes has been to
assess the validity of proposed parentages of cultivars and
progeny. This has been done in apple (Chyi and Weeden, 1984;
Weeden and Lamb, 1985), avocado (Golching et al., 1985;
Torres and Bergh, 1980; Torres et al., 1978a), Citrus
(Torres et al., 1978b), and date palm (Torres and Tisserat,
1980).
Protein and isozyme analyses have proved particularly
helpful in deducing systematic relationships between groups
where morphological and cytological data have been

16
inconclusive. Electrophoretic studies have been used
successfully to establish phylogenetic relationships in
rice, wheat, barley, soybean, and cotton (Ladizinsky and
Hymowitz, 1979). More recently, similar studies have been
conducted with Amaranthus (Hauptli and Jain, 1984) and
Capsicum (Panda et al., 1986).

CHAPTER III
PRODUCTION OF PINEAPPLE PLANTS IN VITRO
Introduction
Pineapple is routinely propagated vegetatively by means
of lateral shoots, basal suckers, or crowns. Plant material
is often limited, especially for the 80,000 plants/ha needed
in the establishment of new plantations, and for the
propagation of improved cultivars or newly discovered
sports.
In vitro propagation of pineapple was first achieved in
1960 by Aghion and Beauchesne (1960), but it was not until
1974, with the simultaneous reports of Laksmi Sita et al.
(1974) in India, Teo (1974) in Malaya, and Mapes (1974) in
Hawaii that in vitro production of pineapple was considered
as a commercial alternative. Most recent reports (Pannetier
and Lanaud, 1976; Wakasa et al., 1978; Mathews and Rangan,
1979; Drew, 1980; Zepeda and Sagawa, 1981) deal only with
the factors involved in the establishment of axillary buds
in culture. They do not consider the number of plants
regenerated or the efficiency of the system for reduced
labor cost. Furthermore, high levels of variability were
observed in regenerated 'Smooth Cayenne' plants by Wakasa
17

18
(1979). Such variability would compromise the commercial use
of in vitro propagation of pineapple, since regenerated
plants could not be assured to be clonal in nature.
This study was conducted using several species and
cultivars to
improve
the
technique of
tissue culture
of
axillary buds for
rapid
pineapple
propagation
and
to
investigate
the possible
variation
occurring
in
the
regenerated
plants.
Materials and Methods
Genotypes Cultured
Two cultivars of the common pineapple Ananas comosus
(L.) Merr. were used: 'PR-1-67', a spiny commercial cultivar
grown for fresh fruit and canning in Puerto Rico and
'Perolera', a smooth-leaved cultivar grown in the Northen
Andes of South America and used for fresh fruit. The third
genotype was Ananas bracteatus var. tricolor Smith, a
small-fruited ornamental characterized by showy colored
bracts and tricolored leaves (Fig. 3.6A). Two plants of
PR-1-67 and 1 plant of the other 2 genotypes were used in
order to determine intracultivar and intercultivar
variability in response to tissue culture.
Sterilization and Initiation of Cultures
Crowns and stems were rinsed in water, defoliated and
surface-sterilized by agitating in a 20% sodium hypochlorite
solution with 2-3 drops of a surfactant (Tween 20 or Tween

19
80) for 20 min, followed by 3 rinses of 10 min each in
sterile deionized water. Terminal and axillary buds were
then excised aseptically and again surface-sterilized in a
2% sodium hypochlorite solution for 10 min followed by 3
more 10 min rinses in sterile deionized water (Fig. 3.1A).
Buds were inoculated onto a modified Murashige and Skoog
(1962) medium (Table 3.1), supplemented with 3% sucrose,
0.8% Difco Bacto-agar, 0.57 mM inositol, 1.2 uM thiamine
HC1, 10.8 uM NAA and 8.8 uM BA. Growth regulators were added
and the medium pH was adjusted to 5.7 prior to autoclaving
at 121 °C, and 1.1 kg cm^ for 20 min. The medium was poured
into 60 X 15 mm sterile Petri dishes for initial bud
explants or into test tubes for subculture (Fig.3.IB).
Cultures were incubated at room temperature (24-27 °C)
-1 -2
with a 16 hr photoperiod of 76 umol s m provided by
cool-white fluorescent lamps. Explants were subcultured onto
fresh medium in test tubes at 6 week intervals until
adequate growth had occurred for transfer to liquid medium
(Fig. 3.2).
In the experiment described, 3 buds from each of the 2
plants of 'PR-1-67' and 1 plant of 'Perolera' and Ananas
bracteatus var. tricolor were established in this manner.
Culture Multiplication
Proliferating explants were multiplied in liquid
cultures consisting of 50 ml of the medium described above
in a 125 ml Erlenmeyer flask, maintained at 100 rpm on

20
-1 -2
gyrorotary shakers under 16 hr photoperiod (76 umol s m )
provided by fluorescent lamps. Liquid shake cultures were
subcultured at approximately 4 week intervals, when flasks
had become tightly packed with plantlets (Fig. 3.3). In this
way, numerous flasks of callus and regenerating plants were
obtained from one original bud.
An experiment was conducted to determine the necessity
of transferring the callus and plantlets to a medium without
hormones to induce root formation and thus plant survival,
and to evaluate the influence of plant growth regulators on
the number and size of plants produced. Ten grams of an
actively dividing 'Perolera' culture were inoculated into
flasks containing 50 ml of basal medium with or without
hormones (10.8 uM NAA and 8.8 uM BA). The 2 treatments were
replicated 9 times.
Establishment in Soil
After each subculture plantlets 2.5 cm and larger were
harvested. They were transferred to individual pots or to
flats containing a commercial soil mixture and enclosed in
plastic bags, creating a greenhouse effect (Fig. 3.4). The
plants were incubated in a growth chamber at 28 °C under
fluorescent lamps and gradúa.ly hardened off by removing the
plastic covers. Acclimated plants were transplanted to
larger pots and placed in a greenhouse under a black cloth
screen to prevent scorching. The screen was removed 3 weeks
later and surviving plants were counted. Fertilization was

21
initially done biweekly with an acid-forming plant
fertilizer and later on with a solution of 1/10 MS salts as
apparent nutrient deficiencies developed. Phenotypic
observations of the plants were made periodically.
Results and Discussion
Production of plantlets from axillary bud cultures
initiated on solid medium and multiplied in liquid shaker
cultures started 9 months after explanting. The total number
of harvestable plantlets doubled with each monthly
subculture after the 11th month (Table 3.2). From 300 to 350
plantlets could be obtained by the 13th month from a single
bud culture (Table 3.3). Approximately 25 plantlets could be
harvested from each 125 ml flask with each additional
subculture. In all genotypes, plantlets larger than 3.0 cm
had a survival rate of nearly 100% when transferred to soil.
A 2-fold increase in the total number of harvestable
plantlets was obtained in 6 weeks when plant growth
regulators were removed from the multiplication medium
(Table 3.4; Fig. 3.5). However, after 2 subcultures (14
weeks), all plantlets had been harvested and no further
callus production or multiplication occurred. Further,
transferring to a medium without plant growth regulators to
induce root formation was not necessary for plantlet
survival. Plantlets without roots produced in multiplication
medium with plant growth regulators also had a high survival
rate and, while initially smaller than plantlets produced in

22
hormone-free medium, attained the same level of growth after
2 months in soil. The cultures periodically transferred into
multiplication medium with plant growth regulators appear to
be capable of indefinite multiplication. Phenotypic
variation using leaf morphology as a marker was assessed in
approximately 2000 regenerated plants and will be discussed
in detail in Chapter VII.
Tissue culture of the species A_;_ bracteatus var.
tricolor produced regenerated plants that were either
tricolor, green or albino (Fig. 3.6). This indicates the
chimeral origin of this genotype. It is clear that these
variegated plants should not be given formal varietal rank.

23
Table 3.1. Murashige and Skoog salts formulation.
Salts
mg 1 1
Major Salts
NH N03
1650.00
kno3
1900.00
MgSO . 7H 0
370.00
CaCl^
440.00
Kh2p64
170.00
Minor Salts
Na EDTA
37.30
FeS04.7H20
27.80
MnS04.4H20
22.30
ZnS04.7H20
8.60
H-.BO-,
6.20
KI
0.83
Na Mo04.2H20
0.25
CoS04 Jh20
0.025
CuS04.5H20
0.025

Table 3.2
In vitro pineapple plantlet production for each initiated bud
Mean number of plantlets harvested per flask2
Subculturey
Monthx
Perolera
Bud 1 Bud 2
Bud 3
Bud 1
PR-1-67
Bud 2
Bud 3
Bud 1
PR-1-67
Bud 2
Bud 3
7
9
2.5 1.0
2.3
1.5
1.7
1.5
0
1.7
0
8
10
2.4 3.3
3.1
3.3
3.5
2.5
3.7
3.8
3.0
9
11
3.7 2.8
3.4
3.1
2.7
3.2
3.2
3.2
2.7
10
12
6.6 7.5
5.6
5.0
1.0W
4.1
6.1
3.8
3.8
11
13
12.8 14.8
14.4
11.2
10.5
9.9
16.2
13.8
13.8
zPlantlets were larger than 2.5 cm.
^Number of subculturing after inoculation.
xNumber of months after culture initiation.
wSeveral flasks became contaminated during the experiment.

Table 3.3. In vitro pineapple plantlet production for
each initiated bud.
Total plantlet production in 5 months of harvesting2
Cultivar
Bud 1
Bud 2
Bud 3
Totals
Perolera
380
237
212
829
PR-1-67
321
42y
344
707
PR-1-67
309
333
112y
754
harvesting began 8 months after culture initiation.
^Several cultures became contaminated during the experiment.

Table 3.4. In vitro pineapple plantlet production in response to the presence
or absence of plant growth regulators in the culture medium.
Total number of plantlets
harvested per flask2
With plant growth
regulators^
Without plant growth
regulators
Replicate
1st harvestx
2nd harvest”
1st harvest 2nd
harvest
1
20
23
7
18
2
21
30
9
17
3
16
29
12
21
4
17
25
7
17
5
21
30
11
14
6
27
28
8
16
7
18
21
7
13
8
17
24
6
14
9
20
22
10
18
Total
177
232
77
148
zPlantlets were 2.5 cm or larger.
^NAA (10.8 uM), BA (8.8 uM).
xSix weeks after inoculation.
wFourteen weeks after inoculation.

27
Figure 3.1. Initiation of axillary bud culture. A. Buds
aseptically removed; B. Bud expansion 8 days
after inoculation.

28
Figure 3.2.
Proliferating culture 4 months after
inoculation and ready to be transferred
to liquid multiplication medium.

29
Figure 3.3. Actively dividing culture ready for
subculture. Plantlets larger than
2.5 cm will be transferred to soil.

Figure 3.4. Regenerated plantlets can be established in individual
12 cm pots or more economically in flats.

31
Figure 3.5. A. Comparison of multiplication cultures
with (right) and without (left) plant
growth regulators.

32
Figure 3.5. continued. B. Plantlets produced in medium
with plant growth regulators; C. Plantlets
produced in medium without plant growth
regulators.

33
Figure 3.6. A. Ananas bracteatus var. tricolor.

34
Figure 3.6. continued. B. In vitro multiplication showing
resolution of chimera; C. Regenerated plants.
Albino plants are incapable of survival in
soil.

CHAPTER IV
ELECTROPHORETIC STUDIES IN ANANAS
Introduction
Electrophoretic characterization of plants based on
differential patterns of protein migration is a powerful
technique to separate and classify genotypes. Isozyme
analysis has aided taxonomists, breeders, and physiologists
in understanding the underlying causes of phenotypic
variation. The plant breeder is particularly interested in
classification and characterization studies in order to
better utilize existing germplasm resources. The purpose of
this study was to determine what enzyme and buffer systems
work well in Ananas and which enzymes show polymorphism. The
analysis was conducted with taxonomy, breeding, and cultivar
improvement in mind.
No previous reports of isozyme studies have been
published for pineapple. For this reason, an initial study
was undertaken using a small number of Ananas genotypes to
determine which enzyme staining systems would give well
resolved banding patterns. Horizontal starch gel
electrophoresis was used for isozyme characterization
because it is non-toxic and yields multiple slices from a
35

36
single gel, thus allowing the rapid screening of a large
number of enzyme staining systems. The procedures followed
were similar to those currently being used with Citrus by
Moore (personal communication, 1985) . The enzyme staining
systems that resulted in well resolved banding patterns were
used to survey a larger number of Ananas genotypes to
identify and characterize isozyme polymorphism. Genetic
studies were performed using seedling populations to
determine the basis of variability observed among genotypes.
Information from these studies was used to formulate a
system by wich cultivars, feral types, and species could be
identified and distinguished, and their phenetic affinities
could be established. These systems will be described in
Chapters V and VI. Selected enzyme staining systems were
used to determine phenotypic stability of tissue
culture-regenerated plants and will be discussed in Chapter
VII.
Materials and Methods
Genotypes Used for Electrophoretic Studies
A list of the genotypes used in this study is given in
Table 4.1. The population included all of the species
presently considered valid in the genus Ananas (Smith and
Downs, 1979; Antoni, 1983) with the exception of A.
fritzmuelleri, which was not available in any of the major
world collections. The A. comosus cultivars surveyed
represent the most important cultivars in use world-wide.

37
The Ananas genotypes indicated by numbers are introductions
made by the author and originally established at the
Facultad de Agronomia, Universidad Central de Venezuela, or
material from the collection at the USDA Subtropical
Horticultural Research Unit, Miami, Florida. These feral
types were collected in the regions of southern Venezuela
and northern Brazil, an area that has been suggested as the
center of origin for the genus Ananas (Leal and Antoni,
1980) . The suspected taxonomic classification of the feral
types follows the introduction number. The identification
was based on the botanical characters considered valid for
each Ananas species by Smith and Downs (1979). High levels
of phenotypic variation were present for several characters
among the collected types, including presence or absence of
spines, leaf color, and fruit shape, size, and color.
Genetic studies were based on isozyme analysis from
both the Ananas germplasm survey and 2 seedling populations.
The seedling populations were obtained from a commercial
planting of 'Smooth Cayenne' and 'Cambray' growing in
adjacent fields in northwestern Ecuador. Since pineapple has
only been reported as self-incompatible within a cultivar
and cross-compatible between cultivars (Brewbaker and
Gorrez, 1967), these seedling populations were assumed to be
2 segregating populations representing reciprocal crosses
between 'Smooth Cayenne' and 'Cambray'. The seeds were taken
from mature fruit, rinsed in water, and air dried. Seeds
were scarified by a treatment of 30-60 sec in concentrated

38
sulphuric acid followed by a rinse in sterile water and were
planted in covered petri dishes containing a sterilized soil
mixture moistened with sterile water. Periodic sprays with a
1% Captan solution were made to avoid fungal contamination.
Germinated seeds were transferred to pots containing a
commercial soil mix composed of sphagnum peat moss,
horticultural grade vermiculite and perlite, composted pine
bark, and washed granite sand. The pots were covered with
polyethylene bags to prevent dessication. Transplants were
initially held in a growth chamber at 27 °C with 16 hr
. -1 —2
fluorescent light (76 umol s m ), and were taken to the
greenhouse after acclimation.
All plants of the collection were kept at Gainesville,
Florida, under greenhouse conditions during the cold months
(Nov.-Apr.) and outside the rest of the year. Plants were
kept in 25 cm pots with the same soil mixture as the
seedlings and were periodically fertilized with a standard
water-soluble 20-20-20 fertilizer.
Starch Gel Preparation
A 10% (w/v) starch gel consisting of a 2:1 (w/w) mixture
of Sigma and Connaught hydrolyzed potato starch was used
throughout this study. All the starch was from the same lot
number. Thirty grams of the starch mixture along with 300 ml
of a gel buffer solution (Table 4.2) were poured into a 1000
ml side arm flask. The mixture was swirled vigorously by
hand and placed on a magnetic stir pad at medium speed for

39
10 min to assure a well-mixed suspension and to avoid the
formation of lumps in the starch. The flask was then placed
on a magnetic hot plate at high heat and stirred at medium
speed. The solution turned translucent as it reached maximum
viscosity. At this point, the flask was again swirled by
hand and the cofactor NADP (10 mg dissolved in 1 ml water)
was added for those staining systems requiring it. The flask
was heated again until large air bubbles appeared on the
surface of the solution.
The solution was degassed immediately by vacuum before
it started to cool, to avoid air bubbles forming in the gel
that could interfere with the migration of proteins. The
vacuum source was provided by attaching an aspirator to a
sink faucet and to the flask side arm. The solution was
ready to be poured when no small bubbles were present.
The molds used had inside dimensions of 19 x 19 x 0.5 cm
and were made from plexigas sealed with methylene chloride.
Any bubbles that formed while pouring the starch solution
into the molds were removed with a spatula before the gel
started to harden. The gel was allowed to cool for
approximately 30 min at room temperature, and then further
cooled by refrigeration (4 °C) for 1 hr prior to loading the
samples. Gels could be kept for 12-16 hr at room temperature
before being placed in the refrigerator and they were
covered with polyethylene film to prevent dessication.

40
Protein Extraction and Gel Loading
Leaf sap was used as the main protein source throughout
this study because it was easily prepared, readily
availabile, and gave good results. All leaf samples were
taken from the fourth leaf at the center of the plant,
counting from the first visible leaf, and were used
immediately or stored in plastic bags at 4 °C for later use.
Samples were used within 7 days of collection, although no
decrease in enzyme activity could be detected for up to 3
weeks in storage. Leaf sap was expressed from the proximal
portion of the adaxial surface of the lamina, using a pestle
and a filter paper wick (Whatman No. 3, 4 x 8 mm). The white
waxy epidermis and underlying layer of fibers were scraped
from the leaf surface to aid in leaf sap absorption into the
wick. The saturated wick was then inserted into a slit 5 cm
from the top (cathodal side) of the chilled gel for anodal
migrating enzymes and at 8 cm from the top for cathodal
migrating enzymes, e.g. peroxidase. Once all samples had
been inserted, glass rods were placed at the top and bottom
of the gel to ensure good contact between wicks and gel
during electrophoresis.
Pollen was collected from flowering plants just prior
to anthesis, dried at room temperature for 24 hr, and frozen
at -4 °C until used in electrophoretic assays. Proteins were
extracted from the pollen using the method outlined by
Weeden and Gottlied (1980). A small amount of frozen pollen

41
was placed in 0.1 ml of extraction buffer for 4 hr. The
supernatant was then absorbed into the wick.
Electrophoretic Buffers and Staining Systems
Five electrophoretic buffer systems were tested:
histidine citrate (H), lithium borate/tris citrate (LBTC),
tris borate (TB), tris citrate (TC), and K buffer (K) [Table
4.2], as were 38 enzyme activity stains. Tested combinations
are listed in Table 4.3.
The formulations used for the enzyme staining solutions
were derived from several authors. Endopeptidase (E.C.
3.4.22.?) was according to Melville and Scandalios (1972).
Formate dehydrogenase (E.C. 1.2.1.2) was from Wendel and
Parks (1982). Creatine kinase (E.C. 2.7.3.2) and hexokinase
(E.C. 2.7.1.1) were as described by Shaw and Prasad (1970).
Malate dehydrogenase (E.C. 1.1.1.37) was from Cardy et al.
(1981). The following were according to Vallejos (1983):
adenylate kinase (E.C. 2.7.4.3), alcohol dehydrogenase (E.C.
1.1.1.1), aldolase (E.C. 4.1.2.13), alpha-amylase (E.C.
3.2.1.1), ascorbate oxidase (E.C. 1.10.3.3), catalase (E.C.
1.11.1.6), diaphorase (E.C. 1.6.4.3), fumarase (E.C.
4.2.1.1), galactose dehydrogenase (E.C. 1.1.1.48), beta-D-
galactosidase (E.C. 3.2.1.23), using alpha-napthyl-D-
galactopyranosidase as a substrate, glucose-6-phosphate
dehydrogenase (E.C. 1.1.1.49), beta-D-glucosidase (E.C.
3.2.1.21), glutathione reductase (E.C. 1.6.4.2), isocitrate
dehydrogenase (E.C. 1.1.1.42), lacease (E.C. 1.10.3.2),

42
lactate dehydrogenase (E.C. 1.1.1.27), 6-phosphogluconate
aenyarogenase (E.C. i.1.1.44), pyruVdte js.xnci£>c; (E.C.
2.7.1.40), shikimate dehydrogenase (E.C. 1.1.1.25), trióse
phosphate isomerase (E.C. 5.3.1.1), urease (E.C. 3.5.1.5),
and xanthine dehydrogenase (E.C. 1.2.1.37). In addition to
these, the following staining systems were modified from
Vallejos (1983) as follows:
Acid phosphatase (E.C. 3.1.3.2)
100 mg alpha-napthyl acid phosphatase, Na salt
50 mg Fast Black salt
100 mg 0.1 M sodium acetate, pH adjusted to 4.7
with glacial acetic acid
Alkaline phosphatase (E.C. 3.1.3.1)
100 mg beta-naphthylacid phosphatase, Na salt
100 mg Fast Blue RR salt
100 mg 0.1 M tris HC1, pH 8.5
Esterase
100
42
40
18
3
(E.C. 3.1.1.2)
mg Fast Blue RR salt
ml H20
ml 0.2 M NaH2P04
ml 0.2 M Na_ HPO.
2 4
ml of the following solution: 1 g alpha-
napthyl acetate and 1 g beta-napthyl acetate
dissolved in acetone and brought to 100 ml
with H2 Glutamate oxaloacetate transaminase (Aspartate amino¬
transferase, E.C. 2.6.1.1)
500 mg L-aspartic acid
70 mg alpha-keto-glutaric acid
50 mg pyridoxal-5'-phosphate
200 mg Fast Blue BB salt
100 ml 0.1 M tris HC1, pH 8.0
Leucine aminopeptidase (E.C. 3.4.11.1)
40 mg L-leucine-beta-napthylamide HC1
100 mg Black K salt
50 ml 0,2 M tris + 0.2 M maleic anhydride
20 ml 0.2 N NaOH
30 ml H20

43
Phosphoglucomutase (E.C. 2.7.5.1)
500 mg glucose-l-phosphate, Na„ salt
20 mg NADP
30 mg MTT
4 mg PMS
100 mg MgCl^
100 ml 0.1 M tris HC1, pH 8.0
40 units glucose-6-phosphate dehydrogenase
Na2 salt
Phosphohexose isomerase (Glucophosphate isomerase,
E.C. 5.3.1.9)
100 mg fructose-6-phosphate,
10 mg NADP
20 mg MTT
4 mg PMS
100 mg MgCl_
100 ml 0.1 M tris HC1, pH 8.0
10 units glucose-6-phosphate dehydrogenase
Malic enzyme was modified from Cardy et al. (1981);
peroxidase and superoxide dismutase were modified from Moore
(unpublished) and Moore and Collins (1983), as follows:
Malic enzyme (E.C. 1.1.4.0)
30 mg NADP
30 mg NBT
4 mg PMS
100 mg MgCl„
20 ml 0.1 M malic acid, pH adjusted to 7.0
with NaOH
1 ml 0.2 M NaH2P04
48 ml 0.2 M Na2HP04
30 ml H20
Peroxidase (E.C. 1.11.1.7)
250 mg para-phenylenediamine
50 mg MnSO^
5 ml 1.0 M sodium acetate, pH adjusted to 4.7
with glacial acetic acid
30 ml 95% ethanol
65 ml H2o
0.5 ml 30% H2C>2 added after above solution is
poured onto gel

44
Superoxide dismutase (E.C. 1.15.1.1)
2 mg Riboflavin
75 mg Na~EDTA
10 mg NBT
100 ml 0.1 M tris HC1, pH 8.0
Electrophoresis
The gel with sample wicks in place was set on top of 2
plexiglass buffer tanks. The electrode consisted of a 19 cm
length of platinum wire. Five hundred milliliter of buffer
solution was added to each electrode buffer tank, covering
the electrode. The bridges between the electrode buffer and
the gel consisted of 4-ply chromatography paper (Whatman No.
3). A glass plate was placed on top of the gel to assure
good contact between the gel and electrode bridges.
Electrophoresis was carried out under constant voltage
in a refrigerator at 4 °C. Power settings and running time
varied with each buffer and enzyme staining system. Gels
were typically run for the initial survey of buffers at 100
to 180 volts and approximately 20 milliamps for 16 to 18 hr
or at 200 to 250 volts and 25 to 50 milliamps for 4 to 6 hr.
Protein migration was monitored using several techniques. In
the gels containing borate buffers, a light brown line was
visible that migrated anodally during electrophoresis. In
gels using K buffer, a sample wick saturated with
bromophenol blue was used to follow the progress of
electrophoresis. Protein migration was monitored in gels
using histidine buffers by noting a pink color in the anode
electrode buffer solution. Protein migration could also be

45
monitored by a decrease in current readings when using a
constant voltage source during electrophoresis.
Enzyme Activity Staining
Gels were sliced horizontally into 4 slices (2 mm
thick), the top slice was discarded. The gel slices were
placed in plastic boxes and refrigerated until staining
(usually less than 2 hr).
Staining solutions were prepared fresh, poured over the
gel slice and swirled lightly to eliminate air bubbles
trapped underneath. Gels were placed in the dark and
observed every 15 to 30 min to follow the progress of
staining. When optimum resolution was observed, the gels
were rinsed in deionized water and examined. Gels were fixed
for long-term storage using a (7:7:1 v/v/v) solution of
methanol, deionized water, and glacial acetic acid. The gels
were then wrapped in polyethylene film and refrigerated.
Analysis of Banding Patterns
The banding patterns (zymograms) were recorded by
measuring the migration rate of each band. An internal
standard was created in each gel system to calculate
relative migration rates. The borate front produced in the
TB and LBTC buffer systems and the bromophenol blue band in
the K buffer systems served as an internal standard for
these systems. Buffer systems that did not produce a front
or a bromophenol blue band (H and TC) were evaluated by

46
placing a sample with a unique banding pattern in the gel,
one band of which served as the internal standard. The
migration of the band relative to the front (R^ value) or to
the unique band (R^ value) was calculated by dividing the
band migration distance by the migration distance of the
internal standard.
A genetic mechanism to explain variability was
hypothesized when zymogram polymorphism was observed among
the genotypes. The 2 segregating populations available were
used to test the genetic mechanism by comparing the observed
ratios with the expected ratios using chi-square analysis.
The possibility of genetic linkage was investigated by
analyzing the segregation ratios at more than 1 locus.
Linkage between loci could be suspected if chi-square
analysis revealed a significant deviation from ratios
expected with independent segregation.
Results and Discussion
Selection of Electrophoretic Buffers and Enzyme Systems
Useful for Studying Isozymes in the Genus Ananas
The results of the survey testing 38 enzyme staining
systems with 5 buffers can be seen in Table 4.3.
Well-resolved banding patterns were obtained with 8 enzyme
activity stains: isocitrate dehydrogenase, malate dehydro¬
genase, peroxidase, phosphoglucomutase, 6-phosphogluconate
dehydrogenase, phosphohexose isomerase, superoxide dismu-
tase, and trióse phosphate isomerase. Table 4.4 lists these

47
enzyme staining systems along with important electrophoretic
parameters. These systems were selected for further study
and are detailed below.
Twelve additional enzyme systems showed limited
resolution or enzyme staining activity and were grouped into
3 categories: those with activity but no discrete banding,
those with poorly resolved banding and no apparent
variability, and those with poorly resolved banding but some
apparent variability. Enzyme staining systems giving no
discrete banding included beta-D-galactosidase, formate
dehydrogenase, and urease. Systems giving poorly resolved
banding with similar activity among genotypes were esterase
and glutathione reductase. Staining systems giving poorly
resolved banding patterns with some variable activity among
genotypes included acid phosphatase, glucose-6-phosphate
dehydrogenase, glutamate oxaloacetate transaminase, leucine
aminopeptidase, malic enzyme, 6-phosphogluconate dehy¬
drogenase, and shikimate dehydrogenase. Although 2
additional protein extraction buffers were tried with these
enzyme staining systems, no improved resolution was
observed.
No activity was detected for the following 18 enzymes:
adenylate kinase, alcohol dehydrogenase, aldolase, alkaline
phosphatase, alpha-amylase, ascorbic oxidase, catalase,
creatine kinase, diaphorase, endopeptidase, fumarase,
galactose dehydrogenase, beta-D-glucosidase, hexokinase,

48
lacease, lactate dehydrogenase, pyruvate kinase, and
xanthine dehydrogenase.
The 8 enzyme activity stains that gave well-resolved
banding patterns were used to survey the 62 genotypes listed
in Table 4.1. In 5 of the staining systems, monomorphic
banding patterns were obtained. The other 3 staining systems
gave polymorphic banding patterns.
Monomorphic Enzymes
These enzymes included isocitrate dehydrogenase,
phosphohexose isomerase, superoxide dismutase and trióse
phosphate isomerase. Banding phenotypes observed for these
monomorphic systems are illustrated in Fig. 4.1. and are
described below:
Isocitrate dehydrogenase. Two anodally migrating bands
were observed with this stain, 1 at 2.0 and the other at 2.5
cm from the origin (Fig. 4.1A). The second band appeared
less intense and was not always detected. No variability; was
observed among the genotypes studied.
6-Phosphogluconate dehydrogenase. The banding pattern
obtained is shown in Fig. 4.IB. Two anodally migrating
bands, 1 major and 1 minor, were observed for all genotypes
investigated. The major band exhibited a mobility of 2.3 cm
from the origin. The fainter minor band migrated 1.1 cm from
the origin. Fainter poorly resolved bands were also present.
Phosphohexose isomerase. The banding pattern for this
enzyme consisted of 3 bands (Fig. 4.1C). The first and

49
second bands stained more intensely and migrated at 2.3 and
2.6 cm, respectively, from the origin. The third band, which
migrated at 3.0 cm, usually stained less intensely and was
not always detected. Another unresolved zone of activity was
present.
Superoxide dismutase. The banding pattern is
illustrated in Fig. 4.2D. A monomorphic pattern consisting
of 2 bands was observed for all the genotypes in this enzyme
staining system. The acromatic bands migrated 3.7 and 4.7 cm
from the origin.
Trióse phosphate isomerase. Three well resolved bands
were observed for this enzyme with relative migrations of
2.0, 2.5, and 2.8 cm from the origin (Fig. 4.2E). No
variability was detected among the genotypes surveyed.
Enzyme Systems with Variable Banding Patterns
Variable banding patterns were observed for 3 enzyme
staining systems: malate dehydrogenase, peroxidase, and
phosphoglucomutase. Hypotheses to explain the observed
variability in these enzyme systems were based on a genetic
analysis of 2 segregating populations of plants from
putative reciprocal crosses between 'Smooth Cayenne' and
'Cambray'. Isozyme patterns for peroxidase and
phosphoglucomutase observed for other genotypes surveyed
will be discussed in detail in Chapter V.
Peroxidase. Three regions, 2 cathodal (Per-1 and Per-2)
and 1 anodal (Per-3), of well resolved variable banding

50
patterns were observed for all the genotypes tested, and are
illustrated in Figs. 4.2 and 4.3. All 3 variable regions
exhibited the same general banding patterns, consisting of
either a single fast-migrating band (FF), a single
slow-migrating band (SS), or a combination of the 2 (FS).
The slow band for Per-1 was at Rf -0.26, while the fast band
was at Rf -0.28. The slow band for Per-2 was at Rf -0.04 and
the fast band was at Rf -0.11. The slow band for the
anodally migrating Per-3 was at Rf 0.04 and the fast band
at Rf 0.07. Zones of activity with no defined bands were
observed between Rf 0.11 to 0.25 and 0.32 to 0.38.
The variability detected for peroxidase may be
explained by a model of a monomeric enzyme system with 2
alleles (F and S) at each of 3 loci (Per-1, Per-2, and
Per-3). The distal band (F) and proximal band (S) are
produced by different alleles. Heterozygous genotypes would
produce both a F and S band, while homozygous genotypes
would produce either an FF or SS band. Progeny produced from
the cross with 'Smooth Cayenne' (SS) as the seed parent gave
all nonparental isozyme phenotypes (FS) for Per-1, which
would result if parents homozygous (SS, FF) for different
alleles at a locus were crossed. 'Cambray', the putative
pollen parent of the seedlings, had a Per-1 phenotype of FF.
Progeny showed a 1:1 ratio of FS and FF phenotypes for both
Per-2 and Per-3, which would be expected if parents of the
seedlings were homozygous (FF) and heterozygous (FS)
respectively for the alleles at the loci. This was the case

51
with 'Smooth Cayenne' (FF) and 'Cambray' (FS) [Tables 4.5
and 4.7; Figs. 4.2 and 4.3A],
Seedlings obtained from open-pollinated crosses with
'Cambray' as the seed parent and 'Smooth Cayenne' as the
putative pollen parent were expected to show a similar type
of segregation, since this would be the reciprocal cross of
the one described above and only the 2 cultivars were
growing in the region. Instead, progeny from the 'Cambray'
female parent exhibited only the 'Cambray' (FF) banding
pattern for Per-1, as would be expected from a cross between
2 plants that were homozygous at this locus (Table 4.6 and
Fig. 4.2). Chi-square values (Table 4.7) showed a very close
fit to a 1:2:1 (FF:FS:SS) ratio for both Per-2 and Per-3,
which indicates a cross between plants heterozygous at these
2 loci (Fig. 4.2). The banding patterns obtained from this
progeny suggested that 'Smooth Cayenne' did not participate
in the cross and that the seedlings are most likely the
result of selfing.
Phosphoglucomutase. Three anodal regions (Pgm-1, Pgm-2,
and Pgm-3) of well resolved variable banding patterns were
observed in all the genotypes tested and are illustrated in
Fig. 4.4. The banding patterns for the Pgm-1 region were
unique in comparison to Pgm-2 and Pgm-3, and consisted of 5
total bands in the wide population of genotypes surveyed; F,
S, M, L, or P were present in various combinations of 2 of
these bands. Both Pgm-2 and Pgm-3 exhibited the same
variable banding pattern consisting of either a single

52
fast-migrating band (FF), a single slow-migrating band (SS),
or combination of the 2 (FS). Values for R, were calculated
b
using P.I. 115, a feral genotype exhibiting a distinctive
banding pattern (Figs. 4.4 and 4.6) which served as an
internal standard. The 5 bands for Pgm-1 were at 0.43
(L), 0.63 (S), 0.73 (M), 0.83(F), and 1.0 (P). The slow band
(S) for Pgm-2 was at Rfa 0.84 and the fast band (F) was at Rfa
0.98. The slow band (S) for Pgm-3 was at Rfc 1.00 and the
fast band (F) was at R, 1.19.
b
The majority of plant species reported in the
literature have contained either 1 or 2 loci governing the
expression of phosphoglucomutase, with the exception of
Camellia japónica, in which a third locus was reported by
Wendel and Parks (1982). In the present study, it was found
that the banding pattern observed in Ananas also may be
explained by hypothesizing a monomeric enzyme with 3 loci
(Pgm-1, Pgm-2, and Pgm-3). Locus Pgm-1 would be composed of
5 alleles, while Pgm-2 and Pgm-3 would each have 2 allelic
forms.
The 2 seedling populations represent only 3 (F, M, and
S) of the 5 possible Pgm-1 allelic forms. Heterozygous
genotypes would show combinations of these 3 bands, i.e.,
FM, FS, or MS, while homozygous genotypes would produce
either a FF, MM or SS band. Likewise, according to the
model, the other 2 loci, Pgm-2 and Pgm-3, produce a distal
band (F) and proximal band (S) as the result of different
alleles. Heterozygous genotypes would produce both a F and S

53
band, while homozygous genotypes would produce either a FF
or SS band (Tables 4.5, 4.6, and 4.7).
The 'Smooth Cayenne'-derived population showed a 1:1
ratio of phenotypes for Pgm-1 (FS:MS), Pgm-2 (FF:FS) and
Pgm-3 (FS:SS), which would be expected if the parents were
heterozygous and homozygous for each locus, i.e., Pgm-1: FM
x SS, Pgm-2: FF x FS, and Pgm-3: SS x FS (Table 4.7).
'Smooth Cayenne' is heterozygous for Pgm-1 (FM) and
homozygous for Pgm-2 (FF) and Pgm-3 (SS). Conversely,
'Cambray' is homozygous for Pgm-1 (SS) and heterozygous for
Pgm-2 and Pgm-3 (FS). The segregation pattern for the 3 loci
indicate that the seedlings are the results of a cross
between 'Smooth Cayenne' x 'Cambray' (Figs. 4.5 and 4.6A).
The 'Cambray'-derived seedlings again exhibited only
the 'Cambray' banding pattern for Pgm-1, as would be
expected for progeny from parents homozygous at this locus
(Table 4.6). Segregation was observed for Pgm-2 and Pgm-3,
and chi-square values (Table 4.7) showed a very close fit to
a 1:2:1 (FF:FS:FS) ratio., indicating a cross between 2
heterozygous types (FS). As discussed above, 'Smooth
Cayenne' is heterozygous for Pgm-1 (FS) and homozygous for
Pgm-2 (FF) and Pgm-3 (SS), so that it could not have been
the pollen parent in this cross. Thus, the
phosphoglucomutase banding patterns obtained for this
population exhibited segregation ratios which corroborate
the hypothesis that the seedlings are indeed the result of
selfing and not outcrossing (Figs. 4.5 and 4.6B).

54
Self-incompatibility in pineapple is due to inhibition
of pollen-tube growth in the upper third of the style. It is
gametophytically controlled by a single S locus with
multiple alleles (Brewbaker and Gorrez, 1967). In general,
pineapples set no seed when self-pollinated, but can set
seed when cultivars are crossed (Collins, 1960). Collins
(1960) reports isolated self-fertile 'Smooth Cayenne'
plants. Seed production was variable and germinated seed
gave rise to seedy offspring. These plants exhibited very
low vigor and continued selfing was not possible.
Pareja (1968) hypothesized that the frequent occurrence
of seeds in 'Cambray' may be due to self-compatibility with
pollination being accomplished by mites. The isozyme banding
patterns displayed by the seedling population obtained from
'Cambray' comfirms the self-compatibility of this cultivar.
Malate dehydrogenase. One large anodal region
consisting of many closely adjacent bands was observed for
all genotypes tested and is illustrated in Fig. 4.5. Six of
the bands were well resolved and migrated to 3.5, 3.8, 4.1,
4.5, 5.0, and 5.5 cm from the origin. The 2 fastest
migrating bands, 5.0 and 5.5, were the only ones to show
clear segregation. Banding patterns for the 'Smooth
Cayenne'-derived seedlings exhibited segregation for these 2
bands (Fig. 4.7A) while no segregation (all seedlings had 2
bands) was observed for the 'Cambray'-derived seedlings
(Fig. 4.7C). When the power setting during electrophoresis

55
was changed from 9 to 16, more separation of the bands was
observed and segregation for other bands seemed to appear
(Fig. 4.7B).
Malate dehydrogenase has been reported as a dimeric
enzyme in numerous diverse plant species, e.g., avocado
(Torres and Bergh, 1980), celery (Orton, 1983c), citrus
(Torres et al., 1982), eucalyptus (Moran and Bell, 1983),
and maize (Goodman and Stuber, 1983). Pollen zymograms from
different cultivars, including 'Smooth Cayenne' and
'Cambray', were compared with their respective leaf
zymograms to determine if intra-allelic heterodimer
formation was present, as would be expected in a polymeric
enzyme. Two bands were absent in pollen zymograms (Fig.
4.7D), implicating intra-allelic heterodimer formation. The
presence of these interaction bands suggests that malate
dehydrogenase functions as a polymeric enzyme in Ananas.
However, more segregating populations must be analyzed
before the genetics of this enzyme system can be clarified.
Linkage Analysis for Enzyme Loci
Linkage analysis was carried out between the
peroxidase and the phosphoglucomutase loci. The results are
given in Tables 4.8 and 4.9. Chi-square analysis by
contingency tables showed that the observed ratios fit
ratios expected for independent segregation.

56
Conclusions
1. Eight enzyme staining systems of the 38 investigated
were found to give well-resolved banding patterns.
2. Well-resolved non-variable isozyme banding patterns
were observed for isocitrate dehydrogenase, 6-phospho-
gluconate dehydrogenase, phosphohexose isomerase,
superoxide dismutase, and trióse phosphate isomerase.
3. Well-resolved variable isozyme banding patterns were
observed for malate dehydrogenase, peroxidase, and
phosphoglucomutase.
4. A genetic model was hypothesized to account for the
variability observed for peroxidase and phosphoglu¬
comutase staining systems in the Ananas germplasm
surveyed and in segregating seedling populations from
'Smooth Cayenne' and 'Cambray' pineapple cultivars. The
model hypothesizes that both peroxidase and phospho¬
glucomutase function as monomeric enzymes coded for
by 3 loci each: Per-1, Per-2, Per-3, Pgm-1, Pgm-2,
and Pgm-3. Each locus contains 2 allelic forms, F
and S, with the exception of Pgm-1, which exhibits
5 allelic forms: F, S, M, L, and P.
5. Independent segregation was found between all of the
peroxidase and phosphoglucomutase loci.
6. The isozyme studies confirmed that the 2 seedling
populations were the products of crosses between
'Smooth Cayenne' x 'Cambray' and 'Cambray' x self.

57
7. The existence of the 'Cambray' x self population calls
into question belief that domestic pineapple cultivars
are obligated self-incompatible.

58
Table 4.1. Ananas genotypes surveyed for isozyme banding
patterns.
Species
Feral
Types2
A. ananassoides
046
A.
ananassoides
A. bracteatus
064
A.
nanus
A. comosus
072-1
A.
comosus
A. lucidus
079
A.
ananassoides
A. nanus
083
A.
ananassoides
A. monstrosus
085
A.
comosus
A. parguazensis
086
A.
comosus
092-1
A.
comosus
A. comosus cultivars
092-2
A.
comosus
Abaka
093
A.
comosus
Black Jamaica
094
A.
comosus
Brecheche
095
A.
comosus
Bumanguesa
097
A.
parguazensis
Cabeza de Mono
108
A.
parguazensis
Cabezona
110
A.
comosus
Cacho de Venado
115
7
Caicara
116
7
Cambray
117
7
Cumanesa
126
A.
comosus
Dupuis Smooth
188
p
Esmeralda
189
7
Injerta
192
A.
comosus
Maipure
193
7
Masmerah
196
A.
comosus
Monte Oscuro
25291
A.
comosus
Morada
487439
A.
comosus
Nacional
487440
A.
comosus
Negrita
487442
A.
comosus
Panare
487443
A.
comosus
Perola
487444
A.
comosus
Perolera
Pina de Brazil
PR-1-67
Queen
Red Spanish
Rondon
Smooth Cayenne
Valera Amarilla
ZNumbers correspond to introductions followed by the
suspected taxonomical classification. "?" indicates that
the botanical characteristics of the plant do not fit any
of the presently described species.

59
Table 4.2. Electrophoretic buffer systems used for isozyme
detection in Ananas.
Buffer
system2
Electrode
buffer^
Gel
buffery
H
0.065 M histidine
0.02 M citric acid
pH adjusted to 5.7
with citric acid
0.009 M histidine
0.003 M citric acid
pH adjusted to 5.7
with citric acid
K
0.18 M trizma base
0.1 M boric acid
0.004 M Na„EDTA
pH 8.6 ¿
0.045 M trizma base
0.025 M boric acid
0.001 M Na EDTA
pH 8.6 Z
LBTC
0.016 M LiOH
0.192 M boric acid
pH 7.2
0.0016 M LiOH
0.019 M boric acid
0.007 M citric acid
0.046 M trizma base
pH 7.7
TB
0.038 M trizma base
0.002 M citric acid
pH 8.6
0.03 M boric acid
pH 8.5
TC
0.05 M trizma base
0.016 M citric acid
pH adjusted to 7.0
with citric acid
0.017 M trizma base
0.005 M citric acid
pH adjusted to 7.0
with citric acid
ZH = Histidine citrate (Cardy et al., 1981); K = K buffer
(Loukas and Pontikis, 1979); LBTC = Lithium borate/Tris
citrate (modified Scandalios, 1969); TB = Tris borate;
TC = Tris citrate (Moore, unpublished).
*10 mg NADP was added to buffer in cathodal tank and to gel
buffer when staining for enzyme system that required NADP
as a cofactor.

60
Table 4.3. Enzyme activity stains and buffer systems tested
for isozyme detection in Ananas.
Buffer system2
Enzyme activity stain
LBTC
H
TC
TB
K
Acid phosphatase
_
+
_
_
+
Adenylate kinase
-
-
-
-
ie
Alcohol dehydrogenase
-
-
-
-
★
Aldolase
-
-
-
-
★
Alkaline phosphatase
-
-
-
-
★
alpha-Amylase
-
-
-
-
★
Ascorbate oxidase
-
-
-
-
★
Catalase
-
-
-
-
★
Creatine kinase
-
-
-
-
★
Diaphorase
-
-
-
-
★
Endopeptidase
-
-
-
-
*
Esterase
-
-
-
+
+
Formate dehydrogenase
+
-
-
-
★
Fumarase
-
-
-
-
★
Galactose dehydrogenase
-
-
-
-
★
beta-D-Galactosidase
-
-
-
-
★
Glucose-6-phosphate dehydrogenase
-
+
-
-
+ +
beta-D-Glucosidase
-
-
-
-
★
Glutamate oxalloacetate transaminase
+ +
-
-
-
+ +
Glutathione reductase
+
-
+
-
★
Hexokinase
-
-
-
-
★
Isocitrate dehydrogenase
+ +
+ + +
-
-
+ +
Lacease
-
-
-
-
★
Lactate dehydrogenase
-
-
-
-
★
Leucine aminopeptidase
-
-
-
+
★
Malate dehydrogenase
+ +
+ + +
+ +
+ +
+ +
Malic enzyme
+
-
-
+
★
Peroxidase
+ +
+ +
+
+ +
+ + +
Phosphoglucomutase
-
+++y
+ +
+ +
★
6-phosphogluconate dehydrogenase
+
+++
+
+
+ +
Phosphohexose isomerase
+ +
+++
+ +
+ +
+ +
Piruvate kinase
-
-
-
-
★
Shikimate dehydrogenase
+ +
-
-
-
+ +
Superoxide dismutase
+ +
-
+ + +
Superoxide dismutase + KCN
+ +
-
+
★
Trióse phosphate isomerase
-
-
-
-
+ + +
Urease
+
-
-
+
★
Xanthine dehydrogenase
-
-
-
-
★
z- = no activity; + = poor migration or resolution;
++ = fair migration or resolution; +++ = well resolved
bands; * = assay not performed.
^Modified to 0.065 M, pH 6.3.

Table 4.4. Well resolved enzyme activity staining systems and electrophoretic
parameters used in Ananas.
Electrophoretic conditions
Enzyme system
Buffer
system
Volts2
Mi11iamps
Tota 1
power
Time
(hr)
Isocitrate dehydrogenase
300
45
13.50
3.5
Malate dehydrogenase
H
300
40
12.00
4.0
Peroxidase
K
250
25
6.25
5.0
Phosphoglucomutase
300
45
13.50
3.5
6-Phosphogluconate dehydrogenase
H
300
40
12.00
4.0
Phosphohexose isomerase
HX
300
45
13.50
4.0
Superoxide dismutase
K
250
34
8.50
4.0
Trióse phosphate isomerase
K
250
25
6.25
4.0
zGels were run at constant voltage, and voltage was changed during electrophoresis
as milliamps increased to maintain the total power indicated.
^pH modified to 6.3.
y __ 1
1.5 g 1 Na2EDTA added to the buffer to reduce the dark background in gels.

62
Table 4.5. Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived from
an open-pollinated 'Smooth Cayenne' fruit.
Isozyme genotypes
Seedlings
Per-1
Per-2
Per-3
Pgm-1
Pgm-2
Pgm-3
1
FS
FS
FF
MS
FS
FS
2
FS
FF
FS
FS
FS
FS
3
FS
FF
FF
FS
FS
SS
4
FS
FF
FS
MS
FF
SS
5
FS
FS
FS
FS
FS
SS
6
FS
FS
FF
MS
FS
FS
7
FS
FF
FS
MS
FF
SS
8
FS
FS
FF
MS
FS
SS
9
FS
FS
FS
MS
FS
SS
10
FS
FF
FF
FS
FS
SS
11
FS
FS
FF
MS
FS
FS
12
FS
FS
FS
MS
FS
SS
13
FS
FF
FS
FS
FS
SS
14
FS
FF
FS
FS
FF
SS
15
FS
FS
FS
MS
FS
SS
16
FS
FF
FF
FS
FS
SS
17
FS
FF
FF
FS
FS
SS
18
FS
FS
FF
FS
FF
FS
19
FS
FF
FS
MS
FS
SS
20
FS
FS
FF
MS
FS
SS
21
FS
FS
FF
FS
FS
SS
22
FS
FF
FF
FS
FF
FS
23
FS
FS
FS
MS
FF
SS
24
FS
FF
FF
FS
FF
FS
25
FS
FS
FF
MS
FF
FS
26
FS
FS
FF
FS
FF
FS
27
FS
FS
FF
MS
FF
FS
28
FS
FS
FF
MS
FF
SS

63
Table 4.6. Peroxidase and phosphoglucomutase banding
patterns observed for seedlings derived
from an open-pollinated 'Cambray' fruit.
Isozyme genotypes
Seedlings
Per-1
Per-2
Per-3
Pgm-1
Pgm-2
Pgm-3
1
FF
FS
FS
SS
FS
FS
2
FF
FS
FF
SS
FS
FF
3
FF
FS
FS
SS
FF
FF
4
FF
FS
SS
SS
SS
FS
5
FF
FF
SS
SS
FF
FS
6
FF
FS
FF
SS
SS
SS
7
FF
FS
SS
SS
FS
FS
8
FF
SS
FS
SS
FS
FS
9
FF
FF
SS
SS
FS
FS
10
FF
FS
SS
SS
SS
FS
11
FF
FS
FS
SS
SS
SS
12
FF
SS
FF
SS
FF
FS
13
FF
FF
FS
SS
FF
SS
14
FF
FF
FS
SS
FS
FS
15
FF
FS
FF
SS
SS
SS
16
FF
SS
FS
SS
FS
FS
17
FF
FF
FS
SS
FF
SS
18
FF
SS
FS
SS
FF
FS
19
FF
FS
FS
SS
FS
FF
20
FF
SS
FS
SS
FS
FF

Table 4.7. Genotypic ratios and chi-square goodness of fit values for 6 isozyme loci
in pineapple.
Loci investigated2 Segregation classes
T0 s t
FF FS MS SS ratio P value
Smooth Cayenne seedlings
Per-1
(SSxFF)
-
28
-
-
Per-2
(FFxFS)
12
16
-
-
1 :1
0.571
.50-.25
Per-3
(FFxFS)
17
11
-
—
1:1
1.285
.50-.25
Pgm-1
(FMxSS)
-
13
15
—
1:1
0.142
.75-.50
Pgm-2
(FFxFS)
10
18
-
-
1:1
2.285
.25-.10
Pgm-3
(SSxFS)
—
10
-
18
1:1
2.285
.25-.10
Cambray seedlings
Per-1
(FFxFF)
20
-
-
-
Per-2
(FSxFS)
5
10
-
5
1:2:1
0.000
1.000
Per-3
(FSxFS)
4
11
-
5
1:2:1
0.100
.90-.75
Pgm-1
(SSxSS)
—
—
-
20
—
Pgm-2
(FSxFS)
6
9
-
5
1:2:1
0.100
.90-.75
Pgm-3
(FSxFS)
4
11
-
5
1:2:1
0.100
.90-.75
zSeedlings from putative crosses of 'Smooth Cayenne' x 'Cambray' and 'Cambray' x self.
Parentheses indicate allelic combinations for parents at the locus.

Table 4.8. Segregation ratios and chi-square goodness of fit values for independent
inheritance of peroxidase and phosphoglucomutase loci in 'Smooth Cayenne'-
derived seedlings.
Loci pair Segregation classes (no. of plants obs.) Test ratio X2 value P value
Per-l/Per-2
Per-2/Per-3
FS/FS(5)
FS/FF(11)
FF/FS(6)
FF/FF(6)
1:1:1:1
0.377
.75-
.50
Per-2/Pgm-l
FS/MS(12)
FS/FS(5)
FF/MS(3)
FF/FS(8)
1:1:1:1
3.447
.10-
.05
Per-2/Pgm-2
FS/FS(10)
FS/FF(6)
FF/FS(8)
FF/FF(4)
1:1:1:1
0.029
.90-
.75
Per-2/Pgrn-3
FS/SS(10)
FS/FS(7)
FF/SS(8)
FF/FS(3)
1:1:1:1
0.120
.75-
.50
Per-3/Pgm-l
FS/MS(7)
FS/FS(4)
FF/MS(9)
FF/FS(9)
1:1:1:1
0.110
.75-
.50
Per-3/Pgm-2
FS/FS(8)
FS/FF(3)
FF/FS(10)
FF/FF(7)
1:1:1:1
0.120
.75-
.50
Per-3/Pgm-3
FS/SS(10)
FS/FS(1)
FF/SS(8)
FF/FS(9)
1:1:1:1
3.846
.05-
.02
Pgm-l/Pgm-2
MS/FS(11)
MS/FF(5)
FS/FS(8)
FS/FF(4)
1:1:1:1
0.085
.90-
.75
Pgm-l/Pgm-3
MS/SS(10)
MS/FS(5)
FS/SS(8)
FS/FS(5)
1:1:1:1
0.013
.95-
.90
Pgm-2/Pgm-3
FS/SS(14)
FS/FS (4)
FF/SS(4)
FF/FS(6)
l:l:lsl
2.528
.25-
.10
z
II _ II
indicates no segregation.

Table 4.9. Segregation ratios and chi-square goodness of fit values for independent
inheritance of peroxidase and phosphoglucomutase loci in 'Cambray'-derived
seedlings.
Loci pair
Segregation classes (no. of plants obs.)
2
Test ratio X value P value
Per-l/Per-2
Per-2/Per-3
FF/FF(0)
FF/FS(3)
FF/SS(2)
FS/FF(3)
1:2:1:
FS/FS(4)
FS/SS(3)
SS/FF(1)
SS/FS (4)
1:2:1:
SS/SS(0)
1:2:1
4.300
.50-
.25
Per-2/Pgm-l
-
Per-2/Pgm-2
FF/FF(3)
FF/FS(2)
FF/SS(0)
FS/FF (1)
1:2:1:
FS/FS(4)
FS/SS(5)
SS/FF(2)
SS/FS (3)
1:2:1:
SS/SS(0)
1:2:1
8.320
.10-
.05
Per-2/Pgm-3
FF/FF(0)
FF/FS(3)
FF/SS(2)
FS/FF(3)
1:2:1:
FS/FS(4)
FS/SS(3)
SS/FF(1)
SS/FS(4)
1:2:1:
SS/SS(0)
1:2:1
4.300
.50-
.25
Per-3/Pgm-l
-
Per-3/Pgm-2
FF/FF(1)
FF/FS(1)
FF/SS(2)
FS/FF(4)
1:2:1:
FS/FS(6)
FS/SS(1)
SS/FF(1)
SS/FS(2)
1:2:1:
SS/SS(2)
1:2:1
3.517
.50-
.25
Per-3/Pgm-3
FF/FF(1)
FF/FS(1)
FF/SS(2)
FS/FF(3)
1:2:1:
FS/FS(5)
FS/SS(3)
SS/FF(0)
SS/FS(5)
1:2:1:
SS/SS(0)
1:2:1
6.000
.25-
.10
Pgm-l/Pgm-2
-
Pgm-l/Pgm-3
-
Pgm-2/Pgm-3
FF/FF(1)
FF/FS(3)
FF/SS(2)
FS/FF(3)
1:2:1:
FS/FS(6)
FS/SS(0)
ss/FF(0)
SS/FS(2)
1:2:1:
SS/SS(3)
1:2:1
7.148
.25-
.10
z
II _ II
indicates no segregation.

67
Figure 4.1. Non-variable isozyme banding patterns
observed for Ananas genotypes. A. Isocitrate
dehydrogenase; B. 6-Phosphogluconate
dehydrogenase. Sample origin is at the bottom
of each photograph and band migration is
anodal.

68
Figure 4.1. continued. C. Phosphohexose isomerase;
D. Superoxide dismutase.

69
E
'«•••••HI «till ti|
Figure 4.1. continued. E. Trióse phosphate isomerase.

70
â–  2 . ,
Per-
1
.3
1
2
3 4
5 6 7 8
9 10 11 12 13
Figure 4.2. Schematic representation of peroxidase isozymes
in 2 pineapple segregating populations.
1. 'Smooth Cayenne'; 2. 'Cambray'; 3-6. Hybrids
of 'Smooth Cayenne' x 'Cambray' cross;
7-13. Hybrids of 'Cambray' x self cross.

71
Figure 4.3. Peroxidase banding patterns observed in
pineapple. A. From right to left, 'Smooth
Cayenne-', ' Cambray' , and progeny from the
putative cross 'Smooth Cayenne' x 'Cambray';
B. 'Smooth Cayenne', 'Cambray', and progeny
from the putative cross 'Cambray' x self.
Sample origin is at the middle of each
photograph and band migration is both
anodal and cathodal.

72
1.2
1.1
1.0
.9
Rb -8
. 7
.6
.5
.4
Figure 4.4. Schematic representation of phosphoglucomutase
isozyme in Ananas. 1. P.I. 115; 2. 'Maipure';
3. 'Smooth Cayenne'; 4. P.I. 072; 5. P.I. 085;
6. 'Black Jamaica'; 7. P.I. 095; 8. P.I. 27281;
9. 'Brecheche'. Black bands = Pgm-1; cross-
hatched bands = Pgm-2; dotted bands = Pgm-3.
+
Pgm-3
1 23456789

73
+
P g m -3
Pg m -2
P g m - 1
1
2 3 4 5 6
7 8 9
Figure 4.5. Schematic representation of phosphoglucomutase
isozyme in 2 pineapple segregating
populations. 1. 'Smooth Cayenne';
2. 'Cambray'; 3-6. Hybrids of 'Smooth
Cayenne' x 'Cambray' cross; 7-9. Hybrids
of 'Cambray' x self cross.

74
B
§
•••###«•••••••#•
Figure 4.6. Phosphoglucomutase banding pattern in
pineapple. A. From right to left, P.I. 115,
'Smooth Cayenne', 'Cambray', and progeny
from the putative cross 'Smooth Cayenne' x
'Cambray'; B. 'Smooth Cayenne', 'Cambray',
and progeny from the putative cross
'Cambray' x self. Sample origin is at the
bottom of each photograph and band migration
is anodal.

75
A
llllltUIHIIIIttltttl
7
Figure 4.7. Malate dehydrogenase patterns observed in
pineapple. A. and B. Gels run at different
power settings (9 and 16), from right to left,
'Smooth Cayenne', 'Cambray', and seedlings
from the putative cross 'Smooth Cayenne'
x 'Cambray'. Sample origin is at the bottom
of each photograph and band migration is
anodal.

76
D
continued. C. Progeny from the putative
cross 'Cambray' x self; D. Pollen and leaf,
from right to left, 'Smooth Cayenne',
'Brecheche', and 'Cambray'.
Figure 4.7.

CHAPTER V
IDENTIFICATION OF ANANAS GENOTYPES
BY ISOZYME PHENOTYPES
Introduction
The utilization of isozyme techniques for cultivar
identification requires high levels of clearly recognizable
inter-cultivar isozyme polymorphism, preferably with
monomorphic cultivar phenotypes. The phenotypes should be
stable over a variety of environmental and developmental
conditions. The techniques used to characterize the
phenotypes should be simple and efficient.
Ananas comosus appears to be an ideal candidate for
electrophoretic characterization using isozyme polymor¬
phisms. The self-incompatible and outcrossing nature of the
species would be expected to generate high levels of
inter-specific genetic variation, while intra-clonal
variability would be expected to be relatively low due to
the apomictic nature of pineapple.
The
taxonomy and origin
of
the genus Ananas has
been
the subject of much debate
and
speculation.
Species
are
generally
distinguished by
the
presence or
absence
and
direction
of leaf spines, petal
morphology, and
fruit
size.
77

78
The purpose of this study was to electrophoretically
characterize the genus Ananas using isozyme polymorphisms
with emphasis on gaining information on the origin of the
commercially important pineapple cultivars of the world.
Materials and Methods
Starch gels were prepared and electrophoresis was
conducted as described in Chapter IV. Two buffer systems
were used for electrophoresis, a histidine system at pH 6.3
and a K buffer at pH 8.6 (Table 4.2). The histidine buffer
was used to assay for phosphoglucomutase activity and the K
buffer for peroxidase activity. At least 2 plants of each
genotype (Table 4.1) were examined electrophoretically for
peroxidase and phosphoglucomutase polymorphisms, except that
no peroxidase determination was made for the feral types.
Assays were repeated until consistent results were obtained.
The banding patterns were recorded using both schematic
drawings and photographs. Band migration distances were
based on R^ and values as calculated in Chapter IV.
Results and Discussion
Considerable isozyme polymorphism was observed among
the genotypes examined for both peroxidase and
phosphoglucomutase. In both systems, bands were
characterized for each locus according to inheritance
studies discussed in Chapter IV. Peroxidase showed either a

79
single or a double banded pattern at 2 migrating regions,
corresponding to the Per-2 and Per-3 loci. No clear bands
were present for Per-1. Phosphoglucomutase exhibited the
most useful isozyme polymorphisms. Bands corresponding to
the Pgm-1 locus gave consistent and clear resolution but
overlapping bands corresponding to Pgm-2 and Pgm-3 loci
complicated the characterization of certain genotypes.
Cultivar Identification
Peroxidase. Limited isozyme polymorphism was detected
for peroxidase. A total of 7 different banding patterns was
observed and designated through A^ (Table 5.1; Figs. 5.1
and 5.2). Patterns A^, A^, and A_, were unique to 'Negrita',
'Pina de Brazil', and 'Maipure' respectively. 'Panare' and
'Queen' shared pattern Ag, while the other cultivars showed
patterns A^, A2 or A<- (Table 5.2).
Phosphoglucomutase. Eleven different phosphoglucomutase
banding patterns were identified and designated B^ through
B^ (Tables 5.1 and 5.2; Figs. 5.1 and 5.2). Unique banding
patterns were observed for 'Black Jamaica', 'Rondon',
'Valera Amarilla', 'Cacho de Venado', and 'Cabezona'. These
patterns were Bg, B^, B^, Bg, and B^q respectively. The rest
of the cultivars were distributed over the patterns B^, B^r
B,-, Bg, Bg, and B^ (Table 5.3).
The polymorphic enzyme systems of peroxidase and
phosphoglucomutase were very useful for cultivar

80
classification. Using only the peroxidase or the
phosphoglucomutase isozyme system, 8 cultivars could be
distinguished by their unique banding patterns (Tables 5.2
and 5.3). However, using information combined from both the
peroxidase and phosphoglucomutase isozyme systems, 20 unique
classes of banding patterns could be distinguished. These
classes permitted the identification of 15 cultivars out of
the total 27 examined (Table 5.4).
The origin and exact relationship of many of the
pineapple cultivars studied remains unclear. A wide variety
of clones and cultivars exists because of the prevalence of
inter-clonal fertility and apomixis accompanied by
self-incompatibility. Certain pineapple cultivars and clones
exhibit high rates of mutation and many unique sports exist;
e.g., 'Smooth Cayenne', the most economically important
cultivar, is really a group of clones (Collins, 1960). These
factors considerably complicate characterization and
classification schemes.
Based on the peroxidase and phosphoglucomutase isozyme
banding patterns, it appears that there is a very close
relationship among many of the cultivars tested. 'PR-1-67',
a reputed hybrid widely grown in Puerto Rico, was obtained
from an open-pollinationed seed of 'Red Spanish' growing
adjacent to a field of 'Smooth Cayenne' (Ramirez et al.,
1970 and 1972). The parental relationship of these 2
cultivars to 'PR-1-67' is corroborated by their isozyme
banding patterns (Table 5.1). 'Esmeralda', a cultivar grown

81
in Mexico (Samuels, 1970), and 'Dupuis Smooth' are presumed
to be sports of 'Smooth Cayenne'. The 3 cultivars exhibited
the same banding pattern, indicating a common origin.
'Cumanesa' is very similar morphologically to 'Red Spanish'
and is believed to be a sport of the latter (Leal and
Antoni, 1980b). The identical banding pattern showed by the
2 cultivars suggests that 'Cumanesa' could indeed have
originated as a sport of 'Red Spanish'.
'Bumanguesa' and 'Perolera' are the 2 cultivars
commonly grown in the Andean regions of Venezuela and
Colombia. Both cultivars have similar morphological
characteristics of plant growth and differ only in mature
fruit color. 'Bumanguesa' produces a bright red fruit, while
'Perolera' produces a yellow fruit. Because of the
similarities of these 2 cultivars, Leal et al. (1979)
hypothesized that 'Bumanguesa' was a sport of 'Perolera'.
However, the peroxidase and phosphoglucomutase banding
pattern observed for these 2 cultivars do not indicate a
close relationship, unless the mutation involved the Per-2,
Per-3, and Pgm-3 loci (Table 5.1).
The cultivars 'Queen' and 'Panare', which represent the
"Queen" group of pineapples, exhibited identical isozyme
banding patterns for both peroxidase and phosphoglucomutase.
They also are very similar morphologically. This is
remarkable since cultivars belonging to this horticultural
group are grown commercially only in South Africa and
Australia (Samuels, 1970). 'Panare' was described by Leal et

82
al. (1979) from a clone being cultivated in southern
Venezuela (Bolivar State). Local people reported that the
original plants were "wild" types found growing nearby.
Since the area including southern Venezuela and northern
Brazil has been suggested as the center of origin for the
Ananas genus (Leal and Antoni, 1980a) it seems likely that
the "Queen" group probably was derived from 'Panare'-like
wild types. This finding confirms the presence of "Queen"
types growing in the Western Hemisphere.
There seems to be some correlation among the Pgm-1
alleles and the horticultural groups of pineapple (Table
5.1). All cultivars within the "Cayenne", "Queen", and
"Maipure" groups exhibit identical banding patterns, i.e.,
FM. Phylogenetic relationships between these groups will be
discussed in Chapter VI. Most of the cultivars in the
"Spanish" group had a common banding pattern for Pgm-1,
i.e., SS. However, the 4 cultivars in the "Abacaki" group,
showed different allelic combinations for Pgm-1, i.e., FM,
MS, SS.
Pineapple cultivars are classified into horticultural
groups without regard to their origin or genetic
relationships, e.g., 'PR-1-67' is included in the "Spanish"
group even though it is a known hybrid (Samuels, 1970; Leal
and Soule, 1977). Similarly, other cultivars could be of
hybrid origin, which would explain the isozyme banding
variability observed between individuals within a group.
'Caicara' is from the same region as
'Panare'
and

83
'Brecheche' and it may have been derived from these. 'Black
Jamaica' may be a hybrid between 'Smooth Cayenne' x 'Red
Spanish', and 'Valera Amarilla' may be a hybrid of
'Perolera' x 'Red Spanish', since the parents and hybrids
are all cultivated in the same regions. 'Cacho de Venado',
which has spiny leaves and conical-shape fruit, has been
included in the "Abacaki" group created by Py and Tisseau
(1965). Based on isozyme analysis of the Pgm-1 locus, it
appears to be more related to the "Spanish" group.
Furthermore, 'Cacho de Venado' is from the same region as
'Monte Oscuro' and the feral type 086 (Monagas State,
Venezuela). The banding pattern of these cultivars also
suggests a hybrid origin for 'Cacho de Venado'.
Species Identification
Peroxidase and phosphoglucomutase banding patterns for
7 Ananas species are shown in Table 5.5, Figs. 5.2 and 5.3.
Based on the banding patterns of peroxidase and
phosphoglucomutase, 5 of the 7 species can be distinguished.
The 2 exceptions are A. comosus, which includes all of the
commercial pineapple cultivars, and A. monstrosus. Ananas
comosus, as indicated in the previous section, exhibited
high levels of isozyme polymorphism, particularly for the
Pgm-1 locus. Ananas monstrosus also exhibited isozyme
polymorphism in the 2 individuals tested.
The amount of polymorphism observed within A. comosus
may be attributed to the present very broad circumscription

84
of this species. Smith (1961) separates A. comosus from the
other Ananas species by the length of the fruit, i.e.,
greater than 15 cm. Thus, in this classification system,
this species includes all the edible pineapples regardless
of other morphological differences such as leaf color,
presence or absence of spines, and fruit morphology. The
same flexibility is not allowed in the classification of the
other species and only individuals that exactly fit the
description, which includes leaf, flower, and fruit
morphology and growth habit, are included.
Ananas monstrosus was originally described by Carriere
(1870) and included in the later classification by Smith
(1961). The description was based on a single individual
plant found. It differs from A. comosus (pineapple) only in
that the fruit lacks a crown. The validity of this species
has been guestioned by Antoni (1983), who reported finding a
mutation of the cultivar 'Negrita' that mimics the A.
montrosus characteristic, i.e., lack of crown. Based on
growth habit and leaf and fruit morphology, the 2 plants
representing A. monstrosus used in this study appear to be
crown mutations of 'Perola' and 'Red Spanish' respectively.
The peroxidase and phosphoglucomutase banding patterns
observed for these plants are also typical of 'Perola' and
'Red Spanish'. The taxon thus seems to be of polyphyletic
origin. The guestionable validity of A. monstrosus as a
species is further corroborated by the fact that crown

85
mutations have commonly been seen in other pineapple
cultivars by the author.
The lack of intra-specific variability observed in the
isozyme banding patterns of A. ananassoides, A. bracteatus
var. tricolor, A. lucidus, A. nanus, and A. parguazensis may
have been due to the low sample number (2) used to
characterize each species. However, when the electrophoretic
data from all 5 of these species are considered together, it
is quite surprising that low inter-specific variability was
detected as well. It may be that this lack of variability
reflects the narrow circumscription of these species. Many
of the feral types used in this study closely resembled
individuals fitting the definition of species recognized by
Smith (1961), but they were not included in any of the
prsently recognized species because they differed in 1 or 2
characters from the species descriptions given by Smith and
Downs (1979). These genotypes may belong within the limits
of some of the presently delimited species, thus giving this
taxa greater intra-specific variability, as in the case of
the different clones of pineapple (A. comosus). Perhaps the
most valid explanation for the high degree of intra-specific
homology observed in all the 'Ananas' species, except for A.
comosus, is that they are self-pollinated. In contrast, A.
comosus is self-incompatible, with obligate out-crossing
favoring a high degree of isozyme and allelic heterogenity.

86
Feral Type Identification
The phosphoglucomutase banding patterns for the 29
feral types characterized electrophoretically are shown in
Table 5.6, Fig. 5.3. The full range of isozymic polymorphism
at each of the 3 phosphoglucomutase loci was represented in
this diverse collection of genotypes. The Pgm-1 locus was
particularly polymorphic, with all of the 5 alleles
represented, i.e., L, S, M, F, and P. Two of these alleles,
L and P, were unique to this collection and had not been
observed in any of the species or pineapple cultivars
previously characterized. Nine different banding patterns
(SL, ML, SS, MS, MM, FM, FS, FF, and PS) were observed at
the Pgm-1 locus. Five of these (SL, ML, MM, FF, and PS) were
new allelic combinations not previously observed.
The FF Pgm-1 genotype was observed in P.I. 095, typical
pineapple plants that produce large, conical 3 kg fruit. The
2 sampled plants of this introduction were collected from a
small plot of plants being cultivated by the Piaroas Indian
River community of El Gavilan. The region is an isolated
portion of dense tropical forest located in the north
central portion of the Amazonian Territory in southern
Venezuela. The cultivated plants were reportedly collected
from the wild by the local Indians.
The Pgm-1 genotype MM was observed in the 2 plants of
the introduction P.I. 487443. These plants were collected
from the tropical rain forest region of San Carlos de Rio
Negro. This region is located in the southernmost portion of

87
Venezuela, on the border with Brazil. Two other
introductions, P.I. 487440 and P.I. 487445, were also
collected from this location. Their genotypes at the Pgm-1
locus were both heterozygous MS. Both the MM and MS plants
from this location were smooth-leafed pineapples with
similar growth habits. It appears likely that hybridization
between plants with 2 MS alleles gave rise to the MM
genotype (Table 5.6).
The Pgm-1 allele L appeared in P.I. 072-1 (ML) and P.I.
085 (SL). These introductions were collected from the
southeastern portion of Venezuela. All of the plants have
leaves with few spines and bear purple-colored fruit. Based
on these morphological characteristics, they would be
included in the "Spanish" horticultural group of pineapples.
The Pgm-1 allele P was observed only in P.I. 115. This
introduction is particularly interesting. Morphologically,
these plants appear intermediate between the primitive A.
ananassoides and the domestic A. comosus (Fig. 5.4) The
plants were collected in the wild from a large homogenous
population of plants growing in an arid sandy region near
the Cunavichito River in the Apure State of southwestern
Venezuela.
Plant introductions 046, 079, and 094; 097 and 108; and
064 were putatively grouped under A. ananassoides, A.
parguazensis, and A. nanus, respectively, because of their
phenotypic similarities to these species (Table 5.6). When
these introductions were electrophoretically characterized,

88
the observed banding patterns at the Pgm-1 locus were all
identical with those observed in the valid individuals of
the species. However, isozyme polymorphisms were recorded in
these putative species members at both the Pgm-2 and the
Pgm-3 loci. The morphological and electrophoretic
similarities of these P.I.s to the genotypes of the
respective species appear to indicate that the plants should
be considered as members of the species.
Plant introductions 116, 117, 188, 189, 193, and 25291
were included in the group "Others" (Table 5.6) because they
exhibited little morphological similarity to any of the
presently delimited Ananas species. All of these genotypes,
with the exception of P.I. 193, produce fruit less than 15
cm long and therefore would not be considered true
pineapples. Plant introduction 193 produces fruit more than
15 cm long and has leaves with retrorse spines similar to
those of A. parguazensis. Plant introduction 189 produces an
11 cm edible fruit with a Brix of 16.5. This diverse
collection of genotypes showed considerable polymorphism for
the 3 phosphoglucomutase loci analyzed.
Conclusions
1. Electrophoretic isozyme determination of peroxidase
and phosphoglucomutase allows the unique identification
of 15 of the 27 pineapple cultivars examined. No
distinction could be made between a cultivar and its
sport.

89
2. The hybrid origin of 'PR-1-67' from a cross of 'Red
Spanish' x 'Smooth Cayenne' was confirmed by their
isozyme banding patterns.
3. The hyphothesis that some cultivars originated as
sports was tested. The isozyme data comfirms the
sport origin of 'Esmeralda' and 'Dupuis Smooth' from
'Smooth Cayenne', and 'Cumanesa' from 'Red Spanish'.
Conversely, 'Bumanguesa' does not appear to be a sport
of 'Perolera'.
4. The isozyme banding pattern 'Panare', and its morpholo¬
gical resemblance to the "Queen" group, indicates the
presence of locally-originated "Queen" types growing in
the Western Hemisphere. The origin of "Queen" pine¬
apples from a 'Panare'-like wild type is suggested.
5. A correlation between the Pgm-1 alleles and the
horticultural groups of pineapple was found. Based
on this fact and on plant locations, a hybrid
origin for 'Caicara', 'Black Jamaica', 'Valera
Amarilla', and 'Cacho de Venado' is indicated.
6. Based of banding patterns of peroxidase and
phosphoglucomutase, 5 of the 7 species could be
distinguished. The 2 exceptions were A. comosus,
which showed high polymorphism in its cultivars, and
A. monstrosus.
7. The A. monstrosus plants were confirmed to be
mutations of pineapple cultivars based on isozyme

90
patterns. The questionable validity of this species
is corroborated.
8. The lack of intra-specific variability observed in iso¬
zyme banding patterns of Ananas species, except A.
comosus, is maybe due to the present restrictive
delimitation of these species, and/or to the self¬
compatible character of these species compared to the
self-incompatible A. comosus.
9. Increased polymorphism for the Pgm-1 locus was observed
in the feral types. This variability was represented
either by 2 different alleles or new allelic
combinations.
10. Plant introductions putatively grouped under species
with similar morphology also showed similarity in
their isozyme banding patterns.

91
Table 5.1. Peroxidase and phosphoglucomutase isozyme
banding patterns observed for pineapple
cultivars.
Hort.Z
group
Cultivar
Per-2
Isozyme
Per-3
v
genotypes^
Pgm-1 Pgm-2
Pgm-3
Abacaki
Abaka
FF
FF
MS
FS
SS
Cacho de Venado
FS
FS
SS
FF
SS
Injerta
FS?
FS
MS
FS
SS
Perola
FF
FS
FM
FF
SS
Cayenne
Dupuis Smooth
FF
FF
FM
FF
SS
Esmeralda
FF
FF
FM
FF
SS
Smooth Cayenne
FF
FF
FM
FF
SS
Queen
Queen
FS
SS
FM
SS
SS
Panare
FS
SS
FM
SS
SS
Maipure
Bumanguesa
FF
FF
MS
FS
FS
Maipure
SS
SS
MS
FS
SS
Perolera
FS
FS
MS
FS
SS
Pina de Brazil
FS
FF
MS
FS
FS
Rondon
FS
FS?
MS
FF
FF
Spanish
Black Jamaica
FF
FF
FS
FS
SS
Cabezona
FF
FF
SS
FS
SS
Caicara
FF
FS?
MS
FS
FS
Cumanesa
FF
FF
SS
SS
SS
Monte Oscuro
FF
FF
SS
FS
FS
Nacional
FF
FF
SS
SS
SS
Negrita
FF
SS?
SS
SS
SS
PR-1-67
FF
FF
MS
FS
SS
Red Spanish
FF
FF
SS
SS
SS
Valera Amarilla
FF
FS
MS
SS
SS
Others
Brecheche
FF
FS
SS
FS
FS
Cambray
FS
FS
SS
FS
FS
Masmerah
FF
FS
SS
SS
SS
zGroups
are based on presence
or
absence
of spines
and
fruit morphology.
Y"?" indicates a doubtful determination.

92
Table 5.2.
Pineapple cultivar classification based on
peroxidase banding patterns.
Per-2/Per-3
genotype
Pattern2
Cultivar
FF/FF
A1
Dupuis Smooth, Esmeralda,
Oscuro, National, PR-1-67
Spanish, Smooth Cayenne
Monte
, Red
FF/FS
A2
Brecheche, Caicara, Masmerah,
Perola, Valera Amarrilla
FF/SS
A3
Negrita (?)^
FS/FF
A4
Pina de Brazil
FS/FS
A5
Cacho de Venado, Cambray,
Perderá, Rondon
Injerta,
FS/SS
A6
Panare, Queen
SS/SS
AV
Maipure
zPatterns are
shown in
Fig. 5.1.
Â¥"?" indicates a doubtful classification.

93
Table 5.3. Pineapple cultivar
phospñoglucomutase
classification based on
banding patterns.
Pgm-l/Pgm-2/ Pattern2
Pgm-3
Cultivar
FM/FF/SS
B1
Dupuis Smooth, Esmeralda,
Smooth Cayenne
Perola,
FM/SS/SS
B2
Queen, Panare
FS/FS/SS
B3
Black Jamaica
MS/FF/FF
B4
Rondon
MS/FS/FS
B5
Bumanguesa, Caicara, Pina
de Brazil
MS/FS/SS
B6
Abaka, Injerta, Maipure,
PR-1-67
Perolera,
MS/SS/SS
B7
Valera Amarilla
SS/FF/SS
w
00
Cacho de Venado
SS/FS/SS
B9
Brecheche, Cambray, Monte
Oscuro
SS/FS/SS
o
rH
CQ
Cabezona
SS/SS/SS
Bn
Cumanesa, Masmerah, Nacional,
Negrita, Red Spanish
2
Patterns are
shown
in Fig. 5.1.

94
Table 5.4. Pineapple cultivar identification based on
peroxidase (Per) and phosphoglucomutase (Pgm)
isozyme banding patterns.
Class
Per
Pgm
Cultivar
1
A1
B1
Dupuis Smooth, Esmeralda, Smooth Cayenne
2
A1
B3
Black Jamaica
3
Aí
B5
Bumanguesa
4
A1
B6
Abaka, PR-1-67
5
A1
B9
Monte Oscuro
6
?10
"21
Cabezona
7
A1
Cumanesa, Nacional, Red Spanish
8
A2
Perola
9
AP
Caicara
10
A2
B7
Valera Amarilla
11
A2
B9
Brecheche
12
A2
l11
k1
Masmerah
13
A3
Negrita (?)Z
14
A4
Pina de Brazil
15
A5
B4
Rondon
16
A5
B6
Injerta, Perolera
17
A5
B
Cacho de Venado
18
A5
Cambray
19
A6
B2
Panare, Queen
20
A7
B6
Maipure
z
"?" indicates a doubtful classification

95
Table 5.5. Peroxidase and phosphoglucomutase isozyme banding
patterns observed for Ananas species.
Species
Per-2
Isozyme genotypes2
Per-3 Pgm-1 Pgm-2 Pgm-3
A.
ananassoides
FF
FS
MS
SS
FS?
A.
bracteatus var
. tricolor FS?
FF
MS
SS
SS
A.
y
comosusJ
FF
FF
FM
FF
FF
FS
FS
FS
FS
FS
SS
SS
MS
SS
SS
SS
A.
lucidus
FF
SS
SS
SS
SS
A.
monstrosusx
FF
FS
FM
FF
SS
FF
FF
SS
SS
SS
A.
nanus
SS
FF?
SS
SS?
SS?
A.
parguazensis
—
—
SS
FS?
SS
z„
?" indicates a
doubtful determination;
indicates
data not available.
^Includes pineapple cultivars.
xPlants from 2 cultivars that resemble this species were
analyzed.

96
Table 5.6. Phosphoglucomutase isozyme banding patterns
observed for various Ananas genotypes.
Species and/or
Hort. group
P.I.Z
v
Isozyme genotypes^
Pgm-1 Pgm-2 Pgm-3
A. comosus
Abacaki
095
FF
FS?
SS
192
MS
FF
FF
Maipure
092-1
FS
FF
SS
093
FM
FS?
SS
126
MS
FF
FF
196
MS
FS
FS
487439
MS
FS
FS
487440
MS
FS
FS
487443
MM
FS
SS
487445
MS
FS
FS
Spanish
072-1
ML
FS
SS
085
SL
FF
FF
086
MS
FF
FS
092-2
SS
FS
SS
110
FS
FS?
FS?
487442
MS
SS
SS
A. ananassoides
046
MS
SS?
FS
079
MS
SS
SS
094
MS
SS
FF
A. parguazensis
097
SS
FS
FS
108
SS
FF
SS
A. nanus
064
SS
SS?
SS
OthersX
115
PS
SS?
FF?
116
SS
FS?
SS
117
SS
FS
FS
188
MS
FS
SS
189
SS
FS
FF
193
FS
FF
FF?
27285
MS
SS
SS
zPlant introduction
numbers.
y”?" indicates a doubtful determination.
xBotanical characteristics of the plant do not fit any of
the presently described species.

97
Figure 5.1. Schematic representation of peroxidase (Per)
and phosphoglucomutase (Pgm) isozymes in
pineapple cultivars.

98
•
p
P
o
fO
r—1
in
>
0
.—.
0)
o
-
in
•H
in
31 '
in
.—.
•H
3
p
3
in
3
-
o
-P
-p
in
•H
in
iX3
W
(0
in
0
c
O
r-H
W
Q)
3
p
fts
p
o
m
-P
T3
-p
a
-P
u
in
c
o
•H
in
to
in
CD
3
(C
(0
Ü
3
3
a.
3
3
p
3
0
T3
o
-
<0
ITS
n
rH
E
(1)
E
—-
3
a
•
•
•
•
•
t
•
<
<
<
<
—
<1
<
<1
Figure 5.2. Schematic representation of peroxidase (Per)
and phosphoglucomutase (Pgm) isozymes in
Ananas species. Ananas comosus is
represented by the cultivars depicted in
Fig. 5.1.
parguazensis

Figure 5.3. Variation in phosphoglucomutase isozyme in
Ananas genotypes leaf extracts. The sequence
is: 1. 'Smooth Cayenne'; 2. 'Perola';
3. 'Caicara'; 4. 'Perolera'; 5 and
16. 'Panare'; 6. 'Bumanguesa'; 7. 'Cambray';
8. P.I. 193; 9, 26 and 38. P.I. 115;
10. P.I. 085; 11. A. monstrosus ('Perola'?);
12. P.I. 072-1; 13. P.I. 487442; 14. 'Rondon';
15. A. bracteatus var. tricolor; 17, 24
and 25. 'Brecheche'; 18. A. parguazensis;
19. P.I. 196; 20. A. lucidus; 21. 'Injerta';
22. 'Cacho de Venado'; 23. 'Monte Oscuro';
27. 'Cabezona'; 28. 'Queen'; 29. P.I. 25291;
30. P.I. 487445; 31. 'Masmerah'; 32. P.I. 097;
33 and 36 'Pina de Brazil'; 34. 'Abaka';
35. P.I. 487440; 37. P.I. 487443. Sample origin
is at the bottom of each photograph and band
migration is anodal.

100

CHAPTER VI
PHENETIC RELATIONSHIPS IN ANANAS BASED
ON ISOZYME ELECTROPHORETIC ANALYSIS
Introduction
Phenetic studies are particularly valuable for the
efficient utilization and preservation of germplasm sources.
When a genus has been well characterized, germplasm
collections can be made that reflect the natural diversity
of the crop. Plant breeders can quickly locate genotypes
that possess those alleles most useful in hybridization
schemes. One way in which electrophoretic isozyme analysis
has been used successfully is in the assessment of the
genetic similarities between populations (Johnson et al.,
1967; Sheen, 1970; Smith et al., 1970; Bassiri, 1977; Loukas
and Pontikis, 1979; Meerow, 1986; Panda et al., 1986). In
general, close relationships are expected in isozyme banding
patterns between 2 or more populations which have common
parentage (Bassiri and Adams, 1978).
Isozymic studies from Chapter IV and V were utilized to
define phenetic relationships between populations of
cultivated pineapples. Special emphasis was placed on
101

102
comparing the commercial cultivars with the genotypes
collected from the center of origin.
The determination of how many individuals and
populations to sample before a confident statement can be
made regarding the amount of genetic divergence between taxa
is an important consideration in the use of
electrophoretical evidence (Gottlieb, 1977) . Marshall and
Brown (1975) showed that, in the extreme case of 20 alleles
at a locus, with frequencies of 0.05 each, a random sample
of 60 individuals will include, with 95% confidence, 1 copy
of each allele. Thus, the sampling number used in this study
(62 genotypes) should include all possible alleles at the
loci analyzed.
A number of statistical coefficients have been devised
to place allelic frequency data into a single statistical
value (Cavalli-Sforza and Edwards, 1967; Hendrick, 1971).
Two such values or statistics are genetic similarity and
genetic distance (Gottlieb, 1977) . Small sample size and low
number of loci are 2 variables which decrease the precision
of the genetic distances estimation (Nei, 1978) .
Unfortunately, the limited number of individuals per
genotype (2) and number of loci (7) analyzed in this study
do not allow any statistically significant explorations of
genetic variation among the genotypes. Another approach for
estimating the genetic distances of conspecific populations
has been suggested by Gottlieb (1975). This method makes use

103
of the presence or absence of alleles rather than their
frequencies and was used in this study.
Material and Methods
A phenetic analysis of 62 Ananas genotypes was
conducted using the genetic isozyme models of Chapter IV and
isozyme polymorphism data of Chapter V. The analysis
consisted of dendographic representations of phospho-
glucomutase and peroxidase isozyme polymorphisms.
The genotypes were numbered from 1-62 as shown in Table
6.1. Alleles from Pgm-1, Pgm-2, Pgm-3, Per-2, and Per-3 loci
were enumerated according to the following system: Pgm-1
L=1, S=2, M=3, F=4, P=5; Pgm-2 F=6, S=7; Pgm-3 F=8, S=9;
Per-2 F = 10, S=ll; Per-3 F=12, S=13. All of the cultivars
were characterized as to the presence or absence of each of
the 13 alleles (Table 6.2). The feral types and species were
analyzed using only the 9 alleles of phosphoglucomutase.
From this information, 3 data sets were constructed. One
data set included the cultivars using both phospho¬
glucomutase and peroxidase data. A second data set was
constructed with the feral types and species using only
phosphoglucomutase data and a third grouping combined the
information from these 2 data sets.
Distance coefficients were calculated based on the
unweighted pair method of Sneath and Sokal (1973), using a
computer program and the data from Table 6.2. The results
are presented as cluster analysis dendograms. Individual

104
genotypes that were found clustered together in the
dendograms are then compared for morphological similarities.
Results and Discussion
The cluster analysis dendogram of the 27 pineapple
cultivars, based on genetic distance coefficients of the
isozyme banding patterns, is shown in Fig. 6.1. Cultivars
that cluster together at a distance coefficient (DC) of 0.0
are assumed to possess nearly identical banding patterns. In
general, the cultivars from a common horticultural group
(Table 5.2) did not exhibit close phenetic relationships as
determined by cluster dendograms and the DCs when the
isozyme banding patterns of both phosphoglucomutase and
peroxidase were considered together. Frequently, cultivars
possessing distinctly different growth habits, leaf
morphologies (including spines), and fruit morphologies
exhibited close isozymic relationships.
The isozyme relationships among the feral types and
species of Ananas are illustrated in the dendogram shown in
Fig. 6.2. As in the previous dendogram few morphological
similarities could be distinguished among the various
individuals or groups of individuals which exhibited close
isozymic relationships.
The dendogram in Fig. 6.3 considers the isozymic
relationships of all the genotypes together. Included are
the Ananas species, the feral types, and the major pineapple
cultivars. The Ananas species do not cluster at close DC

105
coefficients, except for A. nanus (61) and A. lucidus (58)
which cluster at a DC of 0.0.
Most of the cultivars fell into one of several discrete
clusters at a DC of 0.0, indicating a close relationship
between the members of a cluster. This type of clustering
was not observed in the feral types. 'Monte Oscuro' (19), an
isolated small cultivar grown in eastern Venezuela and
characterized by large plants (>2 m) and fruits (>10 kg) was
an exception.
One of the most interesting clusters is formed by a
branch at a DC of 0.204 (indicated by the asterisk in Fig.
6.3). This cluster includes some of the most economically
important pineapple cultivars, including 'Smooth Cayenne'
(6) and its sports 'Esmeralda' (5) and 'Dupuis Smooth' (4)
and 'Perola' (9) and a 'Perola' crown mutation (60).
Slightly less related members of this "Smooth Cayenne" group
include P.I. 093 (32), 'Queen' (7), 'Panare (8), P.I. 48743
(36) and, P.I. 072-1 (38). The relationships contained
within this cluster may provide some evidence on the origin
of 'Smooth Cayenne'. It has been demostrated in Drosophila
(Duke and Glassman, 1968) and in rice (Siddiq et al., 1972)
that the electrophoretic mobility of isozymes tends to be
reduced as the populations become more evolutionarily
advanced. All the genotypes forming the "Smooth Cayenne"
cluster possess the fast mobility isozymes (Tables 5.2 and
5.7), thus suggesting that they may be more ancient than
other cultivated pineapples.

106
'Smooth Cayenne' and its sports are by far the most
important cultivars of pineapple today. Collins (1960) has
traced the early history of 'Smooth Cayenne' back to 5
plants sent by Perrottet from French Guiana to France
sometime before 1840. In France, they were vegetatively
propagated and shipped to England, and then to Australia,
Florida, Jamaica, and Hawaii. Collins (1960) suggests that,
because Perrottet used the name "mai-pouri" for his
pineapple, the original plants may have been obtained from
the Maipure Indians of the upper Orinoco River in south
Venezuela. All the other genotypes forming the cluster with
'Smooth Cayenne' come from this geographic region. ' Panare'
is grown in small areas close to the margin of the Orinoco
River in Bolivar State, Venezuela. 'Queen' seems to have
originated from 'Panare', as discussed in Chapter V.
'Perola' is grown in northern Brazil, and its origin has not
been established, but it presumably comes from the northern
Amazonian region. Plant introductions 095, 477443, and 072-1
were collected in the Amazonian Territory of southern
Venezuela. The fact that 'Smooth Cayenne' clusters with
these types instead of with any other cultivar suggests
that, as Collins believed, it may have been collected in the
basin of the Orinoco River and perpetuated vegetatively, so
that it still resembles the feral types growing in the
region.
The rest of the cultivated varieties of pineapple do
not possess the fast mobility bands observed in the "Smooth

107
Cayenne" cluster. This fact sugggests that these cultivars
probably had a different origin than 'Smooth Cayenne'.
Collins (1960) speculates that many cultivars may have
originated as hybrids in greenhouses in Europe, where
pineapple was very popular in the 19th century.
The feral genotype P.I. 115 (51) exhibits a
particularly interesting and unique isozyme banding pattern
and remains a distant outlayer from all other types, joining
them at a DC of 0.495 (Fig. 6.3). It possesses the fastest
mobility allozyme band (P) at the Pgm-1 locus, suggesting
that it may be the most primitive of the genotypes tested.
Gottlieb (1977) found a high degree of genetic similarity
between conspecific populations. He suggested that if a
population is discovered which has novel alleles, this is
strong evidence that the population should be a distinct
taxon. Thus, P.I. 115 should be further examined with this
in mind, as it may represent an important turning point in
the evolution of domestic pineapples.
A number of genotypes, i.e., P.I.s 072-1 (38), 085
(39), 086 (40), 093 (32), 095 (26), 189 (55), 193 (43), and
487443 (36) appear to be rather isolated individuals. These
genotypes join clusters with other genotypes at DCs greater
than 0.18. The only common character of these feral types is
the fact that they were all collected from southern
Venezuela; otherwise they greatly differ in their
morphological characteristics. This indicates the broad
genetic diversity that exists between feral types and the

108
importance of continuing to collect types from this
as it seems indeed to be the center of origin of the
species.
region,
Ananas

109
Table 6.1. Designation of genotypes
analyzed electrophoretically.
ID no.
Genotype
1
Abaka
2
Cacho de Venado
3
Injerta
4
Dupuis Smooth
5
Esmeralda
6
Smooth Cayenne
7
Queen
8
Panare
9
Perola
10
Bumanguesa
11
Maipure
12
Perolera
13
Pina de Brazil
14
Rondon
15
Blanca
16
Cabezona
17
Caicara
18
Cumanesa
19
Monte Oscuro
20
Nacional
21
Negrita
22
PR-1-67
23
Red Spanish
24
Valera Amarilla
25
Brecheche
26
Cambray
27
Masmerah
28
P.I. 095
29
P.I. 192
30
P.I. 126
31
P.I. 092-1
32
P.I. 093
33
P.I. 196
34
P.I. 487439
35
P.I. 487440

110
Table 6.1. continued.
ID no.
Genotype
36
P. I.
487443
37
P. I.
487445
38
P. I .
072-1
39
P. I .
085
40
P. I.
086
41
P.I.
092-2
42
P.I .
110
43
P.I.
193
44
P.I.
27285
45
P.I.
046
46
P.I.
079
47
P.I.
094
48
P.I.
064
49
P.I.
097
50
P.I.
108
51
P.I.
115
52
P.I.
116
53
P.I.
117
54
P.I.
188
55
P.I.
189
56
A. ananassoides
57
A. bracteatus
58
A. lucidus
59
A. monstrosus (Red Spanish)
60
A. monstrosus (Perola)
61
A. nanus
62
A. parguazensis

Ill
Table 6.2. Presence-absence data matrix for electrophoretic
analysis of Ananas genotypes.
2
Band number
ID no.y 1 2 3 4 5 6 7 8 9 10 11 12 13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
0 110 0
0 10 0 0
0 110 0
0 0 110
0 0 110
0 0 110
0 0 110
0 0 110
0 0 110
0 110 0
0 110 0
0 110 0
0 110 0
0 110 0
0 10 10
0 10 0 0
0 110 0
0 10 0 0
0 10 0 0
0 10 0 0
0 10 0 0
0 110 0
0 10 0 0
0 110 0
0 10 0 0
0 10 0 0
0 10 0 0
0 0 0 1 0
0 110 0
0 110 0
0 10 10
0 0 110
0 110 0
0 110 0
0 110 0
1 1
1 0
1 1
1 0
1 0
1 0
0 1
0 1
1 0
1 1
1 1
1 1
1 1
1 0
1 0
1 0
1 1
0 1
1 0
0 1
0 1
1 1
0 1
0 1
1 1
1 1
0 1
1 1
1 0
1 0
1 0
1 1
1 1
1 1
1 1
0 1
0 1
0 1
0 1
0 1
0 1
0 1
0 1
0 1
1 1
0 1
0 1
1 1
1 0
0 1
0 1
1 1
0 1
1 0
0 1
0 1
0 1
0 1
0 1
1 1
1 1
0 1
0 1
1 0
1 0
0 1
0 1
1 1
1 1
1 1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
0
0
0
1
1
0
0
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
0
0
1
1
0
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
0
1
1
0
0
0
1
1
1
c
1
1
0
1
0
0
1
0
0
0
1
0
0
1
1
1
1

112
Table 6.2. continued.
ID no.
Y 1
2
3
4
5
6
2
Band number
7 8 9 10 11 12 13
36
0
0
1
0
0
1
1
0
1
37
0
1
1
0
0
1
1
1
1
38
1
0
1
0
0
1
1
0
1
39
1
1
0
0
0
1
0
1
0
40
0
1
1
0
0
1
0
1
1
41
0
1
0
0
0
1
1
0
1
42
0
1
0
1
0
1
1
1
1
43
0
1
0
1
0
1
0
1
0
44
0
1
1
0
0
0
1
0
1
45
0
1
1
0
0
0
1
1
1
46
0
1
1
0
0
0
1
0
1
47
0
1
1
0
0
0
1
1
0
48
0
1
0
0
0
0
1
0
1
49
0
1
0
0
0
1
1
1
1
50
0
1
0
0
0
1
0
0
1
51
0
1
0
0
1
0
1
1
0
52
0
1
0
0
0
1
1
0
1
53
0
1
0
0
0
1
1
1
1
54
0
1
1
0
0
1
1
0
1
55
0
1
0
0
0
1
1
1
0
56
0
1
1
0
0
0
1
1
1
57
0
1
1
0
0
0
1
0
1
58
0
1
0
0
0
0
1
0
1
59
0
1
0
0
0
0
1
0
1
60
0
0
1
1
0
1
0
0
1
61
0
1
0
0
0
0
1
0
1
62
0
1
0
0
0
1
1
0
1
zBlank spaces indicate data not included in the study.
^Refer to Table 6.1 for population designations.

DISTANCE
113
GENOTYPE
Figure 6.1. Cluster analysis dendogram of 27 pineapple
cultivar isozyme phenotypes. Refer to Table
6.1 for genotype designations.

114
GENOTYPE
Cluster analysis dendogram of isozyme
phenotypes of Ananas species and feral
types. Refer to Table 6.1 for genotype
designation.
Figure 6.2

DISTANCE
115
GENOTYPE
. Cluster analysis dendogram of the isozyme
phenotypes of 62 Ananas genotypes. Refer
to Table 6.1 for genotype designations.
Figure 6.3

CHAPTER VII
CHARACTERIZATION OF REGENERATED PINEAPPLE PLANTS
Introduction
The usefulness of a pineapple plant propagation system
is judged by its ability to efficiently establish large
numbers of plants in the greenhouse or field and by the
clonal fidelity of these plants. A tissue culture
multiplication system, as discussed in Chapter III, has been
shown to meet this first requirement. The requirement of
clonal fidelity will be addressed in this Chapter.
The reports of somaclonal variation in numerous plant
species and pineapple in particular, were reviewed in
Chapter II. The main purpose of the research reported in
this Chapter was to determine if the large amount of
variation reportedly found in 'Smooth Cayenne'-regenerated
plants (Wakasa, 1979), is a common phenomenon in pineapple
cultivars. If other cultivars exhibit less somaclonal
variation, then tissue culture may be an effective method
for mass propagation of those cultivars. Conversely, if high
levels of somaclonal variation are an inherent part of in
vitro pineapple propagation systems, then tissue culture may
be a useful method of cultivar improvement and development.
116

117
Of particular interest is the possibility of obtaining
spineless mutants from high guality spiny cultivars such as
'PR-1-67 1.
To investigate somaclonal variation, regenerated
pineapple plants from axillary bud cultures of 'Perolera',
'PR-1-67', and A. bracteatus var. tricolor were evaluated
for variation in both morphological and biochemical
characters.
Materials and Methods
Axillary bud cultures were established from a stem of
'Perolera', a slip of A. bracteatus var. tricolor and a
crown and a slip of 'PR-1-67', according to the methods
described in Chapter III. Regenerated plants from these
cultures were used in this study. Plants were grown in 12-cm
pots containing a soil mixture composed of a (2:1:1)
commercial potting soil, sand, and peat moss. Plants were
maintained in a greenhouse under standard practices of
irrigation, fertilization, and pest control.
The vegetative characteristics of the regenerated
plants, particularly leaf morphology, were observed over a
period of 1 year to determine the phenotypic stability of
these traits. Electrophoretical evaluation of the
regenerates was done for 2 isozyme loci of peroxidase and 3
loci of phosphoglucomutase, following procedures described
in Chapter IV.

118
Results and Discussion
Electrophoretic Characterization
A total of 767 regenerated plants were evaluated for
each of the 2 isozyme staining systems (Table 7.1). All of
the regenerates, with 2 possible exceptions, exhibited
identical isozyme banding patterns for each of the enzyme
systems. Two 'PR-1-67' plants gave zymograms with a unique
band corresponding to the Per-2 allele S ('PR-1-67' normally
had a Per-2 FF genotype). An extra faint band also appeared
on the phosphoglucomutase zymograms of these 2 plants. These
aberrant zymograms may have been artifacts, because several
repeat runs using different leaf samples taken at variuos
times produced inconsistent banding patterns. No plants were
found that consistently produced a unique banding pattern.
There have been other reports in the literature of
somaclonal isozyme variation. A peroxidase variant was found
by Vardi (1977) among plants regenerated from Citrus
protoplasts but studies by Gmitter (1985) and Miller (1986)
on a large number of regenerated Citrus plants uncovered no
variant zymograms. Menendez et al. (1986) found variant
peroxidase zymograms in apple plants regenerated from shoot
tip cultures. The enviromental variability of peroxidase is
well documented (Hart and Bathia, 1967). Perhaps the
variability in peroxidase observed in pineapple was due to
developmental or environmental factors rather than genetic
changes.

119
Morphological Characterization
A total of 1900 regenerated plants were evaluated for
morphological variation (Table 7.1). High levels of
phenotypic variation were observed for a number of diverse
vegetative characteristics in the regenerated plants shortly
after they were established in soil. Initially the leaf
variation included the presence or absence of spines,
differences in the amount of surface wax, albino stripes,
and the presence of recurved or narrow leaves. Nearly all of
the regenerated plants had smooth leaves at the time plants
were transferred to soil. As new leaves were formed,
however, leaves characteristic of the genotype appeared,
i.e., spiny ('PR-1-67' and A. bracteatus var. tricolor) or
piping ('Perolera'). During growth in the greenhouse, a
large number of plants from the spiny-leaved genotypes
exhibited sectors along the leaf edges that were smooth. Two
'PR-1-67' plants with narrow leaves were observed (Fig.
7.1A). These plants died 3 months after being transferred to
the soil. Another common aberration in the 'Perolera'
regenerated plants was the presence of recurved leaves. This
character was transient and, with time, new leaves produced
on the plant appeared normal. Four plants, 2 of 'PR-1-67'
and 2 of 'Perolera', developed leaves with 1-2 albino
stripes (Fig. 7.IB). These plants have continued to express
this aberrant phenotype after 1 year of growth in the
greenhouse.

120
No correlation was found in the regenerated plants
between observed variability and the explant source. Plants
regenerated from cultures derived from crowns, slips, or
stems all exhibited similar levels of variation (less than
0.2%). This level of variability was significantly less than
that reported by Wakasa (1979) for 'Smooth Cayenne'
regenerated plants (34% for lateral buds of crowns and
slips). However, the epigenetic or transient variation
observed in the regenerated plants shortly after
establishment in the soil was more in line with the levels
reported by Wakasa (1979). This indicates that the length of
time during which the regenerates are evaluated affects the
amount of somaclonal variation.
Differences in the concentration of BA may have been
responsible for the observed variability between this study
and Wakasa's. Wakasa (1979) reported that certain sub¬
cultures were made on media containing up to 44 uM of BA.
Cultivar differences may have also contributed to the
variability between these 2 studies. Cultivar differences in
somaclonal variability have ben found in sugarcane (Liu and
Chen, 1976) and Pelargonium (Skirvin and Janick, 1976).
'Smooth Cayenne' may be very susceptible to both in-vivo and
in-vitro induced mutation. Collins (1960) reported that
'Smooth Cayenne' is highly mutable, e.g., the frequency of
reversion from smooth leaves to spiny is approximately 1%.
The phenetic studies of Chapter VI indicate that the 'Smooth
Cayenne' group shows significant isozyme differences from

121
the other major groups of pineapple cultivars. The isozyme
banding patterns of 'Smooth Cayenne' appear to possess more
primitive allozymes. One explanation for the observed higher
level of somaclonal variation may be that 'Smooth Cayenne'
is highly heterozygous and has been vegetatively propagated
over a longer period of time than the cultivars examined in
this study. This heterozygous condition would then allow for
the expression of a greater number of recessive mutants than
would be expected in more homozygous cultivars, where a
single base pair mutation would not be expressed in the
heterozygous condition.
In conclusion, the studies of this Chapter indicate
that the clonal fidelity of the tissue culture-regenerated
plants has been maintained in the pineapple cultivars
tested. These results suggest that tissue culture may be a
viable alternative to traditional methods of pineapple
propagation. No definite conclusion can be made in regard to
the reported 34% somaclonal variation observed in 'Smooth
Cayenne' (Wakasa, 1979) relative to the 0.2% observed in
this study.

Table 7.1. Regenerated pineapple plants evaluated for electrophoretic
and morphological variation.
Cultivar2
Electrophoretic
Morphological
Regenerated
plants (no.)
Zymograms
Regenerated
plants (no.)
Variants-*
(no.)
Perolera
189
378
850
2
PR-1-67
201
402
500
3
PR-1-67
310
620
400
1
A. bracteatus
var. tricolor
67
134
150
0
Total
767
1534
1900
6
zTwo different plants of 'PR-1-671 were initiated in culture.
^Plants showing permanent variant character were included.
122

123
Abnormal pineapple regenerated plants.
A. Narrow leaves (left) and normal
leaves (right); B. Albino striped
leaves.
Figure 7.1.

CHAPTER VIII
SUMMARY AND CONCLUSIONS
There is considerable speculation in the literature on
the in vitro culture of pineapple and its usefulness as a
rapid and efficient propagation method; however, few reports
have been published on the number of plants established in
soil. Furthermore, most research was conducted with 'Smooth
Cayenne' which reportedly has high levels of somaclonal
variation in the regenerated plants.
One goal of this research was to develop an efficient
in vitro propagation system for pineapple, using different
cultivars and species, and to determine the amount of
somaclonal variation present in the regenerated plants. High
levels of somaclonal variation would significantly reduce
the attractiveness of tissue culture as a method for mass
propagation. In contrast, high levels of heritable and
stable somaclonal variation may be an important source of
genetic diversity for pineapple cultivar development and
improvement schemes.
A system utilizing liquid shake cultures derived from
axillary buds of crown, slips, and stems was devised.
Cultures were initiated from excised axillary buds on a
124

125
solid MS medium supplemented with 3% sucrose, 0.8% Difco
Bactor-agar, 0.57 mM inositol, 1.2 uM thiamine HC1, 10.8 uM
NAA, and 8.8 uM BA. Explants were subcultured to fresh
medium at 6 week intervals. Proliferating cultures were
transferred to liquid medium for multiplication and
maintenance. Subculturing was done at 4 week intervals.
Transferring to a medium without plant growth regulators to
induce root formation was not necessary for plantlet
survival. Using this method, approximately 350 plantlets
from each bud culture could be obtained in 13 months. No
apparent loss in regeneration ability of the cultures has
been observed over a 2 year period. Plantlets longer than
3.0 cm had a survival rate of nearly 100% when transferred
to soil.
To evaluate the genetic fidelity of the regenerated
pineapple plants, morphological observations and protein
electrophoresis were employed. Phenotypic variability was
observed among the regenerated plants, but the frequency of
the variation was significantly lower than in previous
reports. Although high levels of somaclonal variation were
observed one month after the plants were transferred to
soil, plants that were originally variant developed a normal
phenotype after new leaves were formed.
A method was devised whereby leaf sap proteins could be
assayed for isozyme polymorphism using starch gel
electrophoresis. Using this technique, several enzyme
systems were identified that proved useful for Ananas

126
cultivar and species identification, for the evaluation of
somaclonal variation, and for use as genetic markers.
Consistent and well resolved nonvariable banding patterns
were observed for isocitrate dehydrogenase, 6-phospho-
gluconate dehydrogenase, phosphohexose isomerase, superoxide
dismutase, and trióse phosphate isomerase. Well resolved and
variable banding patterns were observed for peroxidase and
phosphoglucomutase. Genetic analysis revealed that these 2
enzyme systems are both under the control of 3 loci with 2
alleles each in the crosses examined. These systems are
inherited independently and may be used as genetic markers
in pineapple. Linkage studies could be carried out between
these loci and loci governing morphological traits,
especially those of economic importance. If such linkages
are discovered, it could significantly reduce the selection
generation period, which is 5 years with traditional
pineapple breeding methods. In the case of the the Pgm-1
locus, 3 other alleles were present in the genotypes
surveyed. Variability was also detected for malate
dehydrogenase, but no genetic analysis was possible using
the 2 segregating populations available. Until further
segregating populations can be analyzed, this enzyme system
will probably be of limited use. Improved protein extraction
procedures as well as modifications of the existing buffer
systems might be required for the successful analysis of
these enzyme systems.

127
The genus Ananas has received a variety of taxonomic
treatments since the first pineapples were botanically
described. Confusion still exists as to which species should
be recognized. Collections of feral types are difficult to
classify within the presently delimited species, as they
sometimes show characteristics intermediate between species.
Another purpose of this study was to electrophoretically
characterize the genus Ananas and the pineapple cultivars as
an aid to their classification. A taxonomic survey of 67
genotypes was conducted utilizing the peroxidase and
phosphoglucomutase staining systems. Fifteen of the 27
pineapple cultivars could be uniquely distinguished. The
origin of some cultivars was determined. 'PR-1-67' was
confirmed to be a hybrid of 'Red Spanish' x 'Smooth
Cayenne'. Similarly, the origin by sporting of 'Esmeralda'
and 'Dupuis Smooth' from 'Smooth Cayenne' and 'Cumanesa'
from 'Red Spanish' was demonstrated. Five of the 7 Ananas
species could be distinguished based on banding patterns of
peroxidase and phosphoglucomutase. The 2 exceptions were A.
comosus and A. monstrosus. Ananas comosus showed high
polymorphism in its cultivars. Ananas monstrosus plants were
confirmed to be crown mutations of pineapple cultivars and
therefore this should not be considered a valid species.
Most of the feral types could be grouped under species with
similar morphology and isozyme banding patterns.
Phenetic relationships in Ananas based on isozyme
analysis indicate a close relation among the pineapple

128
cultivars in comparison to the feral types. Evidence of the
origin of 'Smooth Cayenne' based on cluster dendogram
analysis indicates that it may have been collected in the
basin of the Orinoco River and that it still resembles the
feral types growing in this region. One of the feral types
showed a unique banding pattern that suggested that it may
be a distinct taxon.
Electrophoretic evaluation of peroxidase and phospho-
glucomutase isozymes from regenerated pineapple plants was
done to determine if genetic changes had occurred. All
regenerated plants were found to be invariable in their
isozyme banding patterns. The presence of some
morphologically variant plants among the regenerated plants
analyzed suggested that somaclonal variation may be
occurring at a low level in these cultivars. However, this
variation has not affected the small part of the genome
which codes for the enzymes used in the analysis. Further
studies should be conducted to determine the extent of the
variation occurring in regenerated plants from these
genotypes. Particularly important is the need to grow the
regenerated plants to maturity and evaluate flower and fruit
characteristics before final conclusions on pineapple
somaclonal variation are drawn. Similar studies should be
undertaken with other cultivars and species to determine
whether the genetic background of the pineapple material
placed into culture has a definitive relation to the level
of phenotypic stability observed. These suggested studies

129
may generate the information necessary to determine whether
uniform or variant plants are produced from pineapple tissue
culture.

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BIOGRAPHICAL SKETCH
Maria Grazia Antoni DeWald was born in Rome, Italy, on
October 16, 1948. At the age of 3, she moved to Venezuela
were she obtained primary and secondary education. She
received the degree of Ingeniero Agronomo from the
Universidad Central de Venezuela in June, 1975. During the
last 2 years as an undergraduate, she was employed as
research asistant working on an alfalfa breeding program.
She was appointed after graduation as instructor
professor in the Department of Agronomy (Fruit Crops) at the
same university and promoted to assistant professor in 1980.
She enrolled as a graduate student in the Fruit Crops
Department of the University of Florida in January, 1981,
sponsored by the Consejo de Desarrollo Científico y
Humanístico and the Universidad Central de Venezuela. She
received the degree of Master of Science in August, 1983,
working in the area of taxonomy and cytogenetics.
In September, 1983, Mrs. DeWald enrolled again in the
Fruit Crops Department at the same university as a Ph.D
student, this time in the area of plant biotechnology.
The author and her husband, Stephen, are parents of a
2-year-old son, Stephen Francis.
141

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'
nd B.
Waynd B. Sherman, Chairman
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.
cl. Co CXST-fi-
Gloria A. Moore, Cochairman
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.
Richard E. Litz 0
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.
Paul M. Lyrene O
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.
IdcjjjL
Walter S.
Associate Professor of Botany

This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
May, 1987
Dean,
cuJt 'cf-
liege of Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08553 9947




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