Citation
In vitro production of colchiploids and mutants of native Florida diploid Vaccinium species and hybrids and evaluation of 12X V. ashei colchiploids

Material Information

Title:
In vitro production of colchiploids and mutants of native Florida diploid Vaccinium species and hybrids and evaluation of 12X V. ashei colchiploids
Creator:
Perry, Julia Lucile Wood, 1955- ( Dissertant )
Lyrene, Paul M. ( Thesis advisor )
Sherman, Wayne B. ( Reviewer )
Moore, Gloria A. ( Reviewer )
Bassett, Mark ( Reviewer )
Quesenberry, Ken ( Reviewer )
Fry, Jack L. ( Degree grantor )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1984
Language:
English

Subjects

Subjects / Keywords:
Blueberries ( jstor )
Cells ( jstor )
Chromosomes ( jstor )
Diameters ( jstor )
Diploidy ( jstor )
In vitro fertilization ( jstor )
Ploidies ( jstor )
Polyploidy ( jstor )
Species ( jstor )
Tetraploidy ( jstor )
Blueberries -- Breeding
Blueberries -- Florida
Dissertations, Academic -- Horticultural Science -- UF
Horticultural Science thesis, Ph.D.
Vaccinium
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )
Spatial Coverage:
United States -- Florida

Notes

Abstract:
In vitro produced colonies of Florida native diploid blueberry species Vaccinium elliottii Chapm., V. darrowi Camp, and their interspecific hybrid were treated with colchicine to double their chromosome number and facilitate interspecific hybridization with tetraploid highbush blueberry, V. corymbosum Lamarck. In vitro colchiploids, identified by increased shoot diameter, were confirmed as tetraploid by stomate guard cell measurement and chromosome counts from shoot tip squashes. V. elliottii produced the greatest number of increased-diameter shoots when 2-node cuttings were treated for 48 hours on a rotary drum in liquid modified Knops medium containing 0.1% colchicine, or for 2-6 weeks without agitation on solid modified Knops medium containing 0.01% colchicine. V. darrowi produced the greatest number of increased-diameter shoots when 2-node cuttings were treated on a rotary drum in .01% colchicine-containing liquid modified Knops medium for 24 hours. The V. elliottii X V. darrowi hybrid produced the greatest number of increased diameter shoots when 2-node cuttings were treated for 4 hours in colchicine-free liquid modified Knops medium on a rotary drum, apparently due to spontaneous doubling. The LD-50 of in vitro V. elliottii colonies treated with gamma radiation was 4.5 krads. No explants receiving a dose greater than 25 krads survived. Two mutant colonies were identified from the 1.5 krad treatment and 1 mutant colony was identified from the 3 krad treatment. These mutant colonies were dwarfed and feathery in appearance. The 12X colchiploid ramets of V. ashei cultivars 'Beckyblue' and 'Bluebelle' apparently are of low fertility as both male and female parents. Pollen germination was low and pollen tubes weak. No fruit was set when the 12X cultivars were intercrossed or crossed with 6X V. ashei 'Bonita'. A few fruit were set when the ramets were crossed with tetraploid V. corymbosum 'Sharpblue' or diploid V. darrowi, possibly due to accidental outcrossing or selfing.
Thesis:
Thesis (Ph.D.)--University of Florida, 1984.
Bibliography:
Includes bibliographical references (leaves 71-79).
General Note:
Vita.
General Note:
Typescript.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
ACN6231 ( LTUF )
11780133 ( OCLC )
0030486413 ( ALEPH )

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IN VITRO PRODUCTION OF COLCHIPLOIDS AND MUTANTS OF NATIVE
FLORIDA DIPLOID Vaccinium SPECIES AND HYBRIDS AND
EVALUATION OF 12X V. ASHEI COLCHIPLOIDS





BY






JULIA LUCILE WOOD PERRY


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


1984
















ACKNOWLEDGEMENTS


Appreciation is extended to the members of my graduate

committee: Dr. Wayne Sherman, Dr. Gloria Moore, Dr. Mark

Bassett and Dr. Ken Quesenberry for their guidance and

assistance in the preparation of this research. Special

appreciation is extended to Dr. Paul Lyrene, my major

professor, whose gentle wit and great intellect taught me

how to formulate a question, properly conduct research, and

analyze results to obtain an answer. His teachings and

friendship will be cherished always.

I also wish to extend deep gratitude to my family, my

patient and loving husband of 7 years, Robert Thomas Perry

with whom I have weathered many storms and shared just as

many rainbows. Loving appreciation also to my supportive

parents, Drs. Charles and Mary Wood, who not only gave me

the finest of role models but a varied and valuable genetic

base on which to build.



















TABLE OF CONTENTS


PAGE


ACKNOWLEDGEMENTS . .

LIST OF TABLES . .

LIST OF FIGURES . .

ABSTRACT . . .


. . . . v

S. . . vii

S. . . viii


CHAPTERS


INTRODUCTION .

LITERATURE REVIEW

Cytology . .
Improvement .
Tissue Culture .
Radiation Induction


. . . . 1

. . . . 5

. . . . 11
. . . . 14
. . . . 17
of Mutants ...... 19


IDENTIFICATION OF OPTIMAL TREATMENT FOR
POLYPLOID INDUCTION .. . .

Introduction . . . .
Materials and Methods (Experiments 1-6)

Experiment 1 . . .
Experiment 2 . . .
Experiment 3 . . .
Experiment 4 . . .
Experiment 5 . . .
Experiment 6 . . .

Results and Discussion (Experiments 1-6)

Experiment 1 . . .
Experiment 2 . . .
Experiment 3 . . .
Experiment 4 . . .
Experiment 5 . . .
Experiment 6 . . .

Conclusions . . . .


iii


I

II







III


. .
. .










ENHANCEMENT OF COLCHICINE ACTION . .

Introduction . . . .
Materials and Methods (Experiments 7-11)


Experiment 7 .
Experiment 8 .
Experiment 9 .
Experiment 10 .
Experiment 11 .

Results and Discussion

Experiment 7 .
Experiment 8 .
Experiment 9 .
Experiment 10 .
Experiment 11 .

Conclusions . .


(Experiments 7-11)


V RADIATION STUDIES . . .

Introduction . . .
Materials and Methods . .
Results and Discussion . .
Conclusions . . .

IV BREEDING BEHAVIOR OF 12 X V. ashei
CHOLCHIPLOIDS . . .

Introduction . . .
Materials and Methods . .
Results and Discussions . .
Conclusions . . .

VII CONCLUSIONS . . .

REFERENCES . . . . .

BIOGRAPHICAL SKETCH . . . .


52



r



















LIST OF TABLES


TABLE PAGE

1 Ploidy Level and Characteristics of
Vaccinium Species Native to Florida . 15

2 Composition of Modified Woody Plant Medium,
Modified Andersons Medium, and Modified
Knops Medium . . . ... 26

3 Ploidy Level and Stomate Length of 10 Large
Diameter Shoot in vitro Colonies of V.
elliottii Screened from Experiment 1 32

4 Growth Response of V. elliottii Following
Treatment with 0.1% Colchicine for Various
Durations . . . ... .34

5 Frequency of Fatal Decline Syndrome
Following in vitro Treatment of V. elliottii
with 0.1% Colchicine on a Rotary Drum for
Various Durations . . ... .35

6 Growth Response of Increased-Diameter Shoots
of V. elliottii when Placed on 3 in vitro
Media for Regeneration . ... 37

7 Growth Response of V. darrowi after Treat-
ment of 2-node Cuttings from in vitro
Colonies with 0.1% Colchicine for Various
Durations . . . ... .38

8 Growth Response of V. darrowi X V. elliottii
in vitro 2-node Cuttings to Treatment with
0.1% Colchicine for Various Durations 39

9 Effects of Colchicine Concentration and
Treatment Duration on Regrowth Vigor of
Treated Vaccinium Explants . .. 41

10 Number of Colonies Containing 1 or More
Shoots of Increased-Diameter Following
Treatment with Various Colchicine Concen-
trations for Various Lengths of Time 43











11 Effects of Colchicine Concentration and
Treatment Duration on Regeneration Vigor
of V. elliottii Explants Treated on Solid
Modified Knops Medium . . 44

12 Number of in vitro Colonies of V. elliottii
Producing 1 or More Shoots of Increased
Diameter 8 Weeks after Planting with
Colchicine-treated Explants . .. 45

13 Growth Response of 3 Vaccinium Clones to
Treatment for 3 Weeks on Solid Modified
Knops Medium Containing 0.01% Colchicine 46

14 Response of Seed of V. elliottii and
V. darrowi to Colchicine Added to the in
vitro Germination Medium .. . 47

15 In vitro Shoot Formation of V. elliottii
when Cultured on 3 Levels of 2iP in
Modified Knops Medium . . .. 53

16 Frequency of Increased-Diameter in vitro
V. elliottii Shoots Following Treatment
with 0.1% Colchicine, Etiolation and G.A. 55

17 Effect of Gamma Radiation on Regrowth
Vigor of in vitro V. elliottii Explants 60

18 Pollen germination of 12X Colchiploid
V. ashei Ramets . . . 65

19 Fertility of 12X V. ashei Colchiploids
when Crossed with Several Vaccinium Species
of Various Ploidy Levels and Intercrossed
between Cultivars . . ... .67


TABLE


PAGE
















LIST OF FIGURES


FIGURE PAGE

1 Normal diameter in vitro shoots of V.
elliottii (center) and increased diameter
colchiploid shoots left and right ... 24

2 Stomate prints from leaves of an increased-
diameter shoot of V. elliottii (top) and a
regular-diameter shoot (bottom) photo-
graphed at the same magnification (X40) 30

3 Chromosomes of a shoot tip from an
increased-diameter shoot of V. elliottii
(top) and an undoubled shoot of regular-
diameter (bottom) photographed at 100X
magnification . . . 31

4 Flowers from 12X 'Beckyblue' subclone 1 (r.)
and from the undoubled "Beckyblue" (1.)
about 1 day before anthesis . ... 64


vii
















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


IN VITRO PRODUCTION OF COLCHIPLOIDS AND MUTANTS OF NATIVE
FLORIDA DIPLOID Vaccinium, SPECIES AND HYBRIDS AND
EVALUATION OF 12X V. ASHEI COLCHIPLOIDS

By

Julia Lucile Wood Perry

April 1984

Chairman: Dr. Paul Lyrene
Major Department: Horticultural Science

In vitro produced colonies of Florida native diploid

blueberry species Vaccinium elliottii Chapm., V. darrowi

Camp, and their interspecific hybrid were treated with

colchicine to double their chromosome number and facilitate

interspecific hybridization with tetraploid highbush blue-

berry, V. corymbosum Lamarck. In vitro colchiploids,

identified by increased shoot diameter, were confirmed as

tetraploid by stomate guard cell measurement and chromosome

counts from shoot tip squashes.

V. elliottii produced the greatest number of increased-

diameter shoots when 2-node cuttings were treated for

48 hours on a rotary drum in liquid modified Knops medium

containing 0.1% colchicine, or for 2-6 weeks without agita-

tion on solid modified Knops medium containing 0.01% colchi-

cine. V. darrowi produced the greatest number of


viii









increased-diameter shoots when 2-node cuttings were treated

on a rotary drum in .01% colchicine-containing liquid

modified Knops medium for 24 hours. The V. elliottii X V.

darrowi hybrid produced the greatest number of increased

diameter shoots when 2-node cuttings were treated for

4 hours in colchicine-free liquid modified Knops medium on a

rotary drum, apparently due to spontaneous doubling.

The LD-50 of in vitro V. elliottii colonies treated

with gamma radiation was 4.5 krads. No explants receiving a

dose greater than 25 krads survived. Two mutant colonies

were identified from the 1.5 krad treatment and 1 mutant

colony was identified from the 3 krad treatment. These

mutant colonies were dwarfed and feathery in appearance.

The 12X colchiploid ramets of V. ashei cultivars

'Beckyblue' and 'Bluebelle' apparently are of low fertility

as both male and female parents. Pollen germination was low

and pollen tubes weak. No fruit was set when the 12X

cultivars were intercrossed or crossed with 6X V. ashei

'Bonita'. A few fruit were set when the ramets were crossed

with tetraploid V. corymbosum 'Sharpblue' or diploid V.

darrowi, possibly due to accidental outcrossing or selfing.
















CHAPTER I

INTRODUCTION



Blueberry, (Vaccinium species), is native to North

America, Europe and several other parts of the world. Many

wild species exist in the United States, with chromosome

ploidys ranging from diploid to hexaploid. Wild relatives

are valuable sources of locally adaptive characteristics

which would be useful if they could be incorporated into

cultivated species.

Most blueberry acreage in Florida is planted with

hexaploid rabbiteye (V. ashei Reade) cultivars. This is due

to the resistance of V. ashei to Phytopthora cinnamon Rands

root rot, cane canker (Botryosphaeria corticis Demaree and

Wilcox) and various other fungal diseases (74, 75) and to

its low chilling requirement (97), high vigor and high

productivity (45). Highbush (V. corymbosum) cultivars are

also widely planted in the South, but several factors impede

their cultivation in Florida, including fungus suscepti-

bility, higher winter chilling requirement and lack of heat

and drought tolerance. Despite these weaknesses, V.

corymbosum has one important advantage over V. ashei early

ripening. With bloom occurring at approximately the same

date, fruit of early highbush clones mature in 60 days while









it takes early rabbiteye cultivars 80 to 90 days post-

pollination to reach maturity (45, 95).

Florida native blueberry species include diploids

(2N=2X=24) V. darrowi Camp, V. elliottii Chapm., V.

atrococcum Heller, V. arboreum Marsh, and V. staminium L.;

tetraploids (2N=4X=48) V. fuscatum Ait., and V. myrsinites

Lam. and the hexaploid (2N=6X=72) V. ashei (45, 73). In the

section Cyanococcus, which includes all of the above species

except V. arboreum and V. staminium, there are only weak

sterility barriers between species with the same ploidy

level (8, 18). Heteroploid crosses give variable results

ranging from partial success to almost complete failure.

Hybrids derived from heteroploid crosses may exhibit ovule

or pollen sterility (27).

Several of the wild diploid Vaccinium species native to

Florida possess characteristics which could complement the

tetraploid highbush genome. Diploid V. elliottii is adapted

to the drier Florida soil, has high resistance to certain

fungal diseases, high berry flavor, low chilling requirement

and high vigor. V. darrowi has low chilling requirement,

heat and drought resistance, light blue fruit color, attrac-

tive lowbush growth form and some cane canker resistance

(45, 75, 96, 109). Because of a triploid block, few hybrids

are obtained from diploid by tetraploid crosses and their

reciprocals (4).









There are several possible methods for bypassing the

triploid block. These methods include production of

haploids from tetraploid V. corymbosum, rescue of triploid

embryos prior to endosperm failure, enhancement of 2N gamete

production in diploid species, and colchicine treatment of

diploid species to convert them to autotetraploids.

Chromosome doubling of woody perennial species by

colchicine treatment has had limited success (29, 37, 38,

40, 48, 56). Treatment in vitro has several advantages over

traditional methods, including a stable, germ-free environ-

ment with provision of optimum light, nutrients, and

humidity which allows survival of weaker autotetraploids.

Shoots produced in tissue culture are very vigorous and have

short internodes and numerous buds which make them highly

suited for chromosome doubling with colchicine. Tissue

culture is also quite space conservative, allowing large

numbers of shoots to be treated in a small area (53, 82).

Polyploid shoots, characterized in vitro by increased shoot

diameter, can be quickly and easily screened by visual

examination (72).

Mutation has played a large part in the development of

fruit cultivars. Mutation induction in vegetatively propa-

gated species can be valuable in altering a few traits in an

otherwise outstanding cultivar or to induce tissue rearrange-

ments of existing periclinal chimeras (31,41,42).

Determination of the fertility of autoploids is essen-

tial to their use in a breeding program. Not only must the









pollen germinate, it must also have the ability to effect

fertilization and allow fully-developed, fertile seed to be

set.

The purpose of this study was to identify the best type

of explant material for blueberry chromosome doubling by in

vitro colchicine treatment as well as optimal colchicine

concentration and treatment duration for enhancement of

doubling. Additional objectives were to find the optimal

radiation treatment for mutant production, and to evaluate

the breeding value of 12X V. ashei colchiploids.
















CHAPTER II

LITERATURE REVIEW



Polyploidy has been an important feature of plant

evolution, and its value in cultivar breeding has long been

recognized. Possible beneficial changes induced by poly-

ploidy include broader, thicker leaves, larger flowers and

fruit and increased fertility of hybrids not fertile as

diploids (35, 48). The evolution of the genus Vaccinium has

occurred through millions of years and polyploidy has played

a large part in the process (29).

Harlan and DeWet suggest that the most common and

widespread form of spontaneous polyploidy in the higher

plants is probably by 2N + N reproduction (52). This occurs

by a two-step process. The first step consists of fusion of

a 2N female gamete with a normal N male gamete giving rise

to a triploid plant. This triploid plant, in turn, may

produce some cytologically unreduced triploid female gametes

that are fertilized by haploid gametes of the diploid

parents resulting in tetraploid offspring (66). This is a

general phenomenon that probably takes place at a low but

significant frequency in nearly all species of sexual plants

(52). Approximately 75% of all monocot species and 40% of

the dicots are considered polyploid (66).









The methods used to induce polyploidy may be divided

into 2 classes, somatic doubling and gametic doubling. In

somatic doubling, the chromosomes which would normally be

distributed to 2 sister somatic nuclei are included in

1 nucleus. If the diploid plant in which this occurs is not

a hybrid, it gives rise to an autopolyploid, with 4 very

similar sets of chromosomes. If the diploid plant is a wide

hybrid, somatic doubling gives rise to an allopolyploid with

2 pairs of identical chromosome sets (5, 48).

In gametic doubling, the chromosomes which would have

been distributed to the 4 nuclei produced by meiosis are

included in 2 nuclei which give rise to diploid gametes.

Most of the natural polyploids produced by this method have

come from hybrids with low fertility. The zygotes which are

produced generally arise from unreduced gametes, male or

female or both (48).

With the discovery of the polyploidizing action of

colchicine in the 1930's, polyploid induction was no longer

left to nature's whim. A number of other agents, chemical

and physical, have been used to induce polyploidy including

brome-acenaphthene (99), temperature shock (34), severe

pruning to encourage adventitious bud break (5, 56), nitrous

oxide, and podophyllin (62). The greatest success in

polyploid induction, however, has been with colchicine

treatment (3, 11, 21, 34, 35, 40, 46, 48, 72, 80, 89).

Colchicine is a mitotic inhibitor which acts by inter-

fering with the equilibrium between microtubules and their









subunits, preventing the assembly of spindle fibers and

ultimately blocking completion of mitosis (65). Colchicine

in aqueous solution is readily diffusible into plant tissue

and acts over a wide range of concentrations with high

specificity. The cellular reaction to colchicine depends on

several conditions: specific concentration, exposure period,

mitotic stage when contacted by the chemical, cell type and

presence of an environment favorable to mitosis (11).

Colchicine acts only on dividing cells; with no effect on

resting cells (99). The mitotic stage at which colchicine

is most effective in lowest concentration is late prophase

(46); however, the effect of colchicine is not confined to a

limited number of cells at a particular stage of cellular

development. Any cell may be affected if the cell goes

through division while containing the chemical (34, 35). As

long as colchicine remains in treated material above thres-

hold concentration, affected cells will repeatedly fail to

divide at the end of each nuclear division cycle resulting

in multipolyploidy (34). When colchicine is removed, the

chromosomes gather into small groups and loose spindles

appear (46). A common aberration following treatment is

aneuploidy, which seems to be due to partial instead of

total arrest of chromosome division or to a multipolar

division in some of the polyploid nuclei (35).

Chromosome duplication may result in an increase in

nuclear and cell volume. Changes in volume and cell









dimensions are often correlated with changes in size of

plant parts (34). New leaves and stems which grow from

doubled material are usually wrinkled, thicker, darker

green, and coarser in texture. Leaf veins of doubled

material become coarser, leaf hairs and glands become

coarser and more abundant, leaf profile may become shorter

and more rounded and flower buds produce shorter, stockier

pedicels. Similarly, the petals may be stunted and

coarsened in texture or larger and the buds more compact.

In general, a polyploid plant has a more rugged appearance,

looks sturdier and has certain giant-like features. Usually

growth is slower, and the plant is usually shorter than its

original diploid counterpart (5, 34, 100).

Colchicine induction of polyploidy has been successful

with many herbaceous species (34, 48, 100). Woody species,

however, are generally unresponsive to conventional methods

of colchicine treatment. Azalea (89), camellia (3), grape

(47), cranberry (36), pear (56) and chestnut (40) have been

doubled with limited success, while plum, blueberry, cherry,

peach and apple are very difficult species in which to

induce polyploidy (40, 48).

A problem common to both herbaceous and woody species

following colchicine treatment is chimera formation due to

the multicellular nature of the bud apex (12). Chimeras may

be periclinal or sectorial. Growing points are usually made

up of 3 independent cell layers termed L-I, L-II, and L-III.









When only a single layer is affected by colchicine, a

periclinal chimera results. When a portion or sector of all

3 layers is doubled, a sectorial chimera is the result.

Utilization of colchiploids depends upon identification of

plants which produce doubled gametes. These include solidly

doubled plants and plants with a doubled L-II layer. The

anticlinal mode of cell division characteristic of growing

shoot tips is not a permanently fixed condition. Periclinal

division occurs occasionally in cells of the L-I, more

frequently in the L-II and commonly in the L-III histogenic

layer with the result of varying the ploidy level of colchi-

ploid plant layers (39).

Identification of the ploidy level of each layer is

desirable. Measurement of stomatal guard cells of the

epidermis of colchicine-treated versus untreated plants has

been used to identify doubled L-I layers in a number of

species including Vaccinium (1, 19, 20), Bromus (104), and

Triticum (93). Increased intensity of green leaf color of

doubled plants has been found to be associated with the

increased depth of leaf tissue and a proportionate increase

in number of chloroplasts (63). Chloroplast numbers in

stomatal guard cells are also found in increased numbers in

doubled plants and are another measurable indicator of

increased ploidy of the L-I layer in some species (66, 94).

The L-II layer, which gives rise to the gametes, is of

greatest interest to plant breeders. Autotetraploids have

larger pollen grains than their diploid counterparts (33).









These grains may take on characteristic shapes providing a

morphological indication of induced polyploidy (34).

The L-III layer gives rise to various internal tissues.

Chromosome doubling of this layer may be measured by chromo-

some counts from root or shoot tip squashes (5, 56).

Naturally amphiploid species generally do not show

consistent increases in these parameters when compared with

parental species. Genetic factors and the nature of the

parental species may be more influential than the change in

chromosome number (46).

Various methods of increasing the effectiveness of

colchicine treatments have been tested. These include

pretreatment of plant material with gibberellic acid to

accelerate the rate of cell division and cell elongation.

This acceleration is postulated to increase cell perme-

ability to colchicine (110). Etiolation has also been used

to induce cell elongation. Exposure of etiolated shoots to

light causes cell division to occur simultaneously in a

large number of cells making the shoots more susceptible to

the doubling action of colchicine (21, 55). Treatment of

Allium root tips with 20 50 ppm of indole-3-acetic acid

has been shown to induce mitosis in a large number of cells.

Tips were treated for 4 hours and a large number of mitotic

figures were recorded 24 hours later (44).

DMSO (dimethyl sulfoxide) has been used as a carrier

for colchicine, increasing treatment effectiveness by

increasing drug penetration (92). Other factors affecting









treatment success are pH of the colchicine solution (66),

aeration of the solution (2), and treatment temperature

(21).



Cytology

Subfamily Vacciniaceae is a taxonomically ancient group

with many cytologically primitive characteristics. The

basic karyotype of the section Cyanococcus has not evolved

much from the ancestral form characterized by high chromo-

some number and small, metacentric chromosomes (17, 23).

The basic haploid complement of Vaccinium is considered to

be 12 chromosomes (2 long, 8 intermediate and 2 short)

metacentrics) (51). The whole section Cyanococcus in

eastern North America forms a large polyploid complex in

which both autoployploidy and allopolyploidy have played

significant roles (17, 18). There are naturally occurring

diploid, tetraploid and hexaploid species and a few penta-

ploid hybrids (28). According to Camp, all but 2 diploid

species (V. elliottii and V. myrtilloides) have given rise

to polyploids. All species, diploid to hexaploid, have been

found to produce unreduced pollen with an especially high

rate in diploid species (22). These unreduced gametes allow

interspecific heteroploid crosses which would not normally

be possible (9, 27). Members of diploid and tetraploid

groups rarely hybridize but are generally interfertile

within any ploidy group (24). Barriers to natural hybrid

production among homoploids include weak incompatibility









barriers within the style which slow pollen tube growth

(106) and, to a limited extent, differing pollinizers (105).

Cytological studies in Vaccinium have revealed regular

pairing in diploid species (68). An exception to this is a

diploid hybrid between V. myrtillus X V. vitis ideaea which

showed a number of meiotic irregularities due to chromosomal

differences. Each parental species alone was quite regular

in meiosis. An autotetraploid created by doubling the

chromosome number of V. myrtillus displayed a number of

multivalent associations at metaphase I (91).

Vander Kloet postulated that V. corymbosum originated

from an ancient hybrid swarm between V. tenellum X V.

darrowi (107). Bivalents only were found in some clones of

V. corymbosum (60, 68), with multivalence and secondary

pairing indicative of segmental allopolyploidy occurring in

others (59, 83). Trivalents and univalents were rare. The

high frequency of bivalents may be due to the almost com-

plete diploidization of the tetraploid species (60). Most

of the bivalents are involved in pseudo-multivalent associa-

tions. Highbush cultivar 'Jersey' produced bivalents and

quadrivalents in meiosis with 0-50% of the chromosome

complement participating in quadrivalent associations (59,

60). The primary factors governing the type and frequency

of multivalent association in polyploids are chiasma fre-

quency and position, size of chromosomes (61), and genes

that regulate pairing.









Tetraploid V. uliginosum, possibly due to its presumed

recent autotetraploid origin, has a number of meiotic

irregularities. When V. uliginosum was crossed with culti-

vated highbush, meiosis was quite regular due perhaps to

autosyndetic pairing (91).

Hexaploid blueberry species studied by Longley included

V. ashei, V. constablaei and V. amoenum and were generally

found to be meiotically stable (68).

Throughout the plant kingdom, autoploids are almost

without exception less fertile than diploids. This is

probably due to multivalent associations, which result in

unbalanced gametes. Great variations in fertility are found

from species to species, ranging from almost complete

sterility to 75% fertility (87).

Among progenies of alloploids, the first generation may

be quite fertile while later generations have reduced

fertility (46).

In a study where pollen germination was used as an

indicator of fertility of Vaccinium species and hybrids,

tetraploid V. corymbosum pollen germination ranged from

22-49%, tetraploid V. myrsinites pollen averaged 42% germi-

nation and 27% of diploid V. darrowi germinated (49).

Non-functional pollen is characterized by tetrad collapse

and large globules of sporopollenin deposit.

A second fertility indicator is seed set (108). Four

types of seed have been described in blueberry fruit: large,

well-filled seed with brown or tan seed coat; large









flattened or concave brown or tan seed which may be par-

tially shriveled; well-filled brown or tan seed half the

size of class 1; or very small white seed which appear

empty. Only the seeds of the first class are fully devel-

oped and capable of germination (7, 25). Among highbush

cultivars, only 21-52% of the seeds were found to be fully

developed, while 36-55% of open-pollinated rabbiteye seeds

were judged to be capable of germination (79).



Improvement

Blueberry improvement was begun by Coville with a

species cross of wild tetraploid V. corymbosum clone

'Brooks' with V. angustifolium clone 'Russell' (24).

Northern tetraploid cultivars lack several important charac-

teristics which would make them optimal southern cultivars

(56, 73, 75, 77). Problems include a high chilling require-

ment and susceptability to cane canker and root rot fungal

diseases. The characteristics of the Vaccinium species

native to Florida are listed in Table 1. As can be seen in

the table, some native Florida, diploid species are disease

resistant, vigorous, and tolerant to Florida soils and

climate (73, 74, 76). Crosses between these 2 ploidy levels

are unsuccessful due to developmental failure of triploid

seed (4, 91). One way to combine the favorable character-

istics of the 2 ploidys is to equalize their chromosomes

numbers by colchicine treatment of diploid species to

produce autotetraploids (78,87,89).









Table 1

Ploidy Level and Characteristics of Vaccinium
Species Native to Florida


Possible desirable
Ploidy Species characteristics


2N=6X=72










2N=4X=48


2N=2X=24


V. ashei










V. fuscatum



V. myrsinites


V. darrowi


V. elliottii





V. atrococcum


V. arboreum






V. staminium


Chilling requirement between
500 and 700 hours
Heat, disease, insect and
drought tolerant
Very productive with firm
large fruit
Vigorous--3-6m at maturity
Small fruit scar
Late ripening
Adaptive to varied soil type

Evergeen foliage
Drought and heat tolerant
Low chilling requirement

Evergreen foliage
Drought and heat tolerant
Low chilling requirement

Heat, and drought tolerant
Low chilling requirement
Evergreen foliage
Attractive lowbush form
Light blue color

Disease resistant
Tolerant of mineral soils
High berry flavor
Vigorous
Early ripening

Early ripening
Concentrated fruit ripening

Tolerant of high pH soils
Drought tolerant
Late ripening; August through
December
Native south to Manatee and
Hardee Counties, Fl.

Heat and drought tolerant
Low chilling requirement
Large fruit









Species hybridization is important in the transfer of

desirable traits from diploid to polyploid species (30).

Another way to transfer genes from native diploid and

hexaploid species into tetraploid highbush, is to produce

intermediate tetraploids by interspecific hybridization (43,

57, 80).

Several heteroploid crosses have been made to create

introgression bridges with variable results. Hexaploids

crossed with diploids generally produce pentaploids when V.

darrowi is used as the diploid parent (9, 43). Crosses have

also been made between hexaploid V. ashei and diploids V.

tenellum and V. elliottii without much success in tetraploid

production (31, 43). Hybrids have also been made between

hexaploid V. ashei and tetraploid highbush in order to

combine favorable traits from each species. These penta-

ploid interspecific hybrids are readily produced and are

vigorous, hardy, and reasonably fertile when pollinated by

hexaploids or tetraploids (30, 31). Chromosome elimination

during meiosis occurs at a high frequency in blueberry

pentaploids and makes them function primarily as tetraploids

(58). These interspecific functional tetraploids should

cross readily with cultivated highbush to give improved

tetraploid breeding lines. This offers one possibility of

combining the vigor, productivity, and broad soil

adaptability of rabbiteye with the earliness, and quality of

highbush (30, 58).









Tissue Culture

Proliferation in tissue culture can occur in 3 ways:

enhanced formation of axillary shoots, production of adven-

titious shoots, and somatic cell embryogenesis (50, 81).

Plant cell culture itself may generate genetic

variability and may be employed in some cases to enhance

recombination in hybrids. It may also generate variants of

commercial cultivars in a high frequency without hybridi-

zation to other genotypes (64, 111). One consequence of

growth in vitro is the appearance of dividing cells with

chromosome numbers and karyotypes not usually found in

growing points of the intact plant. Aberrant plants are

found at rates that approximate mutation rates in field

grown plants among plants derived from axillary shoots

(101). Aberrations are found at increased rates among

plants grown from adventitious shoots, and are even more

frequent among plants derived from somatic embryos. When

plant cells are allowed to proliferate in callus or suspen-

sion culture, there is a high possibility of chromosomal

variation. Conditions which favor callus formation have

been shown to cause nuclear fragmentation, which produces

spontaneous polyploids, aneuploids, and chromosomal

rearrangements (10, 85, 86, 88, 98).

Another factor affecting in vitro variability is

explant source. Murashige and Nakano found constant

diploidy to be confined to meristem tissue, while a mixture

of diploid and polyploid cells comprised the differentiated









and mature tissues of tobacco. Plantlets derived from

regenerated tobacco pith cells were found to vary widely in

ploidy level from diploid to octoploid (82).

Adventitious buds are known to form in over 350 species

(13, 15). The apex of an adventitious shoot is formed by

one or a few vegetative daughter cells of one original cell,

thus, its ultimate origin is from a single cell (14).

Treatment of explants with mutagenic or polyploidizing

agents should result in more whole-plant changes and fewer

chimeral changes than are normally encountered following

conventional treatments (70), as mutation and adventitious

bud formation are both unicellular events. After colchicine

treatment, the often less-vigorous polyploid cells are

unable to complete with the faster-growing original cells.

If a polyploid cell survives, the end product is often a

mixoploid or cytochimera. Colchicine treatment in combina-

tion with the adventitious bud technique should provide a

high percentage of solid, non-cytochimeral polyploids (12,

13, 54).

Adventitious shoot formation has been observed in V.

corymbosum, V. ashei, V. atrococcum, V. constablaei, V.

darrowi and V. elliottii and their interspecific hybrids.

Adventitious shoots and callus have been observed to form

from the edges of leaves in contact with the nutrient

medium. The callus may, in turn, produce adventitious

shoots (71, 84, 112).





19



The addition of colchicine to in vitro systems for

induction of polyploidy has been utilized in sugarcane cell

suspension (53), daylilly callus (21) and Hordeum callus

(86). Polyploids of tobacco have been identified pheno-

typically in vitro by their smaller, thicker, greener leaves

which were distinctly rosetted and pubescent. Stems were

thicker than on the original plants and internodes were

shorter. The style and stigma were larger and the ability

to produce functional pollen was much reduced. Leaf cells

were found to be larger and an additional 2 layers of spongy

mesophyll were observed. Chromosomal laggards were often

observed in meiotic studies (82).



Radiation Induction of Mutants

Induction of mutants by radiation treatment has its

greatest value in vegetatively propagated species in which

one or a few characteristics of an otherwise outstanding

cultivar may be changed without affecting the remaining

genotype (5, 16, 100). Radiosensitivity varies among plant

species and depends mainly on the nuclear volume (greater

DNA content, more sensitive), chromosome number (plants with

fewer large chromosomes are more sensitive than plants with

more, smaller chromosomes), and ploidy level (higher ploidy

level, less radiosensitive) as well as on the developmental

stage of the plant (most resistant seed to dormant plant to

highly susceptible growing plant) (16).









Mutation breeding in woody perennials has great

promise. The long time lapse between generations, the large

space required to grow a plant to maturity in the field, and

the high degree of heterozygosity make it difficult to

produce cultivars by conventional breeding methods. This is

demonstrated by the unusually high ratio of commercial fruit

cultivars that have originated from spontaneous bud muta-

tions compared to the cultivars produced by cross breeding

(16, 26).

There has been no published work on the radiation

induction of mutation in Vaccinium to date. Because some

Vaccinium species are heterozygous and diploid, some mutants

such as albino or varigated leaves, altered leaf shape, or

dwarfed growth form, should be immediately visible following

mutagenesis and propagation (16).

Tissue culture should be quite valuable in radiation

treatment and mutant identification. Hundreds of bud apexes

are contained within a single culture vial and can be

treated with ease. Treated shoots, used as explant sources,

may regenerate mutant shoots from axillary or adventitious

buds. These mutants can easily be screened by visual exam-

ination of the in vitro colonies.

Adventitious shoots formed after radiation treatment of

irradiated Begonia leaves produced mutants in 30% of the

regenerates, 98.5% of which were non-chimeral (14, 90).

Tissue culture has also been used successfully in conjunc-

tion with radiation treatments with detached leaves of





21



Achimenes, Saintpaulia, Streptocarpus (13), and Crysanthemum

(15).

Gamma radiation has been used in pear to obtain geneti-

cally homogeneous shoots by tissue rearrangement of existing

periclinal chimeras. Radiation-induced tissue rearrange-

ments have also been used to uncover cryptic mutations

hidden within the inner cell layers (32, 41, 42).

















CHAPTER III

IDENTIFICATION OF OPTIMAL TREATMENT
FOR POLYPLOID INDUCTION



Introduction

In vitro colonies of various clones of V. elliottii, V.

darrowi and their interspecific hybrid (69) were tested to

determine optimal explant source, colchicine concentration,

treatment duration and mode of treatment for chromosome

doubling.



Materials and Methods (Experiments 1-6)



Experiment 1

To determine optimal explant material, 2 types of V.

elliottii explants from established in vitro colonies of

Clone 13 were compared. Two-node shoot cuttings were

compared with shoot bases which contained scores of axillary

buds as explant sources. Colchicine (0.1%) was dissolved in

liquid modified Knops medium (103). A wheel drum rotating

at about 3 rpm was used to aerate the medium in the

treatment vials. Treatment durations tested were 24, 48,

72, and 120 hours. After the treated explants had been

rinsed in liquid, colchicine-free modified Knops medium for









1 hour on the wheel drum, five 2-node cuttings were placed

in each of 10 new vials containing solid modified Knops

medium from each treated vial. After 45 days, the colonies

were rated for survival and screened for shoots of

increased-diameter, an indicator of increased ploidy (72)

(Fig. 1). Leaf casts were made from these shoots and

stomate measurements taken.

Shoots of increased diameter screened from the colonies

were severed from the shoot bases and rooted in a greenhouse

under mist. These subclones were labeled and planted in a

field nursery. Leaves and buds were collected from each

subclone the following spring for ploidy determination.

Five leaves were collected from each subclone and each leaf

collected was the third leaf from the apical meristem. Leaf

prints were made by brushing fingernail polish on the

abaxial leaf surface and removing the cast. The casts were

examined microscopically and fifteen stomatal guard cells

were measured per leaf cast.

An average of 10 axillary and apical buds were col-

lected from each subclone. After fixing in 1:1 absolute

ethanol: glacial acetic acid for 24 hours, the buds were

stripped of leaves and softened in an enzyme solution of

0.03 g cellulysin, and 0.03 g pectinase in 5 ml distilled

water for 15 minutes. After rinsing, the buds were squashed

in a drop of 1% acetocarmine dye. The material was

destined with 45% acetic acid to facilitate phase contrast

examination of the chromosome number.




















































Figure 1. Normal diameter in vitro shoots of V. elliottii
(center) and increased diameter colchiploid
shoots (left and right).









Experiment 2

To determine optimum treatment duration for treatment

with 0.1% colchicine, 2-node cuttings of V. elliottii Clone

10, V. darrowi Clone 5, and the interspecific V. darrowi X

V. elliottii hybrid from established in vitro colonies were

treated in liquid modified Knops medium on a rotary wheel

for durations of 0, 4, 12, 24, and 48 hours. After rinsing,

19 vials were planted from each treatment with three 2-node

cuttings on solid modified Knops medium. After 45 days

regeneration, the colonies were rated for survival, vigor

and increased shoot diameter.

The increased diameter shoots of V. elliottii

screened from this experiment were planted on 3 media (6,

67, 103) to determine if shoot production could be enhanced.

Vials containing each medium were planted with each of

30 increased diameter subclones. The components of the

3 media used in this experiment are listed on Table 2.

Growth response on each medium was recorded 30 days after

subculture.



Experiment 3

To determine the optimal colchicine concentration/

treatment duration combination, 2-node cuttings from estab-

lished in vitro colonies of V. elliottii, V. darrowi and

their hybrid were tumbled on a rotary drum (3rpm) in colchi-

cine concentrations of 0.00, 0.01, 0.05, 0.10 or 0.20%









Table 2

Composition of Modified Woody Plant Medium, Modified
Andersons Medium, and Modified Knops Medium


Modified Modified
woody Andersons Modified
Compound plant medium medium Knops medium


NH NO3
KNO3
K SO4
K2SO4
KH2PO4
Ca(NO3) 4H20
CAC1I 2H20
MgSO4 7H20
NaH2PO H20
Na EDTA
FeSO 7H2 0
MnSo4 H20
ZnSO 7H 0

H3BO3
Na2MoO4 2H20
CuSO4 5H20
KI
CoC12 6H20
Pyridoxine HC1
Thiamine CH1
Nicotinic Acid
Myo-inositol
Adenine Sulfate
Glycine
Casein Hydrolysate
Sucrose
Agar
2ip


(mg/liter)
400


990
170
556
96
370


74.5
55.6
22.3
8.6
6.2
0.25
0.25



0.5
1.0
0.5
100


2.0
1,000
30,000
4,000
5


(mg/liter)
400
480


(mg/liter)


190


170
1,140


440
370
380
74.5
55.6
16.9
8.6
6.2
0.25
0.025
0.83
0.025


0.4


100
80


1,000
30,000
4,000
5


370


74.5
55.6
22.3
8.6
6.2
0.25
0.025
0.83
0.025
0.5
0.1
0.5
100


2.0
1,000
30,000
4,000
5


pH adjusted to 5.7 with IN NaOH. Autoclaved at 1.05 Kg/cm2
for 15 minutes.









for durations of 6, 12, 24, 48 or 72 hours. A composite of

clones was treated for each taxon. For each treatment

combination, 10 vials containing modified Knops medium were

planted with five 2-node cuttings. Survival, vigor and

frequency of increased-diameter shoots were recorded for

each taxon after 45 days.



Experiment 4

Two-node cuttings of V. elliottii, V. darrowi and their

hybrid were placed on solid modified Knops medium containing

0.00, 0.01, 0.05, 0.10, or 0.20% colchicine to determine

optimum colchicine concentration for production of poly-

ploids. After 2, 4, 6, or 8 weeks of treatment, the cut-

tings were rinsed and replanted on solid modified Knops

medium for regeneration. After 45 days, the colonies were

evaluated for survival, vigor, and increased-diameter

shoots.



Experiment 5

Two-node cuttings from established colonies of 2 clones

of V. elliottii (Clones 121 and 133) and 1 clone of V.

darrowi (Clone 5) were placed on solid modified Knops medium

containing 0.01% colchicine for 3 weeks. The cuttings were

rinsed and placed on modified Knops medium. Ten vials were

planted from each treated vial. After 60 days the colonies

were rated for survival and frequency of increased-diameter

shoots.









Experiment 6

Seeds of V. elliottii and V. darrowi collected from

open-pollinated wild stands in Florida were placed on solid

water/agar medium containing 0.00, 0.10 or 0.25% colchicine

to determine seed tolerence and rate of polyploidization.

Sixteen vials of each treatment were planted with 5 plump

seeds of each species. As soon as germination occurred, the

seeds were rinsed and transferred to solid modified Knops

medium for colonization.



Results and Discussion (Experiments 1-6)



Experiment 1

Comparison of survival rates for the two explant

sources showed a linear decrease in regrowth vigor with

increasing duration of colchicine treatment in both treated

cuttings and shoot bases. Although the 2 explant types did

not differ significantly in survival after treatment, there

was a significant difference between explant sources in

frequency of shoots with increased-diameter (chi-square

probability = .05). The treated cuttings gave rise to

12 vials containing shoots of increased diameter, whereas

treated bases produced none. Nine of the 12 vials

containing increased-diameter shoots were regenerated from

the 48 hour treatment, with the remainder regenerated from

the 72 hour treatment. For cuttings, the 48 hour treatment









gave significantly more increased-diameter shoots than

treatments of 24, 72 or 120 hours (chi-square probabi-

lity = .05).

Stomate measurements of in vitro leaves from increased-

diameter shoots and normal-diameter shoots were not corre-

lated with stomate measurements of the same cuttings later

transplanted to the field nursery. Screening for increased

stomate size as an indicator of increased ploidy is, there-

fore, not recommended at the early in vitro stage.

Stomate guard cell measurements of leaves and chromo-

some counts of shoot tips from greenhouse rooted thick

shoots supported the correlation found earlier between

increased stem diameter and induced polyploidy (Figures 2,

3). Seventy percent of the shoots examined had increased

stomatal guard cell lengths as well as higher ploidy levels

(Table 3). Stomates of Subclone C were significantly larger

than diploid or average autotetraploid stomate lengths.

This could be due to a periclinal chimera (L-I = 8X?

L-III = 4X). If adventitious bud formation were encouraged

by in vitro culture of leaves, solid octoploid plants

derived from colchicine-treated diploids are possible. Some

of the shoots with increased-diameter also showed other

morphological anomalies, such as increased and coarse

appearing pubesence, darker green, thicker leaves or altered

leaf shape. In vitro growth rate was also decreased for

large-diameter shoots.























































Figure 2. Stomate prints from leaves of an increased-
diameter shoot of V. elliottii (top) and a
regular-diameter shoot (bottom) photographed at
the same magnification.
























































Figure 3. Chromosomes of a shoot tip from an increased-
diameter shoot of V. elliottii (2N=ca 48) (top)
and an undoubled shoot of regular-diameter
(2N=24) (bottom) photographed at the same
magnification.














Table 3

Ploidy Level and Stomate Length of 10 in vitro
Subclones of V. elliottii Selected for
Large Shoot diameter in Experiment 1


Stomate length (pm)

Subclone Ploidy levelz Meany Range SD


A 24 104 92-115 11

F 24 115 92-115 15

K 24 118 92-138 12

G 48 172 161-184 12

E 48 173 161-207 12

J 48 181 161-207 14

L 48 184 161-207 15

H 48 187 161-207 12

D 48 189 161-230 15

C 48 240 207-276 14


Counts were determined by shoot tip squashes and are
approximate.

YAverage derived from measurement of 15 stomates per
subclone.









Since the treated plant material has yet to flower,

assessment of the L-II or gamete-producing layer has yet to

be made. Chimeras are common problems in colchicine-

treated plant material; therefore, total assessment of

increased-diameter shoots is not yet complete. Two of the

3 increased-diameter shoots found to be diploid by chromo-

some and stomate measurements did flower, and pollen

measurements fall within the normal range for diploid V.

elliottii.

Aberrant and weak growth was observed in several

increased diameter subclone populations following 1 year of

growth in the field nursery. Colchicine-induced aneuploidy

is a possible explanation for this altered morphology.

Vaccinium chromosomes are quite numerous and small, there-

fore exact counts are not always possible. The chromosome

numbers reported in Table 3 are nearest approximations.



Experiment 2

V. elliottii produced both the greatest total number of

shoots and the greatest number of shoots of increased-

diameter (Table 4). Most of the increased-diameter colonies

regenerated from the 4 hour treatment and several from the

12 hour treatment succumbed to fatal decline syndrome

(Table 5). Fatal decline is characterized by strong initial

growth which ceases when the shoots are 3-5 cm. in height.

A few shoots initially take on a water-soaked appearance,

and this subsequently spreads throughout the colony. Only

colchicine-treated shoots of increased-diameter have been













Table 4

Growth Response of V. elliottii Following Treatment
with 0.1% Colchicine for Various Durations



Surviving Shoots
Treatment Normal- Increased- Increased-
duration Total diameter diameter diameter
(hrs) (no.) (no.) (no.) (%)


0 415 415 0 0

4 410 105 5 1

12 270 181 59 22

24 133 67 66 50

48 135 51 84 62

Chi-square = 337.40

*
Indicates that the ratio of normal-diameter: increased-
diameter shoots varied with duration of colchicine
treatment.














Table 5

Frequency of Fatal Decline Syndrome Following in vitro
Treatment of V. elliottii with 0.1% Colchicine
on a Rotary Drum for Various Durations



Normal-diameter Fatal decline
Treatment duration shoots shoots
(hrs) (no.) (no.)


0 415 0

4 105 300

12 181 30

24 67 0

48 51 0
*
Chi-square = 643.37


Indicates that the ratio of normal-diameter shoots: fatal
decline shoots varied with duration of colchicine
treatment.









observed to be afflicted. Affected colonies were examined

by pathologists, and no fungi or bacteria were found to be

associated with the decline symptoms. The decline is

hypothesized to be physiological. Perhaps the decline

results when some colchicine-induced increased diameter

shoots are altered in their response to some medium compo-

nent. These shoots may be unable to correctly metabolize

some nutrient or they may convert some component of the

medium into a toxic byproduct.

The 30 increased-diameter subclones of V. elliottii

planted on the 3 media varied in growth response and fre-

quency of fatal decline syndrome (Table 6). There is an

obvious interaction between media and frequency of produc-

tion of very vigorous colonies and colonies with fatal

decline. Woody plant medium produced both the greatest

number of colonies afflicted with fatal decline syndrome and

the most very vigorous colonies. Knops and Andersons media

were equal in ability to produce vigorous or average colo-

nies. Very few very vigorous or fatally declined colonies

were produced on these 2 media.

Increases in callus growth and decreases in shoot

production were observed in both V. darrowi (Table 7) and

the interspecific hybrid (Table 8) colonies with increased

colchicine treatment duration. Callus can originate from

leaves, stems, or buds. If the apical and axillary buds

succumb to the phytotoxic effect of colchicine, callus










Table 6

Growth Response of Increased-Diameter Shoots of V. elliottii
when Placed on 3 in vitro Media for Regeneration


Medium


Number of colonies which produced growth that was
> 20 shoots 10-20 shoots 1-10 shoots


Andersons 3

Knops 4

Woody plant 17

Chi-square = 46.49


Number of vials which produced colonies of
Healthy growth Fatal decline syndrome


Medium


Andersons 29 3

Knops 43 2

Woody plant 20 16
**
Chi-square = 23.65


Indicates interaction between medium and colony vigor.

**
Indicates interaction between medium and frequency of
fatal decline syndrome.










Table 7

Growth Response of V. darrowi after Treatment of
2-node Cuttings from in vitro Colonies
with 0.1% Colchicine for Various Durations



Treatment
duration Total Normal- Increased-
(hrs) shoots diameter diameter


169

163


159

141


Chi-square = 4.92 N.S.


Treatment Number of vials which produced
duration Total Callus
(hrs) vials only Shoots


0 19 0 19

4 19 1 18

12 19 5 14

24 19 4 15

48 19 11 8
**
Chi-square = 22.86


Indicates non-significant interaction.


Indicates that the ratio of callus: shoot production
varied with duration of colchicine treatment.









Table 8

Growth Response of V. darrowi X V. elliottii in vitro
2-node Cuttings to Treatment with 0.1%
Colchicine for Various Durations



Treatment
duration Total Normal- Increased-
(hrs) shoots diameter diameter


102


102


Treatment Number of vials which produced
duration Total Callus
(hrs) vials only Shoots


0 19 1 18

4 19 3 16

12 19 6 13

24 19 12 7

48 19 14 5

Chi-square = 28.36


Indicates that the ratio of callus: shoot production
varied with colchicine treatment duration.









proliferation, characteristic of these taxons in vitro, may

still occur.



Experiment 3

Survival rate of explants of the 3 Vaccinium taxons

treated with various concentrations of colchicine for

various durations in liquid medium on a rotary drum

decreased with increasing colchicine concentrations

Table 9). V. elliottii had the highest overall survival

rating and the highest frequency of increased-diameter

shoots (Table 10). Optimal colchicine concentrations and

treatment durations for each of the 3 taxons for polyploid

induction in this experiment were: V. elliottii--0.01% for

72 hours; V. darrowi--0.01% for 48 hours; V. darrowi X V.

elliottii--0.00% for 6 hours. Spontaneous chromosome

doubling in V. darrowi and V. darrowi X V. elliottii

apparently was responsible for the appearance of

large-diameter shoots in the 0.00% colchicine treatments.



Experiment 4

Higher colchicine concentration and longer treatment

duration on solid colchicine containing medium were both

correlated with decreased survival of 2-node cuttings

(Tables 11 and 12). Several regenerated colonies contained

shoots of increased diameter. Colchicine concentration of

0.01% for 2-6 weeks was the most successful treatments for

production of increased-diameter shoots in V. elliottii.










Table 9

Effects of Colchicine Concentration and Treatment Duration
on Regrowth Vigor of Treated Vaccinium Explants



Colchicine Regrowth vigorz
concn. Duration (hrs)
(%) 6 12 24 48 72 Mean


0.00 2.6

0.01 2.3

0.05 1.7

0.10 1.8

0.20 1.6

Mean 2.0

Vigor Score = 1.85 -

r = .56**


0.00

0.01

0.05

0.10

0.20

Mean


3.0

2.4

3.1

2.8

1.4

2.5


V. darrowi

1.4 2.1

1.2 1.2

0.7 1.0

0.6 1.0

0.1 0.9

0.8 1.2

5.13 concn.


V. elliottii

2.2 1.7

2.1 2.6

1.3 1.3

1.7 1.2

0.8 0.2

1.6 1.4


Vigor Score = 2.66 7.61 conc.


- .01 hours


r = .68*


Continued


2.9

0.9

1.0

0.2

0.1

1.0


2.2

1.5

1.2

1.0

0.8


1.8

1.7

1.6

1.4

1.5

1.6


2.6

3.1

1.8

1.2

1.7

2.1


2.1

2.8

0.4

0.2

0.6

1.2


2.3

2.6

1.6

1.4

0.9










Table 9

Continued


Colchicine Regrowth vigorz
concn. Duration (hrs)
(%) 6 12 24 48 72 Mean



V. darrowi x V. elliottii

0.00 2.4 1.7 2.7 1.7 1.6 2.0

0.01 1.8 1.4 2.6 0.8 0.7 1.5

0.05 1.6 2.1 0.7 0.1 0.0 0.9

0.10 1.8 0.8 0.7 0.9 0.0 0.7

0.20 0.7 0.9 0.6 0.9 0.0 0.4

Mean 1.7 1.4 1.5 0.5 0.5

Vigor Score = 2.21 6.78 concn. .02 hrs.
**
r = .81


ZMean vigor score for 10 vials, each planted with five
2-node explants receiving this treatment (4 = very
vigorous; 0 = dead).

YIn these equations, concn. is expressed in parts per
*hundred.
Slope of regression line significantly different from
0.0 at 5% level;
Slope of regression line different from 0.0 at % level.
Slope of regression line different from 0.0 at 1% level.





43



Table 10

Number of Colonies Containing 1 or More Shoots of Increased-
Diameter Following Treatment with Various Colchicine
Concentrations for Various Lengths of Time.z



Colchicine
concn. Treatment duration (hrs)
(%) 6 12 24 48 72 Total


V. darrowi


0.00
0.01
0.05
0.10
0.20
Total


V. elliottii


0.00
0.01
0.05
0.10
0.20
Total


V. darrowi x V. elliottii


0.00
0.01
0.05
0.10
0.20
Total


ZFor each treatment, 10 vials were used, each planted with
five 2-node cuttings.














Table 11

Effects of Colchicine Concentration and Treatment Duration
on Regeneration Vigor of V. elliottii Explants
Treated on Solid Modified Knops Medium



Colchicine Regeneration vigor
concn. Treatment duration (wks)
(%) 2 4 6 8 Mean


0.00 3.5z 3.7 3.9 4.0 3.8

0.01 3.3 1.5 2.5 1.2 2.1

0.05 1.7 0.9 0.7 0.1 0.9

0.10 1.3 0.1 0.4 0.0 0.5

0.20 0.2 0.1 0.0 0.1 0.1

Mean 2.0 1.3 1.5 1.1

Vigor Score = 3.16 14.87 concn. 0.13 wks.


Mean vigor score for 10 vials planted with five 2-node
explants receiving this treatment (4 = very vigorous;
0 = dead).

Slope of regression line different from 0.0 at 5% level.





45



Table 12

Number of in vitro Colonies of V. elliottii Producing 1 or
More Shoots of Increased Diameter 8 Weeks after
Planting with Colchicine-treated Explants



Colchicine No. vials with large diameter shoots
concn. Treatment duration (wks)
(%) 2 4 6 8 Mean


0.01 6 2 2 0 2.5

0.10 2 0 0 0 0.5

Mean 4 1 1 0

ZOf 10 total vials. Five 2-node treated explants were
planted per vial.










Experiment 5

The 2 clones of V. elliottii and 1 clone of V. darrowi

treated for 3 weeks with 0.01% colchicine on solid modified

Knops medium produced increased-diameter shoots. Three

vials of V. elliottii Clone 121 and 9 vials of V. elliottii

Clone 133 contained shoots of increased-diameter. Seven

vials of V. darrowi Clone 5 were found to contain increased-

diameter shoots (Table 13). There was a clonal as well as a

species difference in response to colchicine treatment and

induction of polyploidy.


F_












Table 13

Growth Response of 3 Vaccinium Clones to Treatment
for 3 Weeks on Solid Modified Knops Medium
Containing 0.01% Colchicine


Increased-
Total Normal- Increased- diameter
shoots diameter diameter shoots
Explant source (no.) (no.) (no.) (%)


V. elliottii -
Clone 121 70 62 8 11

V. elliottii -
Clone 133 290 100 190 65

V. darrowi -
Clone 5 41 34 7 19

Chi-square = 87.21


Indicates that the ratio of normal-diameter: increased-
diameter shoots varied with the clone treated.


Experiment 6

Although several seeds germinated from each treatment,

none of the seedlings which were germinated on medium

containing colchicine survived (Table 14). The long time

required for seed germination may have allowed high

concentrations of colchicine to be absorbed, resulting in

phytotoxicity.









Table 14

Response of Seed of V. elliottii and V. darrowi to
Colchicine Added to the in vitro Germination Medium



Colchicine
Explant cone. Total Number of seed that
source (%) seed germinated survived


V. elliottii 0.00 80 24 24

0.10 80 7 0

0.25 80 9 0


V darrowi 0.00 80 26 23

0.10 80 6 0

0.25 80 6 0


Conclusions



Tissue culture appeared useful as a vehicle for

colchicine treatment. Shoots with increased-diameter could

easily be screened by visual examination. The space

conservative feature of in vitro culture allowed treatment

of a great number of rapidly growing shoot tip cuttings,

which enhanced the probability for success. Doubled shoots

could be rapidly cloned in vitro allowing large numbers of

autotetraploid ramets to be grown. This is quite important

because many induced polyploids die before they flower.









The best type of explant to treat is 2-node cuttings.

The optimal colchicine concentration/treatment duration

combination for induction of polyploids varies with species

as well as clone. V. elliottii 2-node cuttings produced the

greatest number of increased-diameter shoots when treated

for 48 hours on a rotary drum with 0.10% colchicine dis-

solved in liquid modified Knops medium. When explants were

treated on solid modified Knops medium, colchicine at 0.01%

for 2-6 weeks was the most successful treatment combination

for this species.

Both V. darrowi and V. darrowi X V. elliottii clones

produced increased-diameter shoots without addition of

colchicine to the treatment medium. Spontaneous doubling

exceeded colchicine-induced doubling in the interspecific

hybrid and was equal in production of increased-diameter

shoots to treatment for 48 hours with 0.01% colchicine in

liquid modified Knops medium. Colchicine is apparently

unneccessary for induction of polyploidy in the clones of

the 2 taxons tested.

Due to the clonal variation in response to the doubling

action of colchicine, it is advisable to treat many clones

of a species. Treatment of many clones is also beneficial

for obtaining a broad gene base and diverse breeding lines.

















CHAPTER IV

ENHANCEMENT OF COLCHICINE ACTION



Introduction

Once optimal in vitro colchicine concentration and

treatment duration are established for a taxon, polyploids

may be easily produced although at a variable frequency.

Various researchers have proposed a variety of techniques to

reduce the phytotoxicity of the colchicine treatment or to

increase the number of cells which are susceptible to the

alkaloid.



Materials and Methods (Experiments 7-11)

Experiment 7

DMSO (dimethyl sulfoxide) is used to increase penetra-

tion of any substance in solution with it. Colchicine at a

concentration of 0.20% was used alone and in combination

with 1.0% DMSO in liquid modified Knops medium. Three shoot

bases of V. elliottii were tumbled on a wheel drum (3 rpm)

in each solution for 7 days. The treated shoot bases were

rinsed in liquid modified Knops medium for 24 hours and

divided to plant 45 vials of each treatment on solid modi-

fied Knops medium. After 30 days, the colonies were rated









for survival, vigor and presence of increased-diameter

shoots.



Experiment 8

The cytokinin 2iP (6-gamma-gamma-dimethyl-allyl amino

purine) encourages axillary bud break and formation of

numerous meristems (81). Young, actively growing meristems

are known to be ideal material for colchicine treatment.

The purpose of this study was to determine the effect of

increased levels of 2iP incorporated into the medium both

before and after colchicine treatment.

Two-node cuttings of V. elliottii Clone 7 from estab-

lished in vitro colonies were tumbled in liquid modified

Knops medium alone or with 0.1% colchicine. After 48 hours,

the cuttings were rinsed and planted on modified Knops

medium containing either 5, 20, or 40 mg/l of 2iP. After

8 weeks, the colonies were transferred to modified Knops

medium containing 5 mg/l of 2iP.

In addition, various concentrations of 2iP (5, 20 or

40 mg/1) were incorporated into the medium on which seed-

lings of V. elliottii were planted. The resultant colonies

were tumbled in 0.10% colchicine dissolved in modified Knops

medium for 48 hours on a rotary drum (3 rpm). The treated

colonies were rinsed and planted on solid modified Knops

medium containing 5 mg/l of 2iP. After 60 days, the

colonies were scored for survival and frequency of

increased-diameter shoots.









Experiment 9

Gibberellic acid has been used in combination with

etiolation to improve colchicine effectiveness in woody

plants (110).

Gibberellic acid concentrations of 10 or 100 ppm were

used to treat colonies of V. elliottii Clone 4 with or

without a 3 day etiolation period. After 3 days the etio-

lated colonies were returned to the light and on the fifth

day, all vials were treated with 0.10% colchicine for

48 hours on a rotary drum (3 rpm). Eighteen vials were

planted from each treatment on solid modified Knops medium.

After 45 days, the colonies were rated for survival and

frequency of increased-diameter shoots.



Experiment 10

The purpose of this experiment was to determine if

colchicine treatment administered before a cold temperature

shock would reduce colchicine phytotoxicity or enhance

polyploid production. Six shoot bases of V. elliottii

Clone 131 were placed in 0.1% colchicine dissolved in liquid

modified Knops medium and tumbled on a rotary drum (3 rpm)

for 96 hours. After rinsing, the shoot bases were placed on

modified Knops medium: 2 at room temperature, 2 in a refrig-

erator at 7 degrees C. for 48 hours and the remaining 2 at

7 degrees C. for 72 hours. After treatment, each shoot base

was divided and 30 vials were planted from each treatment.









After 60 days, the colonies were evaluated for vigor and

increased-diameter shoots.



Experiment 11

Auxins are known to induce cell mitosis (44). The

purpose of this experiment was to determine the effects of

various combinations of colchicine treatment, etiolation and

auxin treatment. Several colonies of V. elliottii Clone 10

were etiolated for 3 days, then returned to the light for

1 day. Colchicine at 0.10% was dissolved in liquid modified

Knops medium alone or with 1 or 10 mg/l of the auxin,

indole-3-butyric acid (IBA), and used to treat 2-node

etiolated or non-etiolated cuttings from established in

vitro colonies on a rotary drum (3 rpm) for 48 hours. After

rinsing, the cuttings were placed on modified Knops medium.

Fifteen vials were planted from each treatment combination,

and the resultant colonies were rated for vigor and fre-

quency of increased-diameter shoots after 60 days.



Results and Discussion (Experiments 7-11)



Experiment 7

The combination of DMSO and colchicine was quite

phytotoxic. Thirty days after treatment, 41 of 45 colonies

treated with both colchicine and DMSO were dead and 45 of

45 were dead after 60 days. Twenty-eight of the 45 colonies

treated with colchicine alone were dead after 30 days and









32 of 45 were dead after 60 days. Of the surviving 13 colo-

nies, none regenerated shoots of increased-diameter.

Although the DMSO did increase the frequency of dead colo-

nies, the high concentration of colchicine (0.20%), the long

duration of treatment (7 days) and the use of unresponsive

shoot bases as explants all were probably contributing

factors to the death rate.



Experiment 8

Growth of colonies on modified Knops medium with 20 ppm

2iP resulted in an increased number of shoots per colony

(Table 15). Colony height was decreased with increased

cytokinin concentration due to shortened internode length.

The 40 ppm 2iP treatment produced a moss-like colony of







Table 15

In vitro Shoot Formation of V. elliottii when Cultured
on 3 Levels of 2iP in Modified Knops Medium



mg/l of 2iP Number of shoots


5 57

20 148

40 95


From total of 10 vials per treatment.









shoots. Fewer shoots were apparent per colony because the

shoots were so compact that they were indistinguishable from

callus.

Due to a high incidence of contamination following

colchicine treatment, frequency of polyploid formation could

not be meaningfully assessed in this experiment. The

increased number of treatable shoot meristems formed with

increased concentration of 2iP would seem to favor increased

production of polyploids.

Placement of 2-node cuttings following colchicine

treatment on media containing various levels of 2iP did not

increase the frequency of increased-diameter shoots over

what was obtained with 2iP at 5 mg/l. No increased-diameter

shoots were detected in the treated colonies in any of the

treatments. Since the clones used in this experiment

differed from those used in experiments that yielded

increased-diameter shoots, possible clonal variation in

susceptibility to the polyplodizing effect of colchicine is

suggested.



Experiment 9

The use of 0.1% colchicine alone and in combination

with 10 ppm or 100 ppm gibberellic acid (G.A.) and/or

etiolation in all combinations resulted in increased-

diameter shoots in all treatments (Table 16). The non-

etiolated, 0 G.A. treatment was as successful in the














Table 16

Frequency of Increased-Diameter in vitro V. elliottii
Shoots Following Treatment with 0.1% Colchicine,
Etiolation, and Gibberellic Acid



Total Normal- Increased Increased-
shoots diameter diameter diameter
(no.) (no.) (no.) (%)


0 ppm G.A. 54 15 39 72

10 ppm G.A. 41 22 19 46

100 ppm G.A. 95 39 56 59

0 ppm G.A. +
etiolation 90 50 40 44

10 ppm G.A. +
etiolation 91 51 40 44

100 ppm G.A. +
etiolation 118 85 33 28

Chi-square = 36.97


Indicates that the ratio of normal-diameter: increased-
diameter shoots varied with treatment.









production of increased diameter shoots as any other treat-

ment combination. Therefore, colchicine treatment of

non-etiolated 2-node cuttings is recommended without G.A.



Experiment 10

The cold shock administered after colchicine treatment

of in vitro shoot bases of V. elliottii did not decrease

phytotoxicity or increase polyploid induction. Increased

refrigeration resulted in increased senescence of shoot

bases. No increased-diameter shoots were identified from

any of the treatments.



Experiment 11

The combination of etiolation and auxin resulted in

increased senescence. The addition of auxin to the treat-

ment medium increased callus formation. After 120 days of

regrowth following treatment, the callus had still not

regenerated shoots. The cultures were monitored until the

medium was depleted, and were then discarded.



Conclusions

None of the enhancement techniques tried was more

successful than the standard procedure. Thus, the recom-

mended technique for inducing polyploidy in the Vaccinium

taxons studied is to treat 2-node cuttings with 0.1%

colchicine dissolved in modified Knops medium. The cuttings





57



should be tumbled on a rotary drum for 48 hours, rinsed, and

replanted on solid modified Knops medium for regeneration of

colonies.

















CHAPTER V

RADIATION STUDIES



Introduction

Mutation induction in vegetatively propagated crops can

be valuable in altering a few traits in an otherwise out-

standing cultivar. Perennial fruit crops with a long

juvenile stage are excellent candidates for this treatment.

In order to treat a species most effectively a survey of

radiation tolerence must be accomplished.

The purpose of this study was to estimate the LD-50 for

in vitro gamma irradiation of diploid V. elliottii colonies

and to screen colonies regenerated from treated cuttings for

mutant phenotypes.



Materials and Methods

Twenty colonies of Clone 7 of V. ellottii growing on

modified Knops medium were subjected to doses of gamma

radiation ranging from 0.5 to 50 kilorads (krads). Each

vial contained 30-40 miniature shoots, each with 10-15

axillary buds. V. elliottii is a highly heterozygous

diploid (2N=24) species.

Immediately after treatment, each colony was divided

into 10 subcultures of five 2-node cuttings each and









replanted on solid modified Knops medium. After 60 days of

regrowth, the colonies were rated for survival.



Results and Discussion

An LD-50 of 4.5 krads was determined for the treated

colonies of V. elliottii. The highest dose which produced

living colonies was 25 krads. There was a correlation

between increasing radiation dosage and decreasing survival

(Table 17).

Three colonies (2 from the 1.5 krad treatment and

1 from the 3 krad treatment) appeared to have an altered

morphology. In vitro growth of all 3 altered colonies was

feathery and dwarfed in appearance compared to the usual

small-leaved vigorous growth typical of the other colonies.

Shoots from the altered colonies and from control

colonies were severed from the shoot bases and rooted in a

greenhouse under mist. About 20 rooted plantlets from each

mutant colony and the control were transplanted to a field

nursery for observation. The feathery-leaved appearance was

not retained in the field. After 1 year in the nursery,

differences were noted in leaf color in 1 of the 2 mutants

screened from the 1.5 krad treatment. Leaves of this mutant

were yellow-green in appearance in contrast with the darker

green of normal V. elliottii in the same nursery. Leaves of

the mutant became darker green as they matured, but remained

paler than leaves of normal plants. The mutant plants were

less vigorous than comparable non-mutants, and several died










Table 17

Effect of Gamma Radiation on Regrowth Vigor
of in vitro V. elliottii Explants



Krads Vigorz


0.5 4.0

1.0 4.0

1.5 3.2

2.0 1.0

2.5 2.5

3.0 3.2

3.5 2.6

4.0 1.2

4.5 1.1

5.0 1.7

6.0 0.6

7.0 0.1

8.0 0.0

9.0 0.0

10.0 0.2

15.0 0.2

20.0 0.2

25.0 0.5

30.0 0.0

50.0 0.0


Mean vigor score 10 vials (4 = very vigorous; 0 = dead).









during the first year. The other 2 mutants appeared normal

after 1 year of growth in the field.



Conclusions

The combination of in vitro plant material and

mutagenesis has great potential. Tissue culture colonies

contain a high concentration of potentially mutable buds and

may also allow mutated cells which would otherwise not

survive to be expressed. Once the range of radiation dosage

is established for a species, mutations should be easily and

readily induced. Only visually apparent mutants were

screened from the treated colonies as indicators of optimal

dosage. It is recognized that all favorable mutants are not

visible.
















CHAPTER VI

BREEDING BEHAVIOR OF 12X V. ASHEI COLCHIPLOIDS



Introduction

V. ashei (2N=6X=72) and V. corymbosum (2N=4X=48) each

possess valuable characteristics, and production of the

interspecific hybrid could be useful. Direct crosses

between the species result in 5X plants which have so far

been of little value in further breeding. In hybridizing

the two genomes, various chromosome manipulations may be

helpful.

Colchicine doubling of 6X V. ashei would result in

12X autopolyploids. When crossed with 4X V. corymbosum,

8X hybrids might be produced. These 8X's could then be

backcrossed to the 4X corymbosum, in an effort to produce

6X plants. If this 6X hybrid required more V. ashei charac-

teristics, they could be introgressed into the hybrid genome

by backcrossing to V. ashei.



Materials and Methods

Three doubled plants of V. ashei were regenerated from

tissue culture treatment with colchicine. Two plants were

derived from the cultivar 'Beckyblue', and one from the

cultivar 'Bluebelle'. Crosses were made between the 12X









plants using each cultivar both as male and female parent.

In addition, the 12X plants were used as male parents in

crosses with 6X V. ashei cultivar 'Bonita', 4X V. corymbosum

cultivar 'Sharpblue' and 2X V. darrowi. The pollen germina-

tion, number of flowers pollinated, fruit set, mean seed

number and mean number percent of viable seed were recorded.

The flowers of the 12X plants were larger and thicker

than those of their undoubled counterparts (Figure 4). The

leaves were larger, darker green and thicker, and the plants

were substantially less vigorous than their 6X counterparts.

Pollen of each 12X ramet was germinated using the

method of Goldy and Lyrene (49). Freshly collected pollen

was dusted on pollen germination medium contained in Petri

plates. After 24 hours at room temperature (25 C), the

plates were examined under the microscope and percent

germination was recorded for 4 fields of vision at 40X

magnification.



Results and Discussions

Pollen tube growth of one 12X 'Beckyblue' ramet was

strong, while that of the other 12X 'Beckyblue' ramet was

weak, with pollen tubes barely protruding from the pollen

grain. The 12X 'Bluebelle' clone also had weak pollen tube

growth and the pollen clumped in 2 tetrads (Table 18).

No fruit was set when the 12X plants were intercrossed.

When used as male parents on 6X V. ashei, only 'Beckyblue'














































Figure 4. Flowers from 12X 'Beckyblue' Subclone l(r.) and
from the undoubled 6X 'Beckyblue' (1.) about
1 day before anthesis.
















Table 18

Pollen Germination of 12X Colchiploid and 6X V. ashei Ramets



Total Number %
grains germinated germination


'Beckyblue' I 12X 113 28 25

'Beckyblue' II 12X 181 159 88

'Bluebelle' 12X 270 56 21

Chi-square = 218.30



'Beckyblue' 6X 157 144 92

'Bluebelle' 6X 211 186 88

*
Indicates that the pollen germination varied with the ramet
tested.









subclone I set 4 fruit from 64 pollinated flowers. The

mature berries contained many plump seeds. The seeds have

not yet germinated, therefore, it is unknown if the

seedlings will be true hybrids or accidental outcrosses.

When 'Sharpblue' was crossed with each 12X male parent,

many fruit set. Many viable-looking seeds were obtained

from the fruit. 'Sharpblue' is highly self-fertile. The

high apparent fertility of the 4X 12X cross seems

incompatible with the low degree of pollen viability from

the 12X parents, and raises the possibility that the set on

'Sharpblue' was a result of unintentional selfing rather

than planned crossing.

A few fruit were set when the 12X males were crossed

with 2X V. darrowi females. Very few viable-looking seed

were obtained from the berries. Most seeds were flattened

and shriveled (Table 19).



Conclusions

The pollen germination and fruit set data suggest that

the 12X plants had low fertility both as males and as

females.





















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CHAPTER VII

CONCLUSIONS



Tissue culture is a valuable tool for plant breeding.

An in vitro colony with its many shoots and numerous buds

allows chemical and physical treatments to take place on a

large scale with limited space requirements. The supportive

environment allows weaker mutated or polyploidized cells to

survive and possibly proliferate. The colonies can be

easily screened visually for mutants or increased-diameter

shoots, a visible indicator of increased ploidy. Once

optimum dosage and duration of treatment are established for

a species, the numerous polyploidized clones required for

introgression of traits into a gene pool can be easily

produced.

The most successful polyploidizing treatment varied

depending on the taxon and the specific clone treated. For

V. elliottii, 0.01% colchicine dissolved in liquid modified

Knops medium was the most successful medium for polyploid

induction. Two-node in vitro cuttings treated in this

medium for 72 hours on a rotary drum (3 r.p.m.) produced the

greatest number of polyploid shoots. Both V. darrowi and









V. darrowi X V. elliottii, produced polyploid shoots spon-

taneously in vitro, without added colchicine. When treat-

ment of 2-node V. elliottii cuttings were treated on solid

colchicine containing, modified Knops medium, 0.01% colchi-

cine for 2 weeks duration was the most effective treatment

for polyploid induction.

Induced autotetraploids in Vaccinium should be valuable

in moving adaptive and economically important traits from

diploid native species into cultivated tetraploid breeding

lines. In vitro colchicine treatment may also be quite

useful in facilitating interspecific gene transfers in other

genera where the species differ in ploidy. This will make

it possible to tap new gene pools in the breeding of some

crop species.

V. elliottii, treated with a wide range of radiation,

produced several visible mutants in vitro. An LD-50 of 4.5

krads was established for the treated clone. The mutants,

2 derived from the 1.5 krad treatment and 1 from the 3 krad

treatment, were visually identified by altered growth form

when compared to control colonies.

Induction of mutations facilitated by radiation treat-

ment of in vitro colonies should be valuable in improvement

of long generation, vegetatively propagated perennial crops.

Alteration of a few traits of an otherwise outstanding

cultivar with mutagenesis may obivate the extensive cycles

of hybridization to wild species and backcrossing ordinarily

needed to restore the horitcultural value of a cultivar.





70



The enhancement of adventitious bud formation is an

important feature of in vitro culture, because it allows

formation of a higher frequency of solid polyploids and

mutants. In some cases, the in vitro system alone is

sufficient to allow genetic alterations to occur in

explants. With, the added impetus of colchicine or radia-

tion, a much higher frequency of mutant genotypes should be

obtained.

















REFERENCES


1. Aalders, L.E., and I.V. Hall. 1962. New evidence on
the cytotaxonomy of Vaccinium species as revealed by
stomatal measurements from herbarium specimens. Nature
196:694.

2. Aalders, L.E., and I.V. Hall. 1963. Note on aeration
of colchicine solution in the treatment of germinating
blueberry seeds to induce polyploidy. Can. J. Plant
Sci. 43:107.

3. Ackerman, W. L., and H. Dermen. 1972. A fertile
colchiploid from an interspecific Camellia hybrid. J.
Hered. 28:56.

4. Ahokas, H. 1971. Notes on polyploidy and hybridity in
Vaccinium species. Ann Bot. Fennici 8:254.

5. Allard R. W. 1960 Principles of Plant Breeding. John
Wiley and Sons, Inc. New York, N.Y.

6. Anderson, W. C. 1975. Propagation of Rhododendrons by
tissue culture. Proc. Intern. Plant Prop. Soc. 25:129.

7. Ballington, J. R., and G. J. Galletta. 1974. Effects
of gibberellin compounds, concentrations and treatment
duration on blueberry seed germination. HortScience
9:283.

8. Ballington, J. R., and G. J. Galletta. 1976.
Potential fertility levels in four diploid Vaccinium
species. J. Amer. Soc. Hort. Sci. 101(5) 507.

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BIOGRAPHICAL SKETCH


Julia Lucile Wood Perry was born on May 6, 1955, in

Bethesda, Maryland. She attended high school in Shreveport,

Louisiana, at St. Vincent's Academy, graduating in 1973.

She attended the University of Colorado, Boulder, for two

years, majoring in archaeology. In 1976, she married Robert

Thomas Perry. She completed her B.S. degree at Eastern

Kentucky University in Richmond in 1977, with a major in

horticulture.

She received her M.S. in 1980 from the University of

Arkansas, Fayetteville, working under the direction of

Dr. James Moore. Her research problem was determination of

self- and cross-compatibility of various tetraploid culti-

vars of blackberry (Rubus Eubatus).

In 1980 she began work with Dr. Paul Lyrene at the

University of Florida, Gainesville, to facilitate hetero-

ploid crosses between Vaccinium species by in vitro

colchicine treatment of native diploids.









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 Docotor of Philosophy.






Paul M. Lyrene, Chairman
Associate Professor of
Horticultural Science





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






Wayne B. Sherman
Professor 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 Docotor of Philosophy.





Gloria A. Moore
Assistant 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 Docotor of Philosophy.


I rA

a A h


Mark Bassett N
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 Docotor of Philosophy.






Ken Quesenberry
Associate Professor of
Agronomy





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.


April, 1984


Dean College of Agiculture


Dean for Graduate Studies and
Research


ec:ti7 1!c


































































UNIVERSITY OF FLORIDA


3 1262 08553 4435




Full Text

PAGE 1

IN VITRO PRODUCTION OF COLCHIPLOIDS AND MUTANTS OF NATIVE FLORIDA DIPLOID Vaccinium SPECIES AND HYBRIDS AND EVALUATION OF 12X V. ASHEI COLCHIPLOIDS BY JULIA LUCILE WOOD PERRY 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 1984

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r ACKNOWLEDGEMENTS Appreciation is extended to the members of my graduate committee: Dr. Wayne Sherman, Dr. Gloria Moore, Dr. Mark Bassett and Dr. Ken Quesenberry for their guidance and assistance in the preparation of this research. Special appreciation is extended to Dr. Paul Lyrene, my major professor, whose gentle wit and great intellect taught me how to formulate a question, properly conduct research, and analyze results to obtain an answer. His teachings and friendship will be cherished always. I also wish to extend deep gratitude to my family, my patient and loving husband of 7 years, Robert Thomas Perry with whom I have weathered many storms and shared just as many rainbows. Loving appreciation also to my supportive parents, Drs. Charles and Mary Wood, who not only gave me the finest of role models but a varied and valuable genetic base on which to build. ii

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TABLE OF CONTENTS PAGE ii ACKNOWLEDGEMENTS .. LIST OF TABLES. LIST OF FIGURES ABSTRACT . . . . . . . . V CHAPTERS I II III INTRODUCTION .... LITERATURE REVIEW Cytology. . . ..... Improvement Tissue Culture. .... Radiation Induction of Mutants .. IDENTIFICATION OF OPTIMAL TREATMENT FOR POLYPLOID INDUCTION ... Introduction ....... Materials and Methods (Experiments 1-6) Experiment 1 . . . . . Experiment 2 . . . Experiment 3 . . . . . Experiment 4 . . . Experiment 5 . . . . Experiment 6 . . . . . Results and Discussion (Experiments 1-6) Experiment 1 . . . . . Experiment 2 . . . . . Experiment 3 . . . . Experiment 4 . . . . Experiment 5 . . . . Experiment 6 . . . . . Conclusions iii . . . vii viii . . . 1 5 11 14 17 19 22 22 22 22 25 25 27 27 28 28 28 33 40 40 45 46 47

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IV V IV VII ENHANCEMENT OF COLCHICINE ACTION. Introduction . . . . . . .. Materials and Methods (E x periments 7-11) E x p e riment 7 . . . . E x perim e nt 8 . . . E x periment 9 . . . E x p e riment 10 . . . E x periment 11 . . Results and Discussion (Experiments 7-11) Experiment 7 . Experiment 8 . Experiment 9 . . E x p e riment 10 . . E x periment 11 . . Conclusions RADIATION STUDIES Introduction .... Materials and Methods .. Results and Discussion Conclusions ....... . . . . . . . B R EEDING BEHAVIOR OF 12 X V. ashei CHOLCHIPLOIDS . . . Introduction ..... M aterials and Methods Results and Discussions Conclusions CONCLUSIONS REFERENCES BIOGRAPHICAL SKETCH iv 49 49 49 49 50 51 51 52 52 52 53 54 56 56 56 58 58 58 59 61 62 62 62 63 66 68 71 75

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TABLE 1 2 3 4 5 6 7 8 9 10 LIST OF TABLES Ploidy Level and Characteristics of Vaccinium Species Native to Florida Composition of Modified Woody Plant Medium, Modified Andersons Medium, and Modified Knops Medium. Ploidy Level and Stomate Length of 10 Large Diameter Shoot in vitro Colonies of V. elliottii Screened from Experiment 1-. Growth Response of V. elliottii Following Treatment with 0.1%-Colchicine for Various Durations ............. Frequency of Fatal Decline Syndrome Following in vitro Treatment of V. elliottii with 0.1% Colchicine on a Rotary-Drum for Various Durations ....... Growth Response of Increased-Diameter Shoots of V. elliottii when Placed on 3 in vitro MedTa for Regeneration. .. Growth Response of V. darrowi after Treat ment of 2-node CuttTngs from in vitro Colonies with 0.1 % Colchicinefor Various Durations ........... Growth Response of V. darrowi XV. elliottii in vitro 2-node Cuttings to Treatment with 0.1% Colchicine for Various Durations Effects of Colchicine Concentration and Treatment Duration on Regrowth Vigor of Treated Vaccinium Explants Number of Colonies Containing 1 or More Shoots of Increased-Diameter Following Treatment with Various Colchicine Concen trations for Various Lengths of Time. V PAGE 15 26 32 34 35 37 38 39 41 43

PAGE 6

TABLE 11 12 13 14 15 16 17 18 19 Effects of Colchicine Concentration and Treatment Duration on Regeneration Vigor of V. elliottii Explants Treated on Solid PAGE Modified Knops Medium . . 44 Number of in vitro Colonies of V. elliottii Producing lor More Shoots of Increased Diameter 8 Weeks after Planting with Colchicine-treated Explants ... Growth Response of 3 Vaccinium Clones to Treatment for 3 Weeks on Solid M odified Knops Medium Containing 0.01% Colchicine Response of Seed of V. elliottii and V. darrowi to Colchicine Added to the in vitro Germination Medium ... In vitro Shoot Formation of V. elliottii when Cultured on 3 Levels of-2iP in Modified Knops Medium .......... Frequency of Increased-Diameter in vitro V. elliottii Shoots Following Treatment with 0.1 % Colchicine, Etiolation and G.A . Effect of Gamma Radiation on Regrowth Vigor of in vitro V. elliottii Explants Pollen germination of 12X Colchiploid V. ashei Ramets . .. Fertility of 12X V. ashei Colchiploids when Crossed with-Several Vacciniurn Species of Various Ploidy Levels and Intercrossed between Cultivars . . . .. vi 45 46 47 53 55 60 65 67

PAGE 7

FIGURE 1 2 3 4 LIST OF FIGURES Normal diameter in vitro shoots of V. elliottii (center) and increased diameter colchiploid shoots left and right ..... Stomate prints from leaves of an increased diameter shoot of V. elliottii (top) and a regular-diameter shoot (bottom) photo graphed at the same magnification (X40) Chromosomes of a shoot tip from an increased-diameter shoot of V. elliottii (top) and an undoubled shoot-of regulardiameter (bottom) photographed at lOOX magnification .............. Flowers from 12X 'Beckyblue' subclone 1 (r.) and from the undoubled "Beckyblue" (1.) about 1 day before anthesis ....... vii PAGE 24 30 31 64

PAGE 8

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IN VITRO PRODUCTION OF COLCHIPLOIDS AND MUTANTS OF NATIVE FLORIDA DIPLOID Vaccinium, SPECIES AND HYBRIDS AND EVALUATION OF 12X V. ASHEI COLCHIPLOIDS By Julia Lucile Wood Perry April 1984 Chairman: Dr. Paul Lyrene Major Department: Horticultural Science In vitro produced colonies of Florida native diploid blueberry species Vaccinium elliottii Chapm., ~darrowi Camp, and their interspecific hybrid were treated with colchicine to double their chromosome number and facilitate interspecific hybridization with tetraploid highbush blue berry,~corymbosum Lamarck. In vitro colchiploids, identified by increased shoot diameter, were confirmed as tetraploid by stomate guard cell measurement and chromosome counts from shoot tip squashes. V. elliottii produced the greatest number of increased diameter shoots when 2-node cuttings were treated for 48 hours on a rotary drum in liquid modified Knops medium containing 0.1% colchicine, or for 2-6 weeks without agita tion on solid modified Knops medium containing 0.01% colchi cine. V. darrowi produced the greatest number of viii

PAGE 9

increased-diameter shoots when 2-node cuttings were treated on a rotary drum in .01 % colchicin e -containing liquid modified Knops medium for 24 hours. The V. elliottii XV. darrowi h y brid produced the greatest number of increased diameter shoots when 2-node cuttings were treated for 4 hours in colchicine-free liquid modified Knops medium on a rotary drum, apparently due to spontaneous doubling. The LD-50 of in vitro V. elliottii colonies treated with gamma radiation was 4.5 krads. No e x plants receiving a dose greater than 25 krads survived. Two mutant colonies were identified from the 1.5 krad treatment and 1 mutant colony was identified from the 3 krad treatment. These mutant colonies were dwarfed and feathery in appearance. The 12X colchiploid ramets of~ashei cultivars 'Beckyblue' and 'Bluebelle' apparently are of low fertility as both male and female parents. Pollen germination was low and pollen tubes weak. No fruit was set when the 12X cultivars were intercrossed or crossed with 6X V. ashei 'Bonita'. A few fruit were set when the ramets were crossed with tetraploid ~corymbosum 'Sharpblue' or diploid V. darrowi, possibly due to accidental outcrossing or selfing. ix

PAGE 10

CHAPTER I INTRODUCTION Blueberry, (Vaccinium species), is native to North America, Europe and several other parts of the world. Many wild species exist in the United States, with chromosome ploidys ranging from diploid to hexaploid. Wild relatives are valuable sources of locally adaptive characteristics which would be useful if they could be incorporated into cultivated species. Most blueberry acreage in Florida is planted with hexaploid rabbiteye (V. ashei Reade) cultivars. This is due to the resistance of V. ashei to Phytopthora cinnamoni Rands root rot, cane canker (Botryosphaeria corticis Demaree and Wilcox) and various other fungal diseases (74, 75) and to its low chilling requirement (97), high vigor and high productivity (45). Highbush (~. corymbosum) cultivars are also widely planted in the South, but several factors impede their cultivation in Florida, including fungus suscepti bility, higher winter chilling requirement and lack of heat and drought tolerance. Despite these weaknesses, V. coryrnbosum has one important advantage over~ashei early ripening. With bloom occurring at approximately the same date, fruit of early highbush clones mature in 60 days while 1

PAGE 11

I ~it takes early rabbiteye cultivars 80 to 90 days post pollination to reach maturity (45, 95}. 2 Florida native blueberry species include diploids (2N=2X=24) V. darrowi Camp,~elliottii Chapm., atrococcum Heller,~arboreum Marsh, and~staminium L.; tetraploids (2N=4X=48) ~fuscatum Ait., and V. myrsinites Lam. and the hexaploid (2N=6X=72) ~ashei (45, 73). In the section Cyanococcus, which includes all of the above species except y. arboreum and~staminium, there are only weak sterility barriers between species with the same ploidy level (8, 18). Heteroploid crosses give variable results ranging from partial success to almost complete failure. Hybrids derived from heteroploid crosses may exhibit ovule or pollen sterility (27). Several of the wild diploid Vaccinium species native to Florida possess characteristics which could complement the tetraploid highbush genome. Diploid V. elliottii is adapted to the drier Florida soil, has high resistance to certain fungal diseases, high berry flavor, low chilling requirement and high vigor. V. darrowi has low chilling requirement, heat and drought resistance, light blue fruit color, attrac tive lowbush growth form and some cane canker resistance (45, 75, 96, 109}. Because of a triploid block, few hybrids are obtained from diploid by tetraploid crosses and their reciprocals (4).

PAGE 12

3 There are several possible methods for bypassing the triploid block. These methods include production of haploids from tetraploid ~corymbosum, rescue of triploid embryos prior to endosperm failure, enhancement of 2N gamete production in diploid species, and colchicine treatment of diploid species to convert them to autotetraploids. Chromosome doubling of woody perennial species by colchicine treatment has had limited success (29, 37, 38, 40, 48, 56). Treatment in vitro has several advantages over traditional methods, including a stable, germ-free environ ment with provision of optimum light, nutrients, and humidity which allows survival of weaker autotetraploids. Shoots produced in tissue culture are very vigorous and have short internodes and numerous buds which make them highly suited for chromosome doubling with colchicine. Tissue culture is also quite space conservative, allowing large numbers of shoots to be treated in a small area (53, 82). Polyploid shoots, characterized in vitro by increased shoot diameter, can be quickly and easily screened by visual examination (72). Mutation has played a large part in the development of fruit cultivars. Mutation induction in vegetatively propa gated species can be valuable in altering a few traits in an otherwise outstanding cultivar or to induce tissue rearrange ments of existing periclinal chimeras (31,41,42). Determination of the fertility of autoploids is essen tial to their use in a breeding program. Not only must the

PAGE 13

4 pollen germinate, it must also have the ability to effect fertilization and allow fully-developed, fertile seed to be set. The purpose of this study was to identify the best type of e x plant material for blueberry chromosome doubling by in vitro colchicine treatment as well as optimal colchicine concentration and treatment duration for enhancement of doubling. Additional objectives were to find the optimal radiation treatment for mutant production, and to evaluate the breeding value of 12X V. ashei colchiploids.

PAGE 14

CHAPTER II LITERATURE REVIEW Polyploidy has been an important feature of plant evolution, and its value in cultivar breeding has long been recognized. Possible beneficial changes induced by poly ploidy include broader, thicker leaves, larger flowers and fruit and increased fertility of hybrids not fertile as diploids (35, 48). The evolution of the genus Vaccinium has occurred through millions of years and polyploidy has played a large part in the process (29). Harlan and DeWet suggest that the most common and widespread form of spontaneous polyploidy in the higher plants is probably by 2N + N reproduction (52). This occurs by a two-step process. The first step consists of fusion of a 2N female gamete with a normal N male gamete giving rise to a triploid plant. This triploid plant, in turn, may produce some cytologically unreduced triploid female gametes that are fertilized by haploid gametes of the diploid parents resulting in tetraploid offspring (66). This is a general phenomenon that probably takes place at a low but significant frequency in nearly all species of sexual plants (52). Approximately 75% of all rnonocot species and 40% of the dicots are considered polyploid (66). 5

PAGE 15

The methods used to induce polyploidy may be divided into 2 classes, somatic doubling and gametic doubling. In somatic doubling, the chromosomes which would normally be distributed to 2 sister somatic nuclei are included in 6 1 nucleus. If the diploid plant in which this occurs is not a hybrid, it gives rise to an autopol y ploid, with 4 very similar sets of chromosomes. If the diploid plant is a wide hybrid, somatic doubling gives rise to an allopolyploid with 2 pairs of identical chromosome sets (5, 48). In gametic doubling, the chromosomes which would have been distributed to the 4 nuclei produced b y meiosis are included in 2 nuclei which give rise to diploid gametes. Most of the natural polyploids produced by this method have come from hybrids with low fertility. The zygotes which are produced generally arise from unreduced gametes, male or female or both (48). With the discovery of the polyploidizing action of colchicine in the 1930's, polyploid induction was no longer left to nature's whim. A number of other agents, chemical and physical, have been used to induce polyploidy including brome-acenaphthene (99), temperature shock (34), severe pruning to encourage adventitious bud break (5, 56), nitrous oxide, and podophyllin (62). The greatest success in polyploid induction, however, has been with colchicine treatment ( 3, 11, 21, 3 4, 3 5, 4 0, 4 6, 4 8, 7 2, 8 0, 8 9) Colchicine is a mitotic inhibitor which acts by inter fering with the equilibrium between microtubules and their

PAGE 16

---7 subunits, preventing the assembly of spindle fibers and ultimately blocking completion of mitosis (65). Colchicine in aqueous solution is readily diffusible into plant tissue and acts over a wide range of concentrations with high specificity. The cellular reaction to colchicine depends on several conditions: specific concentration, exposure period, mitotic stage when contacted by the chemical, cell type and presence of an environment favorable to mitosis (11). Colchicine acts only on dividing cells; with no effect on resting cells (99). The mitotic stage at which colchicine is most effective in lowest concentration is late prophase (46); however, the effect of colchicine is not confined to a limited number of cells at a particular stage of cellular development. Any cell may be affected if the cell goes through division while containing the chemical (34, 35). As long as colchicine remains in treated material above thres hold concentration, affected cells will repeatedly fail to divide at the end of each nuclear division cycle resulting in multipolyploidy (34). When colchicine is removed, the chromosomes gather into small groups and loose spindles appear (46). A common aberration following treatment is aneuploidy, which seems to be due to partial instead of total arrest of chromosome division or to a multipolar division in some of the polyploid nuclei (35). Chromosome duplication may result in an increase in nuclear and cell volume. Changes in volume and cell

PAGE 17

dimensions are often correlated with changes in size of plant parts (34). New leaves and stems which grow from doubled material are usually wrinkled, thicker, darker green, and coarser in texture. Le a f veins of doubled material become coarser, leaf hairs and glands become coarser and more abundant, leaf profile ma y become shorter and more rounded and flower buds produce shorter, stockier pedicels. Similarly, the petals may be stunted and coarsened in te x ture or larger and the buds more compact. 8 In general, a polyploid plant has a more rugged appearance, looks sturdier and has certain giant-like features. Usually growth is slower, and the plant is usually shorter than its original diploid counterpart (5, 34, 100). Colchicine induction of polyploidy has been successful with many herbaceous species (34, 48, 100). Woody species, however, are generally unresponsive to conventional methods of colchicine treatment. Azalea (89), camellia (3), grape (47), cranberry (36), pear (56) and chestnut (40) have been doubled with limited success, while plum, blueberry, cherry, peach and apple are very difficult species in which to induce polyploidy (40, 48). A problem common to both herbaceous and woody species following colchicine treatment is chimera formation due to the multicellular nature of the bud apex (12). Chimeras may be periclinal or sectorial. Growing points are usually made up of 3 independent cell layers termed L-I, L-II, and L-III.

PAGE 18

9 When only a single layer is affected by colchicine, a periclinal chimera results. When a portion or sector of all 3 la y ers is doubled, a sectorial chim e ra is the result. Utilization of colchiploids depends upon identification of plants which produce doubled gam e tes. These include solidly doubled plants and plants with a doubled L-II layer. The anticlinal mode of cell division characteristic of growing shoot tips is not a permanently fixed condition. Periclinal division occurs occasionally in cells of the L-I, more frequently in the L-II and commonly in the L-III histogenic la y er with the result of varying the ploidy level of colchi ploid pl a nt la y ers (39). Identification of the ploidy level of each layer is desirable. M easurement of stomatal guard cells of the epidermis of colchicine-treated versus untreated plants has been used to identify doubled L-I layers in a number of species including Vaccinium (1, 19, 20), Bromus (104), and Triticum (93). Increased intensity of green leaf color of doubled plants has been found to be associated with the increased depth of leaf tissue and a proportionate increase in number of chloroplasts (63). Chloroplast numbers in stomatal guard cells are also found in increased numbers in doubled plants and are another measurable indicator of increased ploidy of the L-I layer in some species (66, 94). The L-II layer, which gives rise to the gametes, is of greatest interest to plant breeders. Autotetraploids have larger pollen grains than their diploid counterparts (33).

PAGE 19

These grains may take on characteristic shapes providing a morphological indication of induced polyploidy (34). 10 The L-III layer gives rise to various internal tissues. Chromosome doubling of this layer may be measured by chromo some counts from root or shoot tip squashes (5, 56). Naturally amphiploid species generally do not show consistent increases in these parameters when compared with parental species. Genetic factors and the nature of the parental species may be more influential than the change in chromosome number (46). Various methods of increasing the effectiveness of colchicine treatments have been tested. These include pretreatment of plant material with gibberellic acid to accelerate the rate of cell division and cell elongation. This acceleration is postulated to increase cell perme ability to colchicine (110). Etiolation has also been used to induce cell elongation. E x posure of etiolated shoots to light causes cell division to occur simultaneously in a large nurr~er of cells making the shoots more susceptible to the doubling action of colchicine (21, 55). Treatment of Allium root tips with 20 50 ppm of indole-3-acetic acid has been shown to induce mitosis in a large number of cells. Tips were treated for 4 hours and a large number of mitotic figures were recorded 24 hours later (44). DMSO (dimethyl sulfoxide) has been used as a carrier for colchicine, increasing treatment effectiveness by increasing drug penetration (92). Other factors affecting

PAGE 20

treatment success are pH of the colchicine solution (66), aeration of the solution (2), and treatment temperature ( 21) Cytology 11 Subfamily Vacciniaceae is a taxonomically ancient group with many cytologically primitive characteristics. The basic karyotype of the section Cyanococcus has not evolved much from the ancestral form characterized by high chromo some number and small, metacentric chromosomes (17, 23). The basic haploid complement of Vaccinium is considered to be 12 chromosomes (2 long, 8 intermediate and 2 short) metacentrics) (51). The whole section Cyanococcus in eastern North America forms a large polyploid complex in which both autoployploidy and allopolyploidy have played significant roles (17, 18). There are naturally occuring diploid, tetraploid and hexaploid species and a few penta ploid hybrids (28). According to Camp, all but 2 diploid species (~. elliottii and V. myrtilloides) have given rise to polyploids. All species, diploid to hexaploid, have been found to produce unreduced pollen with an especially high rate in diploid species (22). These unreduced gametes allow interspecific heteroploid crosses which would not normally be possible (9, 27). Members of diploid and tetraploid groups rarely hybridize but are generally interfertile within any ploidy group (24). Barriers to natural hybrid production among homoploids include weak incompatibility

PAGE 21

12 barriers within the style which slow pollen tube growth (106) and, to a limited extent, differing pollinizers (105). Cytological studies in Vaccinium have revealed regular pairing in diploid species (68). An exception to this is a diploid hybrid between~myrtillus X ~vitis ideaea which showed a number of meiotic irregularities due to chromosomal differences. Each parental species alone was quite regular in meiosis. An autotetraploid created by doubling the chromosome number of~myrtillus displayed a number of multivalent associations at rnetaphase I (91). Vander Kloet postulated that V. corymbosum originated from an ancient hybrid swarm between V. tenellum XV. darrowi (107). Bivalents only were found in some clones of ~corymbosum (60, 68), with multivalence and secondary pairing indicative of segmental allopolyploidy occurring in others (59, 83). Trivalents and univalents were rare. The high frequency of bivalents may be due to the almost com plete diploidization of the tetraploid species (60). Most of the bivalents are involved in pseudo-multivalent associa tions. Highbush cultivar 'Jersey' produced bivalents and quadrivalents in meiosis with 0-50% of the chromosome complement participating in quadrivalent associations (59, 60). The primary factors governing the type and frequency of multivalent association in polyploids are chiasma fre quency and position, size of chromosomes (61), and genes that regulate pairing.

PAGE 22

13 Tetraploid ~uliginosum, possibly due to its presumed recent autotetraploid origin, has a number of meiotic irregularities. When~uliginosum was crossed with culti vated highbush, meiosis was quite regular due perhaps to autosyndetic pairing {91). Hexaploid blueberry species studied by Longley included V. ashei, ~constablaei and V. amoenum and were generally found to be meiotically stable (68). Throughout the plant kingdom, autoploids are almost without exception less fertile than diploids. This is probably due to multivalent associations, which result in unbalanced gametes. Great variations in fertility are found from species to species, ranging from almost complete sterility to 75 % fertility {87). Among progenies of alloploids, the first generation may be quite fertile while later generations have reduced fertility (46). In a study where pollen germination was used as an indicator of fertility of Vaccinium species and hybrids, tetraploid ~corymbosum pollen germination ranged from 22-49%, tetraploid ~myrsinites pollen averaged 42% germi nation and 27% of diploid~darrowi germinated (49). Non-functional pollen is characterized by tetrad collapse and large globules of sporopollenin deposit. A second fertility indicator is seed set {108). Four types of seed have been described in blueberry fruit: large, well-filled seed with brown or tan seed coat; large

PAGE 23

flattened or concave brown or tan seed which may be par tially shriveled; well-filled brown or tan seed half the size of class 1; or very small white seed which appear empty. Only the seeds of the first class are fully devel oped and capable of germination (7, 25). Among highbush cultivars, only 21-52 % of the seeds were found to be fully developed, while 36-55 % of open-pollinated rabbiteye seeds were judged to be capable of germination (79). Improvement 14 Blueberry improvement was begun by Coville with a species cross of wild tetraploid V. corymbosum clone 'Brooks' with V. angustifolium clone 'Russell' (24). Northern tetraploid cultivars lack several important charac teristics which would make them optimal southern cultivars (56, 73, 75, 77). Problems include a high chilling requirement and susceptability to cane canker and root rot fungal diseases. The characteristics of the Vaccinium species native to Florida are listed in Table 1. As can be seen in the table, some native Florida, diploid species are disease resistant, vigorous, and tolerant to Florida soils and climate (73, 74, 76). Crosses between these 2 ploidy levels are unsuccessful due to developmental failure of triploid seed (4, 91). One way to combine the favorable character istics of the 2 ploidys is to equalize their chromosomes numbers by colchicine treatment of diploid species to produce autotetraploids (78,87,89).

PAGE 24

, --Ploidy 2N=6X=72 2N=4X=48 2N=2X=24 15 Table 1 Ploidy Level and Characteristics of Vaccinium Species Native to Florida Species V. ashei V. fuscatum V. myrsinites V. darrowi V. elliottii V. atrococcum V. arboreum V. staminium Possible desirable characteristics Chilling requirement between 500 and 700 hours Heat, disease, insect and drought tolerant Very productive with firm large fruit Vigorous--3-6m at maturity Small fruit scar Late ripening Adaptive to varied soil type Evergeen foliage Drought and heat tolerant Low chilling requirement Evergreen foliage Drought and heat tolerant Low chilling requirement Heat, and drought tolerant Low chilling requirement Evergreen foliage Attractive lowbush form Light blue color Disease resistant Tolerant of mineral soils High berry flavor Vigorous Early ripening Early ripening Concentrated fruit ripening Tolerant of high pH soils Drought tolerant Late ripening; August through December Native south to Manatee and Hardee Counties, Fl. Heat and drought tolerant Low chilling requirement Large fruit

PAGE 25

16 Species hybridization is important in the transfer of desirable traits from diploid to polyploid species (30). Another way to transfer genes from native diploid and hexaploid species into tetraploid highbush, is to produce intermediate tetraploids by interspecific hybridization (43, 57, 80). Several heteroploid crosses have been made to create introgression bridges with variable results. Hexaploids crossed with diploids generally produce pentaploids when V. darrowi is used as the diploid parent (9, 43). Crosses have also been made between hexaploid ~ashei and diploids~ tenellum and V. elliottii without much success in tetraploid production (31, 43). Hybrids have also been made between hexaploid ~ashei and tetraploid highbush in order to combine favorable traits from each species. These penta ploid interspecific hybrids are readily produced and are vigorous, hardy, and reasonably fertile when pollinated by hexaploids or tetraploids (30, 31). Chromosome elimination during meiosis occurs at a high frequency in blueberry pentaploids and makes them function primarily as tetraploids (58). These interspecific functional tetraploids should cross readily with cultivated highbush to give improved tetraploid breeding lines. This offers one possibility of combining the vigor, productivity, and broad soil adaptability of rabbiteye with the earliness, and quality of highbush (30, 58).

PAGE 26

17 Tissue Culture Proliferation in tissue culture can occur in 3 ways: enhanced formation of axillary shoots, production of adven titious shoots, and somatic cell embryogenesis (50, 81). Plant cell culture itself may generate genetic variability and may be employed in some cases to enhance recombination in hybrids. It may also generate variants of commercial cultivars in a high frequency without hybridi zation to other genotypes (64, 111). One consequence of growth in vitro is the appearance of dividing cells with chromosome numbers and karyotypes not usually found in growing points of the intact plant. Aberrant plants are found at rates that approximate mutation rates in field grown plants among plants derived from axillary shoots (101). Aberrations are found at increased rates among plants grown from adventitious shoots, and are even more frequent among plants derived from somatic embryos. When plant cells are allowed to proliferate in callus or suspen sion culture, there is a high possibility of chromosomal variation. Conditions which favor callus formation have been shown to cause nuclear fragmentation, which produces spontaneous polyploids, aneuploids, and chromosomal rearrangements (10, 85, 86, 88, 98). Another factor affecting in vitro variability is explant source. Murashige and Nakano found constant diploidy to be confined to meristem tissue, while a mixture of diploid and polyploid cells comprised the differentiated

PAGE 27

18 and mature tissues of tobacco. Plantlets derived from regenerated tobacco pith cells were found to vary widely in ploidy level from diploid to octoploid (82). Adventitious buds are known to form in over 350 species (13, 15). The apex of an adventitious shoot is formed by one or a few vegetative daughter cells of one original cell, thus, its ultimate origin is from a single cell (14). Treatment of explants with mutagenic or polyploidizing agents should result in more whole-plant changes and fewer chimeral changes than are normally encountered following conventional treatments (70), as mutation and adventitious bud formation are both unicellular events. After colchicine treatment, the often less-vigorous polyploid cells are unable to complete with the faster-growing original cells. If a polyploid cell survives, the end product is often a mixoploid or cytochimera. Colchicine treatment in combina tion with the adventitious bud technique should provide a high percentage of solid, non-cytochimeral polyploids (12, 13, 54). Adventitious shoot formation has been observed in V. corymbosum, V. ashei, ~atrococcum, V. constablaei, darrowi and V. elliottii and their interspecific hybrids. Adventitious shoots and callus have been observed to form from the edges of leaves in contact with the nutrient medium. The callus may, in turn, produce adventitious shoots (71, 84, 112).

PAGE 28

19 The addition of colchicine to in vitro systems for induction of polyploidy has been utilized in sugarcane cell suspension (53), daylilly callus (21) and Hordeum callus (86). Pol y ploids of tobacco have been identified pheno typically in vitro by their smaller, thicker, gr e ener leaves which were distinctly rosetted and pubescent. Stems were thicker than on the original plants and internodes were shorter. The style and stigma w e re larger and the ability to produce functional pollen was much reduced. Leaf cells were found to be larger and an additional 2 layers of spongy mesophyll were observed. Chromosomal laggards were often observed in meiotic studies {82). Radiation Induction of M utants Induction of mutants by radiation treatment has its greatest value in vegetatively propagated species in which one or a few characteristics of an otherwise outstanding cultivar may be changed without affecting the remaining genotype (5, 16, 100). Radiosensitivity varies among plant species and depends mainly on the nuclear volume (greater DNA content, more sensitive), chromosome number (plants with fewer large chromosomes are more sensitive than plants with more, smaller chromosomes), and ploidy level (higher ploidy level, less radiosensitive) as well as on the developmental stage of the plant (most resistant seed to dormant plant to highly susceptible growing plant) ( 16).

PAGE 29

20 Mutation breeding in woody perennials has great promise. The long time lapse between generations, the large space required to grow a plant to maturity in the field, and the high degree of heterozygosity make it difficult to produce cultivars by conventional breeding methods. This is demonstrated by the unusually high ratio of commercial fruit cultivars that have originated from spontaneous bud muta tions compared to the cultivars produced by cross breeding (16, 26). There has been no published work on the radiation induction of mutation in Vaccinium to date. Because some Vaccinium species are heterozygous and diploid, some mutants such as albino or varigated leaves, altered leaf shape, or dwarfed growth form, should be immediately visible following mutagenesis and propagation (16). Tissue culture should be quite valuable in radiation treatment and mutant identification. Hundreds of bud apexes are contained within a single culture vial and can be treated with ease. Treated shoots, used as explant sources, may regenerate mutant shoots from axillary or adventitious buds. These mutants can easily be screened by visual exam ination of the in vitro colonies. Adventitious shoots formed after radiation treatment of irradiated Begonia leaves produced mutants in 30% of the regenerates, 98.5% of which were non-chimera! (14, 90). Tissue culture has also been used successfully in conjunc tion with radiation treatments with detached leaves of

PAGE 30

21 Achimenes, Saintpaulia, Streptocarpus (13), and Crysanthemum ( 15) Gamma radiation has been used in pear to obtain geneti cally homogeneous shoots by tissue rearrangement of existing periclinal chimeras. Radiation-induced tissue rearrange ments have also been used to uncover cryptic mutations hidden within the inner cell layers (32, 41, 42).

PAGE 31

CHAPTER III IDENTIFICATION OF OPTIMAL TREATMENT FOR POLYPLOID INDUCTION Introduction In vitro colonies of various clones of V. elliottii, darrowi and their interspecific hybrid (69) were tested to determine optimal explant source, colchicine concentration, treatment duration and mode of treatment for chromosome doubling. Materials and Methods (E x periments 1-6) E x periment 1 To determine optimal explant material, 2 types of~ elliottii explants from established in vitro colonies of Clone 13 were compared. Two-node shoot cuttings were compared with shoot bases which contained scores of axillary buds as explant sources. Colchicine (0.1%) was dissolved in liquid modified Knops medium (103). A wheel drum rotating at about 3 rpm was used to aerate the medium in the treatment vials. Treatment durations tested were 24, 48, 72, and 120 hours. After the treated explants had been rinsed in liquid, colchicine-free modified Knops medium for 22

PAGE 32

23 1 hour on the wheel drum, five 2-node cuttings were placed in each of 10 new vials containing solid modified Knops medium from each treated vial. After 45 days, the colonies were rated for survival and screened for shoots of increased-diameter, an indicator of increased ploidy (72) (Fig. 1). Leaf casts were made from these shoots and stomate measurements taken. Shoots of increased diameter screened from the colonies were severed from the shoot bases and rooted in a greenhouse under mist. These subclones were labeled and planted in a field nursery. Leaves and buds were collected from each subclone the following spring for ploidy determination. Five leaves were collected from each subclone and each leaf collected was the third leaf from the apical meristem. Leaf prints were made by brushing fingernail polish on the abaxial leaf surface and removing the cast. The casts were examined microscopically and fifteen stomatal guard cells were measured per leaf cast. An average of 10 axillary and apical buds were col lected from each subclone. After fixing in 1:1 absolute ethanol: glacial acetic acid for 24 hours, the buds were stripped of leaves and softened in an enzyme solution of 0.03 g cellulysin, and 0.03 g pectinase in 5 ml distilled water for 15 minutes. After rinsing, the buds were squashed in a drop of 1% acetocarmine dye. The material was destained with 45% acetic acid to facilitate phase contrast examination of the chromosome number.

PAGE 33

Figure 1. Normal diameter in vitro shoots of V. elliottii (center) and increased diameter colchiploid shoots (left and right). 24

PAGE 34

~ ~ -------------------------25 Experiment 2 To determine optimum treatment duration for treatment with 0.1% colchicine, 2-node cuttings of V. elliottii Clone 10, ~darrowi Clone 5, and the interspecific V. darrowi X ~elliottii hybrid from established in vitro colonies were treated in liquid modified Knops medium on a rotary wheel for durations of 0, 4, 12, 24, and 48 hours. After rinsing, 19 vials were planted from each treatment with three 2-node cuttings on solid modified Knops medium. After 45 days regeneration, the colonies were rated for survival, vigor and increased shoot diameter. The increased diameter shoots of V. elliottii screened from this experiment were planted on 3 media (6, 67, 103) to determine if shoot production could be enhanced. Vials containing each medium were planted with each of 30 increased diameter subclones. The components of the 3 media used in this experiment are listed on Table 2. Growth response on each medium was recorded 30 days after subculture. Experiment 3 To determine the optimal colchicine concentration/ treatment duration combination, 2-node cuttings from estab lished in vitro colonies of V. elliottii, ~darrowi and their hybrid were tumbled on a rotary drum (3rpm) in colchi cine concentrations of 0.00, 0.01, 0.05, 0.10 or 0.20%

PAGE 35

26 Table 2 Composition of Modified Woody Plant Medium, Modified Andersons Medium, and Modified Knops Medium Modified Modified woody Andersons Modified Compound plant medium medium Knops medium (mg/liter) (mg/liter) (mg/liter) NH 4 No 3 400 400 KNO 3 480 190 K 2 so 4 990 KH 2 Po 4 170 170 Ca(NO 3 ) 4H 2 O 556 1,140 CAC1 2 2H 2 O 96 440 MgSO 4 7H 2 O 370 370 370 NaH 2 Po 4 H 2 O 380 Na 2 EDTA 74.5 74.5 74.5 Feso 4 7H 2 O 55.6 55.6 55.6 Mnso 4 H 2 O 22.3 16.9 22.3 ZnSO 7H 2 O 8.6 8.6 8.6 H 3 Bo 3 6.2 6.2 6.2 Na 2 MoO 4 2H 2 O 0.25 0.25 0.25 Cuso 4 5H 2 O 0.25 0.025 0.025 KI 0.83 0.83 CoC1 2 6H 2 O 0.025 0.025 Pyridoxine HCl 0.5 0.5 Thiamine CHl 1.0 0.4 0. 1 Nicotinic Acid 0.5 0.5 Myo-inositol 100 100 100 Adenine Sulfate 80 Glycine 2.0 2.0 Casein Hydrolysate 1,000 1,000 1,000 Sucrose 30,000 30,000 30,000 Agar 4,000 4,000 4,000 2i 5 5 5 pH adjusted to 5.7 with lN NaOH. Autoclaved at 1.05 Kg/cm 2 for 15 minutes.

PAGE 36

27 for durations of 6, 12, 24, 48 or 72 hours. A composite of clones was treated for each taxon. For each treatment combination, 10 vials containing modified Knops medium were planted with five 2-node cuttings. Survival, vigor and frequency of increased-diameter shoots were recorded for each ta x on after 45 days. Experiment 4 Two-node cuttings of~elliottii, ~darrowi and their hybrid were placed on solid modified Knops medium containing 0.00, 0.01, 0.05, 0.10, or 0.20 % colchicine to determine optimum colchicine concentration for production of polyploids. After 2, 4, 6, or 8 weeks of treatment, the cut tings were rinsed and replanted on solid modified Knops medium for regeneration. After 45 days, the colonies were evaluated for survival, vigor, and increased-diameter shoots. Experiment 5 Two-node cuttings from established colonies of 2 clones of V. elliottii (Clones 121 and 133) and 1 clone of V. darrowi (Clone 5) were placed on solid modified Knops medium containing 0.01% colchicine for 3 weeks. The cuttings were rinsed and placed on modified Knops medium. Ten vials were planted from each treated vial. After 60 days the colonies were rated for survival and frequency of increased-diameter shoots.

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28 E x periment 6 Seeds of V. elliottii and V. darrowi collected from open-pollinated wild stands in Florida were placed on solid water/agar medium containing 0.00, 0.10 or 0.25 % colchicine to determine seed tolerence and rate of polyploidization. Si x teen vials of each treatment w e re planted with 5 plump seeds of each species. As soon as germination occurred, the seeds were rinsed a nd transferred to solid modified Knops medium for colonization. Results and Discussion (E x periments 1-6) E x periment 1 Comparison of survival rates for the two e x plant sources showed a linear decrease in regrowth vigor with increasing duration of colchicine treatment in both treated cuttings and shoot bases. Although the 2 e x plant types did not differ significantly in survival after treatment, there was a significant difference between explant sources in frequency of shoots with increased-diameter (chi-square probability= .05). The treated cuttings gave rise to 12 v ials containing shoots of increased diameter, whereas treated bases produced none. Nine of the 12 vials cont a ining increased-diameter shoots were regenerated from the 48 hour treatment, with the remainder regenerated from the 72 hour treatment. For cuttings, the 48 hour treatment

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gave significantly more increased-diameter shoots than treatments of 24, 72 or 120 hours (chi-square probabi lity= .05). 29 Stomate measurements of in vitro leaves from increased diameter shoots and normal-diameter shoots were not corre lated with stomate measurements of the same cuttings later transplanted to the field nursery. Screening for increased stomate size as an indicator of increased ploidy is, there fore, not recommended at the early in vitro stage. Stomate guard cell measurements of leaves and chromo some counts of shoot tips from greenhouse rooted thick shoots supported the correlation found earlier between increased stem diameter and induced polyploidy (Figures 2, 3). Seventy percent of the shoots examined had increased stomatal guard cell lengths as well as higher ploidy levels (Table 3). Stomates of Subclone C were significantly larger than diploid or average autotetraploid stomate lengths. This could be due to a periclinal chimera (L-I = 8X? L-III = 4X). If adventitious bud formation were encouraged by in vitro culture of leaves, solid octoploid plants derived from colchicine-treated diploids are possible. Some of the shoots with increased-diameter also showed other morphological anomalies, such as increased and coarse appearing pubesence, darker green, thicker leaves or altered leaf shape. In vitro growth rate was also decreased for large-diameter shoots.

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I I Figure 2. Stomate prints from leaves of an increased diameter shoot of V. elliottii (top) and a regular-diameter shoot (bottom) photographed at the same magnification. 30

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Figure 3. Chromosomes of a shoot tip from an increased diameter shoot of V. elliottii (2N=ca 48) (top) and an undoubled shoot of regular-diameter (2N=24) (bottom) photographed at the same magnification. 31

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Table 3 Ploid y Level and Stomate Length of 10 in vitro Subclones of V. elliottii Selectedfor Large Shoot diameter in E x periment 1 Stomate leng:th ( m) Subclone Ploidy level 2 Meany Range A 24 104 92-115 F 24 115 92-115 K 24 118 92-138 G 48 172 161-184 E 48 173 161-207 J 48 181 161-207 L 48 184 161-207 H 48 187 161-207 D 48 189 161-230 C 48 240 207-276 2 counts were determined by shoot tip squashes and are approximate. YAverage derived from measurement of 15 stomates per subclone. 32 SD 11 15 12 12 12 14 15 12 15 14

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33 Since the treated plant material has yet to flower, assessment of the L-II or gamete-producing layer has yet to be made. Chimeras are common problems in colchicine treated plant material; therefore, total assessment of increased-diameter shoots is not yet complete. Two of the 3 increased-diameter shoots found to be diploid by chromo some and stomate measurements did flower, and pollen measurements fall within the normal range for diploid V. elliottii. Aberrant and weak growth was observed in several increased diameter subclone populations following 1 year of growth in the field nursery. Colchicine-induced aneuploidy is a possible e x planation for this altered morphology. Vaccinium chromosomes are quite numerous and small, there fore exact counts are not always possible. The chromosome numbers reported in Table 3 are nearest appro x imations. Experiment 2 V. elliottii produced both the greatest total number of shoots and the greatest number of shoots of increased diameter (Table 4). Most of the increased-diameter colonies regenerated from the 4 hour treatment and several from the 12 hour treatment succumbed to fatal decline syndrome (Table 5). Fatal decline is characterized by strong initial growth which ceases when the shoots are 3-5 cm. in height. A few shoots initially take on a water-soaked appearance, and this subsequently spreads throughout the colony. Only colchicine-treated shoots of increased-diameter have been

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Table 4 Growth Response of V. elliottii Following Treatment with 0.1 % ColchTcine for Various Durations Surviving Shoots 34 Treatment NormalIncreasedIncreasedduration Total diameter diameter diameter (hrs) (no. ) 0 415 4 410 12 270 24 133 48 135 Chi-square= 337.40 (no.) (no.) ( % ) 415 0 0 105 5 1 181 59 22 67 66 50 51 84 62 Indicates that the ratio of normal-diameter: increaseddiameter shoots varied with duration of colchicine treatment.

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Table 5 Frequency of Fatal Decline Syndrome Following in vitro Treatment of V. elliottii with 0.1% Colchicine on a Rotary Drum for Various Durations 35 Treatment duration (hrs) Normal-diameter shoots (no.) Fatal decline shoots (no.) 0 4 12 24 48 Chi-square= 643.37 415 105 181 67 51 0 300 30 0 0 Indicates that the ratio of normal-diameter shoots: fatal decline shoots varied with duration of colchicine treatment.

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observed to be afflicted. Affected colonies were examined by pathologists, and no fungi or bacteria were found to be associated with the decline symptoms. The decline is hypothesized to be physiological. Perhaps the decline results when some colchicine-induced increased diameter shoots are altered in their response to some medium compo nent. These shoots may be unable to correctly metabolize some nutrient or they may convert some component of the medium into a toxic byproduct. 36 The 30 increased-diameter subclones of V. elliottii planted on the 3 media varied in growth response and fre quency of fatal decline syndrome (Table 6). There is an obvious interaction between media and frequency of produc tion of very vigorous colonies and colonies with fatal decline. Woody plant medium produced both the greatest number of colonies afflicted with fatal decline syndrome and the most very vigorous colonies. Knops and Andersons media were equal in ability to produce vigorous or average colo nies. Very few very vigorous or fatally declined colonies were produced on these 2 media. Increases in callus growth and decreases in shoot production were observed in both V. darrowi (Table 7) and the interspecific hybrid (Table 8) colonies with increased colchicine treatment duration. Callus can originate from leaves, stems, or buds. If the apical and axillary buds succumb to the phytotoxic effect of colchicine, callus

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37 Table 6 Growth Response of Increased-Diameter Shoots of V. elliottii when Placed on 3 in vitro Media for Regeneration Number of colonies Medium > 20 shoots Andersons 3 Knops 4 Woody plant 17 Chi-square= 46.49 which produced 9:rowth that was 10-20 shoots 1-10 shoots 17 9 27 12 3 0 Medium Number of vials which produced colonies of Healthy growth Fatal decline syndrome Andersons 29 43 20 Knops Woody plant ** Chi-square= 23.65 3 2 16 Indicates interaction between medium and colony vigor. ** Indicates interaction between medium and frequency of fatal decline syndrome.

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Table 7 Growth Response of V. darrowi after Treatment of 2-node Cuttings from in vitro Colonies with 0.1 % Colchicine forVarious Durations Treatment 38 duration Total NormalIncreased(hrs) shoots diameter diameter 0 169 159 10 4 163 141 15 12 89 83 6 24 52 50 2 48 32 32 0 Chi-square = 4.92 N.S. Treatment Number of vials which 2roduced duration Total Callus (hrs) vials only Shoots 0 19 0 19 4 19 1 18 12 19 5 14 24 19 4 15 48 19 11 8 ** Chi-square = 22.86 Indicates non-significant interaction. ** Indicates that the ratio of callus: shoot production varied with duration of colchicine treatment.

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Table 8 Growth Response of V. darrowi XV elliottii in vitro 2-node Cuttings to Treatment with 0.1% Colchicine for Various Durations Treatment 39 duration Total Normal Increased(hrs) shoots diameter diameter 0 102 102 0 4 52 52 0 12 33 33 0 24 27 27 0 48 11 11 0 Treatment Number of vials which produced duration Total Callus (hrs) vials only Shoots 0 19 1 18 4 19 3 16 12 19 6 13 24 19 12 7 48 19 14 5 Chi-square = 28.36 Indicates that the ratio of callus: shoot production varied with colchicine treatment duration.

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40 proliferation, characteristic of these taxons in vitro, may still occur. E x periment 3 Survival rate of explants of the 3 Vaccinium taxons treated with various conc e ntrations of colchicine for various durations in liquid m e dium on a rotar y drum decreased with increasing colchicin e c o ncentr a tions Table 9). ~elliottii had the highest overall survival rating and the high e st frequency of increased-diameter shoots (Table 10). Optimal colchicine concentrations and treatment durations for each of the 3 taxons f or polyploid induction in this experiment w e re: V. elliottii--0.01% for 72 hours; V. darrowi--0.01 % for 48 hours;~darrowi XV. elliottii--0.00 % for 6 hours. Spontaneous chromosome doubling in~darrowi and V. darrowi XV. elliottii apparently w a s responsible for the appearance of large-diameter shoots in the 0.00 % colchicine treatments. E x periment 4 Higher colchicine concentration and longer treatment duration on solid colchicine containing medium were both correlated with decreased survival of 2-node cuttings (Tables 11 and 12). Several regenerated colonies contained shoots of increased diameter. Colchicine concentration of 0.01 % for 2-6 weeks was the most successful treatments for production of increased-diameter shoots in V. elliottii.

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41 Table 9 Effects of Colchicine Concentration and Treatment Duration o n Regrowth Vigor of Trea t ed Vacciniurn E xp l ants Colchicine Regrowth vigor z concn. Duration (hrs) ( % ) 6 1 2 24 48 72 Mean v. darrowi 0.00 2.6 1. 4 2.1 1. 8 2. 9 2.2 0.01 2. 3 1. 2 1. 2 1. 7 0.9 1.5 0.05 1. 7 0. 7 1.0 1. 6 1.0 1. 2 0.10 1. 8 0.6 1.0 1. 4 0.2 1.0 0. 2 0 1. 6 0. 1 0. 9 1.5 0.1 0.8 Mean 2.0 0.8 1. 2 1. 6 1.0 Vigor Score == 1.85 5.13 concn. r == .5 6** V. elliottii 0.00 3.0 2 .2 1. 7 2.6 2. 1 2.3 0.01 2 .4 2. 1 2. 6 3.1 2.8 2.6 0.05 3. 1 1. 3 1. 3 1. 8 0.4 1. 6 0.10 2.8 1. 7 1.2 1.2 0.2 1. 4 0.20 1. 4 0.8 0.2 1. 7 0.6 0.9 Mean 2 .5 1.6 1.4 2.1 1.2 Vigor Score == 2.66 7.61 cone. 01 hours r == .68* Continu e d

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Col chi cine concn. ( % ) 0.00 0.01 0.05 0.10 0.20 Mean 6 v. 2.4 1. 8 1. 6 1. 8 0.7 1. 7 Table 9 Continued Regrowth vigor 2 Duration (hrs) 12 24 48 darrowi x V. elliottii 1. 7 2.7 1. 7 1. 4 2.6 0.8 2. 1 0.7 0. 1 0.8 0.7 0.9 0.9 0.6 0.9 1. 4 1.5 0.5 Vigor Score= 2.21 6.78 concn. .02 hrs. ** r = .81 72 1. 6 0.7 0.0 0.0 0.0 0.5 2 Mean vigor score for 10 vials, each planted with five 2-node explants receiving this treatment (4 = very vigorous; 0 =dead). Yrn these equations, concn. is expressed in parts per *hundred. Slope of regression line significantly different from 0.0 at 5% level; ** 42 Mean 2.0 1.5 0.9 0.7 0.4 Slope of regression line different from 0.0 at 1% level.

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43 Table 10 Number of Colonies Containing 1 or More Shoots of Increased Diameter Following Treatment with Various Colchicine Concentrations for Various Lengths of Time.z Colchicine concn. Treatment duration (hrs) ( % ) 6 12 24 48 72 Total V. darrowi 0.00 0 0 0 2 1 3 0.01 0 0 0 3 0 3 0.05 1 0 0 0 0 1 0.10 0 0 0 1 0 1 0.20 0 0 0 0 0 0 Total 1 0 0 6 1 v. elliottii 0.00 0 0 0 0 0 0 0.01 0 0 0 1 7 8 0.05 5 0 0 2 1 8 0.10 1 5 1 2 1 10 0.20 0 0 1 1 1 3 Total 6 5 2 6 10 V. darrowi x V. elliottii 0.00 3 1 1 0 0 5 0.01 0 0 2 0 0 2 0.05 0 2 0 0 0 2 0.10 0 1 0 0 0 1 0.20 1 0 0 0 0 1 Total 4 4 3 0 0 z For each treatment, 10 vials were used, each planted with five 2-node cuttings.

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44 Table 11 Effects of Colchicine Concentration and Treatment Duration on Regeneration Vigor of V. elliottii Explants Treated on Solid Modiiied Knops Medium Colchicine Regeneration vigor concn. Treatment duration (wks) ( % ) 2 4 6 8 0.00 3.5 2 3.7 3.9 4.0 0.01 3.3 1.5 2.5 1. 2 0.05 1. 7 0.9 0.7 0.1 0.10 1. 3 0.1 0.4 o.o 0.20 0.2 0.1 o.o 0.1 Mean 2.0 1. 3 1.5 1.1 Vigor Score = 3.16 14.87 concn. 0.13 wks. 2 Mean vigor score for 10 vials planted with five 2-node explants receiving this treatment (4 = very vigorous; 0 = dead) Mean 3.8 2.1 0.9 0.5 0.1 Slope of regression line different from 0.0 at 5% level.

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45 Table 12 Number of in vitro Colonies of V. elliottii Producing 1 or l~re Shoots of Increased fiiameter 8 Weeks after Planting with Colchicine-treated Explants Colchicine No. vials with larg:e diameter shoots concn. Treatment duration (wks) ( % ) 2 4 6 8 0.01 6 2 2 0 0.10 2 0 0 0 Mean 4 1 1 0 2 Of 10 total vials. Five 2-node treated explants were planted per vial. Experiment 5 z Mean 2.5 0.5 The 2 clones of V. elliottii and 1 clone of V. darrowi treated for 3 weeks with 0.01% colchicine on solid modified Knops medium produced increased-diameter shoots. Three vials of V. elliottii Clone 121 and 9 vials of V. elliottii Clone 133 contained shoots of increased-diameter. Seven vials of V. darrowi Clone 5 were found to contain increased diameter shoots (Table 13). There was a clonal as well as a species difference in response to colchicine treatment and induction of polyploidy.

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Table 13 Growth Response of 3 Vaccinium Clones to Treatment for 3 Weeks on Solid Modified Knops Medium Containing 0.01 % Colchicine 46 Total shoots (no. } Normal diameter (no.} Increased diameter (no.} Increased diameter shoots Explant source V. elliottii Clone 121 v. elliottii Clone 133 V. darrowi Clone 5 70 290 41 Chi-square= 87.21 ( % } 62 8 11 100 190 65 34 7 19 Indicates that the ratio of normal-diameter: increaseddiameter shoots varied with the clone treated. Experiment 6 Although several seeds germinated from each treatment, none of the seedlings which were germinated on medium containing colchicine survived (Table 14}. The long time required for seed germination may have allowed high concentrations of colchicine to be absorbed, resulting in phytotoxicity.

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V. V 47 Table 14 Response of Seed of V. elliottii and V. darrowi to Colchicine Added to the in vitro Germination Medium Colchicine E x plant cone. 'I'otal Number of seed that source ( % ) seed germinated survived elliottii 0.00 80 24 24 0.10 80 7 0 0.25 80 9 0 darrowi 0.00 80 26 23 0.10 80 6 0 0.25 80 6 0 Conclusions Tissue culture appeared useful as a vehicle for colchicine treatment. Shoots with increased-diameter could easily be screened by visual examination. The space conservative feature of in vitro culture allowed treatment of a great number of rapidly growing shoot tip cuttings, which enhanced the probability for success. Doubled shoots could be rapidly cloned in vitro allowing large numbers of autotetraploid rarnets to be grown. This is quite important because many induced polyploids die before they flower.

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48 The best type of explant to treat is 2-node cuttings. The optimal colchicine concentration/treatment duration combination for induction of polyploids varies with species as well as clone. ~elliottii 2-node cuttings produced the greatest number of increased-diameter shoots when treated for 48 hours on a rotary drum with 0.10% colchicine dis solved in liquid modified Knops medium. When explants were treated on solid modified Knops medium, colchicine at 0.01% for 2-6 weeks was the most successful treatment combination for this species. Both V. darrowi and V. darrowi XV. elliottii clones produced increased-diameter shoots without addition of colchicine to the treatment medium. Spontaneous doubling exceeded colchicine-induced doubling in the interspecific hybrid and was equal in production of increased-diameter shoots to treatment for 48 hours with 0.01% colchicine in liquid modified Knops medium. Colchicine is apparantly unneccessary for induction of polyploidy in the clones of the 2 taxons tested. Due to the clonal variation in response to the doubling action of colchicine, it is advisable to treat many clones of a species. Treatment of many clones is also beneficial for obtaining a broad gene base and diverse breeding lines.

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CHAPTER IV ENHANCEMENT OF COLCHICINE ACTION Introduction Once optimal in vitro colchicine concentration and treatment duration are established for a taxon, polyploids may be easily produced although at a variable frequency. Various researchers have proposed a variety of techniques to reduce the phytotoxicity of the colchicine treatment or to increase the number of cells which are susceptible to the alkaloid. Materials and Methods (Experiments 7-11) Experiment 7 DMSO (dimethyl sulfoxide) is used to increase penetra tion of any substance in solution with it. Colchicine at a concentration of 0.20% was used alone and in combination with 1.0% DMSO in liquid modified Knops medium. Three shoot bases of V. elliottii were tumbled on a wheel drum (3 rpm) in each solution for 7 days. The treated shoot bases were rinsed in liquid modified Knops medium for 24 hours and divided to plant 45 vials of each treatment on solid modi fied Knops medium. After 30 days, the colonies were rated 49

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for survival, vigor and presence of increased-diameter shoots. Experiment 8 50 The cytokinin 2iP (6-gamma-gamma-dimeth y l-allyl amino purine) encourag e s ax illary bud break and formation of numerous meri s tems (81). Young, actively gro w ing m eristems are known to be id e al material for colchicine treatment. The purpose of this stud y was to determine the effect of increased levels of 2iP inco r porated into the medium both before and after colchicine tre a tment. Two-node cuttings of~elliottii Clone 7 from estab lished in vitro colonies were tumbled in liquid modified Knops medium alone or with 0.1 % colchicine. After 48 hours, the cuttings w e re rinsed and planted on modified Knops medium containing either 5, 20, or 40 mg/1 of 2iP. After 8 weeks, the colonies were transferred to modifi e d Knops medium containing 5 mg/1 of 2iP. In addition, various concentrations of 2iP (5, 20 or 40 mg/1) were incorporated into the medium on which seedlings of V. elliottii were planted. The resultant colonies were tumbled in 0.10 % colchicine dissolved in modified Knops medium for 48 hours on a rotary drum (3 rpm). The treated colonies were rinsed and planted on s olid modified Knops medium containing 5 mg/1 of 2iP. After 60 da y s, the colonies were scored for survival and frequency of increased-diameter shoots.

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Experiment 9 Gibberellic acid has been used in combination with etiolation to improve colchicine effectiveness in woody plants (110). Gibberellic acid concentrations of 10 or 100 ppm were used to treat colonies of V. elliottii Clone 4 with or without a 3 day etiolation period. After 3 days the etio lated colonies were returned to the light and on the fifth day, all vials were treated with 0.10% colchicine for 51 48 hours on a rotary drum (3 rpm). Eighteen vials were planted from each treatment on solid modified Knops medium. After 45 days, the colonies were rated for survival and frequency of increased-diameter shoots. Experiment 10 The purpose of this experiment was to determine if colchicine treatment administered before a cold temperature shock would reduce colchicine phytotoxicity or enhance polyploid production. Six shoot bases of V. elliottii Clone 131 were placed in 0.1% colchicine dissolved in liquid modified Knops medium and tumbled on a rotary drum (3 rpm) for 96 hours. After rinsing, the shoot bases were placed on modified Knops medium: 2 at room temperature, 2 in a refrig erator at 7 degrees C. for 48 hours and the remaining 2 at 7 degrees C. for 72 hours. After treatment, each shoot base was divided and 30 vials were planted from each treatment.

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After 60 days, the colonies were evaluated for vigor and increased-diameter shoots. Experiment 11 52 Auxins are known to induce cell mitosis (44). The purpose of this experiment was to determine the effects of various combinations of colchicine treatment, etiolation and auxin treatment. Several colonies of V. elliottii Clone 10 were etiolated for 3 days, then returned to the light for 1 day. Colchicine at 0.10% was dissolved in liquid modified Knops medium alone or with 1 or 10 mg/1 of the auxin, indole-3-butyric acid (IBA), and used to treat 2-node etiolated or non-etiolated cuttings from established in vitro colonies on a rotary drum (3 rpm) for 48 hours. After rinsing, the cuttings were placed on modified Knops medium. Fifteen vials were planted from each treatment combination, and the resultant colonies were rated for vigor and fre quency of increased-diameter shoots after 60 days. Results and Discussion (Experiments 7-11) Experiment 7 The combination of DMSO and colchicine was quite phytotoxic. Thirty days after treatment, 41 of 45 colonies treated with both colchicine and DMSO were dead and 45 of 45 were dead after 60 days. Twenty-eight of the 45 colonies treated with colchicine alone were dead after 30 days and

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53 32 of 45 were dead after 60 days. Of the surviving 13 colo nies, none regenerated shoots of increased-diameter. Although the D M SO did incr e ase the frequency of dead colo nies, the high concentration of colchicine (0.20 % ), the long duration of treatment (7 da y s) and the use of unresponsive shoot bases as explants all were probably contributing factors to the death rate. E x periment 8 Growth of colonies on modified Knops medium with 20 ppm 2iP resulted in an increased number of shoots per colony (Table 15). Colony height was decreased with increased cytokinin concentration due to shortened internode length. The 40 ppm 2iP treatment produced a moss-like colony of z Table 15 In vitro Shoot Formation of V. elliottii when Cultured on 3 Levels of 2iP in Modified Knops Medium mg/1 of 2iP 5 20 40 z Number of shoots 57 148 95 From total of 10 vials per treatment.

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54 shoots. Fewer shoots were apparent per colony because the shoots were so compact that they were indistinguishable from callus. Due to a high incidence of contamination following colchicine treatment, frequency of polyploid formation could not be meaningfully assessed in this experiment. The increased number of treatable shoot meristems formed with increased concentration of 2iP would seem to favor increased production of polyploids. Placement of 2-node cuttings following colchicine treatment on media containing various levels of 2iP did not increase the frequency of increased-diameter shoots over what was obtained with 2iP at 5 mg/1. No increased-diameter shoots were detected in the treated colonies in any of the treatments. Since the clones used in this experiment differed from those used in experiments that yielded increased-diameter shoots, possible clonal variation in susceptibility to the polyplodizing effect of colchicine is suggested. Experiment 9 The use of 0.1% colchicine alone and in combination with 10 ppm or 100 ppm gibberellic acid (G.A.) and/or etiolation in all combinations resulted in increased diameter shoots in all treatments (Table 16). The non etiolated, 0 G.A. treatment was as successful in the

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0 10 100 0 10 100 Table 16 Frequency of Increased-Diameter in vitro V. elliottii Shoots Following Treatment with0.1 % Colchicine, Etiolation, and Gibberellic Acid 55 Total NormalIncreased Increasedshoots diameter diameter diameter (no.) (no.) (no.) ( % ) ppm G.A. 54 15 39 72 ppm G.A. 41 22 19 46 ppm G.A. 95 39 56 59 ppm G.A. + etiolation 90 so 40 44 ppm G.A. + etiolation 91 51 40 44 ppm G.A. + etiolation 118 85 33 28 Chi-square = 36.97 Indicates that the ratio of normal-diameter: increaseddiameter shoots varied with treatment.

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56 production of increased diameter shoots as any other treatment combination. Therefore, colchicine treatment of non-etiolated 2-node cuttings is recommended without G.A. Experiment 10 The cold shock administered after colchicine treatment of in vitro shoot bases of V. elliottii did not decrease phytotoxicity or increase polyploid induction. Increased refrigeration resulted in increased senescence of shoot bases. No increased-diameter shoots were identified from any of the treatments. Experiment 11 The combination of etiolation and auxin resulted in increased senescence. The addition of auxin to the treatment medium increased callus formation. After 120 days of regrowth following treatment, the callus had still not regenerated shoots. The cultures were monitored until the medium was depleted, and were then discarded. Conclusions None of the enhancement techniques tried was more successful than the standard procedure. Thus, the recom mended technique for inducing polyploidy in the Vaccinium taxons studied is to treat 2-node cuttings with 0.1% colchicine dissolved in modified Knops medium. The cuttings

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57 should be tumbled on a rotary drum for 48 hours, rinsed, and replanted on solid modified Knops medium for regeneration of colonies.

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CHAPTER V RADIATION STUDIES Introduction Mutation induction in vegetatively propagated crops can be valuable in altering a few traits in an otherwise out standing cultivar. Perennial fruit crops with a long juvenile stage are excellent candidates for this treatment. In order to treat a species most effectively a survey of radiation tolerence must be accomplished. The purpose of this study was to estimate the LD-50 for in vitro gamma irradiation of diploid V. elliottii colonies and to screen colonies regenerated from treated cuttings for mutant phenotypes. Materials and Methods Twenty colonies of Clone 7 of V. ellottii growing on modified Knops medium were subjected to doses of gamma radiation ranging from 0.5 to 50 kilorads (krads). Each vial contained 30-40 miniature shoots, each with 10-15 axillary buds. V. elliottii is a highly heterozygous diploid (2N=24) species. Immediately after treatment, each colony was divided into 10 subcultures of five 2-node cuttings each and 58

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59 replanted on solid modified Knops medium. After 60 days of regrowth, the colonies were rated for survival. Results and Discussion An LD-50 of 4.5 krads was determined for the treated colonies of V. elliottii. The highest dose which produced living colonies was 25 krads. There was a correlation between increasing radiation dosage and decreasing survival (Table 17). Three colonies (2 from the 1.5 krad treatment and 1 from the 3 krad treatment) appeared to have an altered morpholog y In vitro growth of all 3 altered colonies was feathery and dwarfed in appearance compared to the usual small-leaved vigorous growth typical of the other colonies. Shoots from the altered colonies and from control colonies were severed from the shoot bases and rooted in a greenhouse under mist. About 20 rooted plantlets from each mutant colony and the control were transplanted to a field nursery for observation. The feathery-leaved appearance was not retained in the field. After 1 year in the nursery, differences were noted in leaf color in 1 of the 2 mutants screened from the 1.5 krad treatment. Leaves of this mutant were yellow-green in appearance in contrast with the darker green of normal~elliottii in the same nursery. Leaves of the mutant became darker green as they matured, but remained paler than leaves of normal plants. The mutant plants were less vigorous than comparable non-mutants, and several died

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z Mean Table 17 Effect of Gamma Radiation on Regrowth Vigor of in vitro V. elliottii Explants Krads Vigor z 0.5 4.0 1.0 4. 0 1.5 3.2 2.0 1.0 2.5 2.5 3.0 3.2 3.5 2.6 4. 0 1. 2 4.5 1.1 5.0 1. 7 6. 0 0.6 7.0 0. 1 8.0 0.0 9.0 0.0 10.0 0.2 15.0 0.2 20.0 0.2 25.0 0.5 30.0 0.0 50.0 0.0 vigor score 10 vials (4 = very vigorous; 0 = dead) 60

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61 during the first year. The other 2 mutants appeared normal after 1 year of growth in the field. Conclusions The combination of in vitro plant material and mutagenesis has great potential. Tissue culture colonies contain a high concentration of potentially mutable buds and may also allow mutated cells which would otherwise not survive to be expressed. Once the range of radiation dosage is established for a species, mutations should be easily and readily induced. Only visually apparent mutants were screened from the treated colonies as indicators of optimal dosage. It is recognized that all favorable mutants are not visible.

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CHAPTER VI BREEDING BEHAVIOR OF 12X V. ASHEI COLCHIPLOIDS Introduction V. ashei (2N=6X=72) and~corymbosum (2N=4X=48) each possess valuable characteristics, and production of the interspecific hybrid could be useful. Direct crosses between the species result in SX plants which have so far been of little value in further breeding. In hybridizing the two genomes, various chromosome manipulations may be helpful. Colchicine doubling of 6X ~ashei would result in 12X autopolyploids. When crossed with 4X ~coryrnbosum, BX hybrids might be produced. These 8X's could then be backcrossed to the 4X coryrnbosum, in an effort to produce 6X plants. If this 6X hybrid required more~ashei charac teristics, they could be introgressed into the hybrid genome by backcrossing to V. ashei. Materials and Methods Three doubled plants of~ashei were regenerated from tissue culture treatment with colchicine. Two plants were derived from the cultivar 'Beckyblue', and one from the cultivar 'Bluebelle'. Crosses were made between the 12X 62

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63 plants using each cultivar both as male and female parent. In addition, the 12X plants were used as male parents in crosses with 6X V. ashei cultivar 'Bonita', 4X ~corymbosum cultivar 'Sharpblue' and 2X V. darrowi. The pollen germina tion, number of flowers pollinated, fruit set, mean seed number and mean number percent of viable seed were recorded. The flowers of the 12X plants were larger and thicker than those of their undoubled counterparts (Figure 4). The leaves were larger, darker green and thicker, and the plants were substantially less vigorous than their 6X counterparts. Pollen of each 12X rarnet was germinated using the method of Goldy and Lyrene (49). Freshly collected pollen was dusted on pollen germination medium contained in Petri plates. After 24 hours at room temperature (25 C), the plates were examined under the microscope and percent germination was recorded for 4 fields of vision at 40X magnification. Results and Discussions Pollen tube growth of one 12X 'Beckyblue' ramet was strong, while that of the other 12X 'Beckyblue' ramet was weak, with pollen tubes barely protruding from the pollen grain. The 12X 'Bluebelle' clone also had weak pollen tube growth and the pollen clumped in 2 tetrads (Table 18). No fruit was set when the 12X plants were intercrossed. When used as male parents on 6X V. ashei, only 'Beckyblue'

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Figure 4. Flowers from 12X 'Beckyblue' Subclone l(r.) and from the undoubled 6X 'Beckyblue' (1.) about 1 day before anthesis. 64

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-------------------------65 Table 18 Pollen Germination of 12X Colchiploid and 6X V. ashei Ramets 'Beckyblue' I 12X 'Bec kyblue' II 'Bluebelle' 12X Chi-square 'Beckyblue' 6X 'Bluebelle' 6X 12X = Total grains 113 181 270 218.30 157 211 Number germinated 28 159 56 144 186 % germination 25 88 21 92 88 Indicates that the pollen germination varied with the ramet tested.

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subclone I set 4 fruit from 64 pollinated flowers. The mature berries contained many plump seeds. The seeds have not yet germinated, therefore, it is unknown if the seedlings will be true hybrids or accidental outcrosses. 66 When 'Sharpblue' was crossed with each 12X male parent, many fruit set. Many viable-looking seeds were obtained from the fruit. 'Sharpblue' is highly self-fertile. The high apparent fertility of the 4X. 12X cross seems incompatible with the low degree of pollen viability from the 12X parents, and raises the possibility that the set on 'Sharpblue' was a result of unintentional selfing rather than planned crossing. A few fruit were set when the 12X males were crossed with 2X V. darrowi females. Very few viable-looking seed were obtained from the berries. Most seeds were flattened and shriveled (Table 19). Conclusions The pollen germination and fruit set data suggest that the 12X plants had low fertility both as males and as females.

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Table 19 Fertility of 12X V. ashei Colchiploids when Crossed with Several Vaccinium Species of Various Ploidy Levels and Intercrossed between Cultivars Female parent 12X 'Beckyblue' 12X 'Bluebelle' 6X 'Bonita' 4X 'Sharpblue' 2X V. darrowi 6X 'Bonita' 4X 'Sharpblue' 2X v. darrowi 12X 'Beckyblue' 12X 'Beckyblue' 6X 'Bonita' 4X 'Sharpblue' 2X V. darrowi II II Male # flowers parent pollinated 12X 'Beckyblue' I 12 10 64 38 47 12X 'Beckyblue' II 73 88 63 12X 'Bluebelle' 8 2 38 52 58 # berries harvested 0 0 4 21 0 0 26 3 0 0 0 17 1 X # per seed berry 0 0 32 53 0 0 40 18 0 0 0 32 20 X # seed which appear viable/berry 0 0 1. 3 15.8 0 0 2. 4 1.0 0 0 0 3.8 4.0

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CHAPTER VII CONCLUSIONS Tissue culture is a valuable tool for plant breeding. An in vitro colony with its many shoots and numerous buds allows chemical and physical treatments to take place on a large scale with limited space requirements. The supportive environment allows weaker mutated or polyploidized cells to survive and possibly proliferate. The colonies can be easily screened visually for mutants or increased-diameter shoots, a visible indicator of increased ploidy. Once optimum dosage and duration of treatment are established for a species, the numerous polyploidized clones required for introgression of traits into a gene pool can be easily produced. The most successful polyploidizing treatment varied depending on the taxon and the specific clone treated. For ~elliottii, 0.01% colchicine dissolved in liquid modified Knops medium was the most successful medium for polyploid induction. Two-node in vitro cuttings treated in this medium for 72 hours on a rotary drum (3 r.p.m.) produced the greatest number of polyploid shoots. Both V. darrowi and 68

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69 V. darrowi X ~elliottii, produced polyploid shoots spon taneously in vitro, without added colchicine. When treat ment of 2-node V. elliottii cuttings were treated on solid colchicine containing, modified Knops medium, 0.01% colchi cine for 2 weeks duration was the most effective treatment for polyploid induction. Induced autotetraploids in Vaccinium should be valuable in moving adaptive and economically important traits from diploid native species into cultivated tetraploid breeding lines. In vitro colchicine treatment may also be quite useful in facilitating interspecific gene transfers in other genera where the species differ in ploidy. This will make it possible to tap new gene pools in the breeding of some crop species. ~elliottii, treated with a wide range of radiation, produced several visible mutants in vitro. An LD-50 of 4.5 krads was established for the treated clone. The mutants, 2 derived from the 1.5 krad treatment and 1 from the 3 krad treatment, were visually identified by altered growth form when compared to control colonies. Induction of mutations facilitated by radiation treat ment of in vitro colonies should be valuable in improvement of long generation, vegetatively propagated perennial crops. Alteration of a few traits of an otherwise outstanding cultivar with mutagenesis may obivate the extensive cycles of hybridization to wild species and backcrossing ordinarily needed to restore the horitcultural value of a cultivar.

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70 The enhancement of adventitious bud formation is an important feature of in vitro culture, because it allows formation of a higher frequency of solid polyploids and mutants. In some cases, the in vitro system alone is sufficient to allow genetic alterations to occur in explants. With, the added impetus of colchicine or radia tion, a much higher frequency of mutant genotypes should be obtained.

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REFERENCES 1. Aalders, L.E., and I.V. Hall. 1962. New evidence on the cytotaxonomy of Vaccinium species as revealed by stomatal measurements from herbarium specimens. Nature 196:694. 2. Aalders, L.E., and I.V. Hall. 1963. Note on aeration of colchicine solution in the treatment of germinating blueberry seeds to induce polyploidy. Can. J. Plant Sci. 43:107. 3. Ackerman, W. L., and H. Dermen. 1972. A fertile colchiploid from an interspecific Camellia hybrid. J. Hered. 28:56. 4 Ahokas, H. 1971. Vaccinium species. Notes on polyploidy and hybridity in Ann Bot. Fennici 8:254. 5. Allard R. W. 1960 Principles of Plant Breeding. John Wiley and Sons, Inc. New York, N.Y. 6. Anderson, W. C. tissue culture. 1975. Propagation of Rhododendrons by Proc. Intern. Plant Prop. Soc. 25:129. 7. Ballington, J. R., and G. J. Galletta. 1974. Effects of gibberellin compounds, concentrations and treatment duration on blueberry seed germination. HortScience 9:283. 8. Ballington, J. R., and G. J. Galletta. 1976. Potential fertility levels in four diploid Vaccinium species. J. Amer. Soc. Hort. Sci. 101(5) 507. 9. Ballington, J. R., and G. J. Galletta. 1978. Comparative crossability of four diploid Vaccinium species. J. Amer. Soc. Hort. Sci. 103:554. 10. Bayliss, M. W. 1980. Chromosomal variation in plant tissues in culture. International Review of Cytology Suppl llA p. 113. Academic Press, New York, N.Y. 11. Blakeslee, F. A., and A. G. Avery. 1937. Methods of inducing doubling of chromosomes in plants by treatment with colchicine. J. Hered. 28:393. 71

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I I 72 12. Broertjes, C. 1974. Production of polyploids by the adventitious bud technique. FAO/IAEA Eucarpia Conf. on Polyploidy and Induced Mutations in Plant Breeding. Bari 1972:29. 13. Broertjes, C., B. Haccius, and S. Weidlich. 1968. Adventitious bud formation on isolated leaves and its significance for mutation breeding. Euphytica 17:321. 14. Broertjes, C., and A. Keen. 1930. Adventitious shoots: do they develop from one cell? Euphytica 29:73. 15. Broertjes, C., S. Roest, and G. C. Bokelmann. 1976 .Mutation breeding of Crysanthemum morifolium using in vitro and in vitro adventitious bud techniques. Euphytica 25:11. 16. Broertjes, c., and A. M. Van Harten. 1978. Application of Mutation Breeding Methods in the Improvement of Vegetatively Propagated Crops. Elsevier Scientific Pub. Co., New York, N. Y. 17. Camp, W. H. 1942. On the structure of populations in the genus Vaccinium. Brittonia 4:189. 18. Camp. W. H. 1945. The North American blueberries. Brittonia 5:203. 19. Chandler, C. K. 1980. Guard Cell Length and Leaf Thickness as Indicators of Induced Polyploidy in Vaccinium. M. S. Thesis, University of Florida, Gainesville, Fl. 20. Chandler, C. K., and P. M. Lyrene. 1982. Relationship between guard cell length and ploidy in Vaccinium. HortScience 17(1) 53. 21. Chen, C.H., and Y. C. Goeden-Kallemeyn. 1979. In vitro induction of tetraploid plants from colchicine treated diploid daylilly callus. Euphytica 28:705. 22. Cockerham. L. E., and G. J. Galletta. 1976. A survey of pollen characteristics in certain Vaccinium species. J. Amer. Soc. Hort. Sci. 101(...) 671. 23. Coville, F. V. 1927. Blueberry chromosomes. Science 66:564. 24. Coville, F. V. 1937. Improving the wild blueberry. u. s. Dept. Agri. Yearbook of Agri. p. 559. U.S. Government Printing Office. Washington, D.C.

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73 25. Darrow G. M. 1941. Seed size in blueberry and related species. Proc. Amer. Soc. Hort. Sci. 38:438. 26. Darrow, G. M. 1949. Polyploidy in fruit improvement. Proc. Amer. Soc. Hort. Sci. 54:523. 27. Darrow, G. M., and W. H. Camp. 1945. Vaccinium hybrids and the development of new horticultural material. Torrey Bot. Club Bull. 72:1. 28. Darrow, G. M., W. H. Camp, H. E. Fischer, and H. Dermen. 1944. Chromosome numbers in Vaccinium and related groups. Torrey Bot. Club Bull. 71:498. 29. Darrow, G. A., H. Dermen, and D. H. Scott. 1949. A tetraploid blueberry from a cross of diploid and hexaploid species. J. Hered. 40:304. 30. Darrow, G. M., E. B. Morrow, and D. H. Scott. 1952. An evaluation of interspecific blueberry crosses. Proc. Arner. Soc. Hort. Sci. 59:277. 31. Darrow, G. M., D. H. Scott, and H. Dermen. 1954. Tetraploid blueberries from he x aploid X diploid species crosses. Proc. Amer. Soc. Hort. Sci. 63:266. 32. Decourt y e, L. 1964. The use of radiation in apple and pear breeding. In: Proc. Balsgard Fruit Symp. Balsgard Fruit Breeding Institute, Fjalkestad. p.40. 33. Dermen, H. 1937. Detection of polyploidy by pollen grain size. Proc. Amer. Soc. Hort. Sci. 35:96. 34. Dermen, H. 1938. Cytological analysis of polyploidy induced by colchicine and by extremes of temperature. J. Hered. 29:211. 35. Dermen, H. 1940. Colchicine polyploidy and technique. Bot. Rev. 6:599. 36. Derrnen, H. 1945. The mechanism of colchicine induced cytohistological changes in cranberry. Arn. J. Bot. 32:387-394. 37. Dermen, H. varieties. 1947. Inducing polyploidy in peach J. Hered. 38:77. 38. Derrnen, H. 1954. Colchiploidy in grapes. J. Hered. 45:159. 39. Derrnen, H. 1967. Colchiploidy and cytochimeras in the study of ontogenic problems. Proc. XVII International Horticultural Congress 2:3.

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40. Dermen, H., chestnuts. and J. D. Diller. 1962. Forest Sci. 8(1) 43. Colchiploidy of 41. Donnini, B. 1976. The use of radiation to induce useful mutations in fruit trees. In: Improvement of Veg e tatively Propagated Plants and Tree Crops Through Induced Mutations. Wageningen, Vienna. p. 50. 74 42. Doorenbos, J. 1977. Spontaneous mutations as a source of clonal variation in deciduous fruit. Acta Hortic. 75:13. 43. Draper, A. D. 1977. Tetraploid hybrids from crosses of diploid, tetraploid and h ex aploid Vaccinium species. Acta Hortic. 61:33. 44. Dyer, A. F. 1979. Investigating Chromosomes. John Wiley & Sons, New York, N.Y. 138p. 45. Eck, P., and N. F. Childers. 1966. Blueberry Culture. Rutgers Univ. Press, New Brunwsick, N.J. 46. Eigsti, O. J. 1938. A cytological study of colchicine effects in the induction of polyploidy in plants. Proc. Nat. Acad. Sci. 24:56. 47. Fry, B. o. 1963. Production of tetraploid muscadine grapes by gamma radiation. Proc. Am. Soc. Hort. Sci. 83:388. 48. Fyfe, J. L. 1939. The Action and Use of Colchicine in the Production of Polyploid Plants. Cambridge School of Agri. Press, Cambridge, England. 49. Goldy, R. G., and P. M. Lyrene. 1983. Pollen germination in interspecific Vaccinium hybrids. HortScience 18(1) :54. 50. Hall, I. V. sections of cultured on regulators. Callus formation in stem internode lowbush blueberry (Vaccinium angustifolium) a medium containing plant growth Hort. Res. 16:29. 51. Hall, S. H., and G. J. Galletta. 1971. Comparative chromosome morphology of diploid Vaccinium species. J. Amer. Soc. Hort. Sci. 96:289. 52. Harlan, J. R., and J.M. J. Dewet. 1975. and a prayer: the origins of polyploidy. 41:361. On O Winge Bot. Rev. 53. Heinz, D. J., and G. W. P. Mee. 1970. Colchicine induced polyploids from cell suspension cultures of sugarcane. Crop Sci. 10:696.

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54. Hermsen, J. G., M. S. Ramanna, S. Roest, and G. S. Bokelmann. 1981. Chromosome doubling through shoot formation on in vitro cultivated leaf explants from diploid interspecific potato hybrids. Euphytica 30:239. 75 55. Iyer, C. P.A., and G. S. Randhawa. 1965. Increasing colchicine effectiveness in woody plants with special reference to fruit crops. Euphytica 14:293. 56. Janick, J., and J. N. Moore. 1975. Advances in Fruit Breeding. Purdue Univ. Press, West Lafayette, Ind. 57. Jelenkovic, G., and A. D. Draper. 1970. Fertility and chromosome behavior of a derived decaploid of Vaccinium. J. Am. Soc. Hort. Sci. 95(6) :816. 58. Jelenkovic, G., and A. D. Draper. 1973. Breeding value of pentaploid interspecific hybrids of Vaccinium. Jugoslovensko Vocarstvo God. VII br. 25-26 st 237. 59. Jelenkovic, G., and E. Harrington. 1971. Non-random chromosome associations at diplotene and diakinesis in a tetraploid clone of Vaccinium australe Small. Can. J. Genet. Cytol. 13:270. 60. Jelenkovic, G., and L. F. Hough. 1970. Chromosome associations in the first meiotic division in 3 tetraploid clones of V. corymbosum L. Can. J. Genet. Cytol. 12:316. 61. John, B. and K. R. Lewis 1965. The meiotic system. Protoplasmalogia 6 (Fl) :1. 62. Kasha, K. J. 1974. Haploids in Higher Plants Advances and Potential. Proc. First International Syrop. University of Guelph. 63. Kostoff, D. 1938. The size and number of the chloroplasts and the chlorophyll content in eupolyploid forms experimentally produced. Current Sci. 7:270. 64. Larkin, P. J., and W. R. Snowcroft. 1981. Somaclonal variation: a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60:197. 65. Lewin, B. 1980. Gene Expression Vol II. Wiley and Sons. New York, N.Y. p.30. 66. Lewis, H. 1980. Proceedings of the International Conference on Polyploidy. Washington Univ., St. Louis, Mo. Plenum Press, New York, N.Y.

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67. Lloyd, G. M., and B. H. Mccown. 1980. Use of microculture for production and improvement of Rhododendron spp. HortScience 15:416. 68. Longley, A. E. 1927. Chromosomes in Vaccinium. Science 66:566. 69. Lyrene, P. M. 1978. Blueberry callus and shoot tip culture. Proc. Fla. State Hort. Sci. 91:171. 76 70. Lyrene, P. M. 1979. In vitro propagation of rabbiteye blueberries. In Proc.of the Fourth North American Blueberry Research Workers Conference. (Ed. 71. J. N. Moore). University of Arkansas, Fayetteville, Ar. Ed. J. N. Moore. p.249. Lyrene, P. M. blueberries. 1980. Micropropagation of rabbiteye HortScience 15(1) :80. 72. Lyrene, P. M., and J. L. Perry. 1982. Production and selection of blueberry polyploids in vitro. J. Hered. 73:377. 73. Lyrene, P. M., and W. B. Sherman. 1980. Horticultural characteristics of native Vaccinium darrowi, V. elliottii, ~fuscatum and~myrsinites in Alachua County, Florida. J. Amer. Soc. Hort. Sci. 105(1_):393. 74. Lyrene, P. M., and W. B. Sherman. 1977. Breeding blueberries for Florida: accomplishments and goals. Proc. Fla. State Hort. Sci. 90:215. 75. Lyrene, P. M., and W. B. Sherman. 1980. The occur ranee and severity of cane canker (Botryosphaeria corticis) on Vaccinium species native to Florida. HortScience 15:150. 76. Lyrene, P. M., and W. B. Sherman of southern highbush blueberries. 1981. Breeding value HortSci. 16(4) :528. 77. Moore, J. N. 1965. Improving highbush blueberries by breeding and selection. Euphytica 14:39. 78. Moore, J. N., and J. Janick. 1983. Methods in Fruit Breeding. Purdue Univ. Press, West Lafayette, Ind. 79. Moore, J. N., B. D. Reynolds, and G. R. Brown. 1972. Effects of seed number, size and development on fruit size in cultivated blueberries. HortScience 7:268. 80. Moore, J. N., D. H. Scott, and H. Dermen. 1964. Development of a decaploid blueberry by colchicine treatment. Proc. Amer. Soc. Hort. Sci. 84:274.

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I 77 81. Murashige, T. 1978. Principles of rapid propagation. In: Propagation of Higher Plants Through Tissue Culture. Ed. P. Henke and M. Constantin. University of Tennessee, Knoxville. 82. 83. Murashige, T., and R. Nakano. 1966. Tissue culture as a potential tool in obtaining polyploid plants. J. Hered. 57:114. Newcomer, E. H. 1941. Chromosome numbers of some species and varieties of Vaccinium and related genera. Proc. Arner. Soc. Hort. Sci. 38:468. 84. Nickerson, N. L. 1978. In vitro shoot formation in lowbush blueberry seedlingexplants. HortScience 13:698. 85. Orton, T. J. 1980. Chromosomal variability in tissue culture and regenerated plants of Hordeum. Theor. Appl. Genet. 56:101. 86. Orton, T. J., and R. P. Steidl. 1981. Cytogenetic analysis of plants regenerated from colchicine treated callus cultures of an interspecific Hordeum hybrid. Theor. Appl. Genet. 57:89. 87. Parthasarathy, N. 1953. The use of induced autopolyploidy in plant breeding. Ind. J. Genet. and Plant Breeding 13:1. 88. Phillips, G. C., and G. B. Collins. 1980. Somatic embryogenesis from cell suspension cultures of red clover. Crop Sci. 20:323. 89. Pryor, R. L., and L. C. Frazier. 1968. Colchicine induced tetraploid azaleas. HortScience 3(4) :283. 90. Roest, S., M. A. E. Van Berkel, G. S. Bokelmann, and C. Broertjes. 1981. The use of an in vitro adventitious bud technique for mutation breeding of Begonia X Hiemalis. Euphytica 30:381. 91. Rousi, A. 1966. Cytological observations on some species and hybrids of Vaccinium. Zuchter 36:352. 92. Sanders, H., and J. W. Hull. 1970. Dimethyl sulfoxide as an adjuvant of colchicine in the treatment of Rubus seeds and shoot apices. HortScience 5:111. 93. Sapra, V. T., J. L. Hughes, and G. C. Sharma. 1975. Frequency, size and distribution of stomata in Triticale leaves. Crop Sci. 15:356.

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94. Sax, H. J., and K. Sax. 1937. Stomata size and distribution in diploid and polyploid plants. Jour. Arnold Arb. 18:164. 9 5. Sharpe, R. H. 19 5 4. Florida blueberries. 66:188. Horticultural development of Proc. Fla. State Hort. Soc. 78 96. Sharpe, R.H., and G. M. Darrow. 1959. Breeding blueberries for the Florida climate. Proc. Fla. State Hort. Soc. 72:308. 97. Sharpe, R. H., and W. B. Sherman. 1971. blueberries for low-chilling requirement. 6:145. Breeding HortScience 98. Shepard, J. F. D. Bidney, and E. Shahin. April 1980. Potato protoplasts in crop improvement. Science V. 208:17. 99. Shmuck, A., and D. Kostoff. 1939. Brome-acenaphthene and brome-naphthaline as agents inducing chromosome doubling in rye and wheat. Compt. Rend. (Doklady) Acad. Sci. URSS 23:263. 100. Simmonds, N.W. 1979. Principles of Crop Improvement. Longman Group Limited, London, England. 101. Skirvin, R. M. tissue culture. 1978. Natural and induced variation in Euphytica 27:241. 102. Stushnoff, C., and B. F. Palser. 1969. Embryology of 5 Vaccinium taxa including diploid, tetraploid and he x aploid species or cultivars. Phytomorphology 19:312. 103. Tabachnick, L., and D. E. Kester. 1977. Shoot culture for almond and almond-peach hybrid clones in vitro. Hortscience 12:545. 104. Tan, G., and G. M. Dunn. 1973. Relationship of stomatal length and frequency and pollen grain diameter to ploidy level in Bromus inermis Leyss. Crop Sci. 13:332. 105. Vander Kloet, S. P. 1976. Nomenclature, taxonomy and biosystematics of Vaccinium section Cyanococcus (the blueberries) in North America. I. Natural barriers to gene exchange between Vaccinium angustifolium Ait. and ~corymbosum L .. Rhodera V.78 815:503. 106. Vander Kloet, S. P. 1977. Potential and actual gene exchange among 3 sympatric species of Vaccinium and Cyanococcus in Highlands County, Florida. Can. J. Bot. 55:2668.

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79 107. Vander Kloet, S. P. 1979. The taxonomy of the highbush blueberry, Vaccinium corymbosum. Can. J. Bot. 58:1187. 108. Vander Kloet, s. P. 1983. The relationship between seed number and pollen viability in Vaccinium corymbosum L. HortScience 18(2) :225. 109. Ward, D. B. 1974. Contributions to the flora of Florida -6, Vaccinium (Ericaceae). Castanea 39:205. 110. Zatyko, J.M., and I. Simon. 1964. Possibilities in the use of gibberellic acid for increasing colchicine induced polyploidy ration. Naturwissenschaften 51 (16) :392. 111. Zimmerman, R. H., 1978. and other fruit plants. 28:539. Tissue culture of fruit trees Proc. Intern. Plant Prop. Soc. 112. Zimmerman, R.H., and O. C. Broome. 1980. Blueberry micropropagation. In: Proc. Conference on Nursery Production of Fruit Plants Through Tissue Culture. U.S. Department of Agriculture. Beltsville, Md.

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BIOGRAPHICAL SKETCH Julia Lucile Wood Perry was born on May 6, 1955, in Bethesda, Maryland. She attended high school in Shreveport, Louisiana, at St. Vincent's Academy, graduating in 1973. She attended the University of Colorado, Boulder, for two years, majoring in archaeology. In 1976, she married Robert Thomas Perry. She completed her B.S. degree at Eastern Kentucky University in Richmond in 1977, with a major in horticulture. She received her M.S. in 1980 from the University of Arkansas, Fayetteville, working under the direction of Dr. James Moore. Her research problem was determination of selfand cross-compatibility of various tetraploid culti vars of blackberry (Rubus Eubatus). In 1980 she began work with Dr. Paul Lyrene at the University of Florida, Gainesville, to facilitate hetero ploid crosses between Vaccinium species by in vitro colchicine treatment of native diploids. 80

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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 Docotor of Philosophy. Paul M. Lyrene, Chairman Associate Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Docotor of Philosophy. Wayne B. Sherman Prof~ sor 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 Docotor of Philosophy. Gloria A. Moore Assistant Professor of Horticultural Science

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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 Docotor of Philosophy. Mark Bassett 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 Docotor of Philosophy. IL Q L/4,~ ; ~ Ken Quesenberry Associate Professo;: Agronomy This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fullfillment of the requirements for the degree of Doctor of Philosophy. April, 1984 Dean College of A riculture (/ Dean for Graduate Studies and Research

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UNIVERSITY OF FLORIDA 111111111111111111111111111 II IIIIII IIII II IIIIII IIII II IIIIII Ill I I 3 1262 08553 4435