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MULTIFACTOR ANALYSIS OF ENVIRONMENTAL PRE-CONDITIONING OF
TOMATO SEEDLINGS ON PROTOPLAST CULTURE AND DEVELOPMENT

BY
RANDALL PAUL NIEDZ

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Plant Breeding and Genetics Inter-Departmental Program

1987

:8 1r

ABSTRACT

MULTIFACTOR ANALYSIS OF ENVIRONMENTAL PRE-CONDITIONING OF
TOMATO SEEDLINGS ON PROTOPLAST CULTURE AND DEVELOPMENT

BY
Randall Paul Niedz

Leaf mesophyll protoplasts were enzymatically isolated
from 24-30 day old seedlings of Lycopersicon esculentum P.I.
367942 grown either in a controlled environment chamber (CEO)
or i_ giggg (IV). Prior to protoplast isolation, donor
seedlings were pre—conditioned in a 3x3 factorial arrangement
of 0, 12 or 24 hours at 10°C and O, 24 or 48 hours in the
dark. The variables measured included protoplast yield, 244
hour viability, 30—day microcalli diameter and shoot
morphogenesis. Multifactor fixed effect linear models were
constructed to determine the influence of seedling source and
cold/dark pre-conditioning on the observed variables.

Protoplasts were cultured in the dark (27°C) in a liquid
modified Kao and Micahyluk (KM) medium over a semi-solid
modified Murashige and Skoog (MS) medium. Both media
contained the growth regulators (mg/L): 2,4-
dichlorophenoxyacetic acid (1AM, alpha—naphthaleneacetic
acid (1.0) and 6-benzylaminopurine (01”. To stimulate shoot
morphogenesis protoplast—derived calli were cultured in the
light (cool white fluorescent lamps; 30 uEm'zs'l) on modified
MS medium with 2 mg/L zeatin.

Protoplasts from CEC donor seedlings pre—conditioned with

RANDALL PAUL NIEDZ
0 hours dark/12 and 24 hours cold died in 24-48 hours.

Protoplast yield from CEC seedlings preconditioned with
12 and 24 hours of cold was reduced an average of 27.4%
(compared to 0 hours cold over all levels of dark). However,
the subclass 12 hours cold/48 hours dark was not
significantly different from 0 hours cold/all levels of dark.
In.contrast, yield from IV seedlings was not significantly
reduced by cold preconditioning, but decreased an average
43.9% by a 48 hour dark treatment over all levels of cold.
Protoplast viability was improved an average of 14.1%
of CEO seedlings receiving a 48 hour dark treatment, and was
0% for the 2 treatments 12 and 24 hours cold/O hours dark.
For IV seedlings preconditioned with 12 and 24 hours cold/0
hours dark protoplast viability was 6.9% greater than
compared to 0 hours cold.

CEC microcalli diameter of 12 and 24 hour cold were
12.5% less than 0 hour cold, In contrast, IV microcalli from
12 hour cold/0 hour dark:were~11.8% larger than microcalli
from all other IV preconditioning treatments.

Shoot morphogenesis from CEC seedlings was 10.3% less
from 0 hours dark compared to 24 and 48 hours dark, and 25.4%
less from 24 hour cold compared to O and 12 hour cold

preconditioning of IV seedlings.

TABLE OF CONTENTS

Page

LIST OF TABLES ---------------------------------------- v
LIST OF FIGURES --------------------------------------- vi
ABBREVIATIONS ----------------------------------------- vii
INTRODUCTION ------------------------------------------ 1
LITERATURE REVIEW ————————————————————————————————————— 4
I. History of the tomato -------------------------- 5
II. In vitro culture ------------------------------- 5
A. Tissue culture ------------------------------- 5

B. Genetic control of shoot morphogenesis ------- 8

C. Protoplast culture --------------------------- 11

D. Anther culture ------------------------------- 13

E. Uses ----------------------------------------- 14

III. Preconditioning of donor tissue ---------------- 18
A. Genotype ------------------------------------- 18

B. Greenhouse, CEC, _I_n_ y_ tr_q ------------------ 19

C. Light ---------------------------------------- 20

D. Nutrition ------------------------------------ 21

E. Tissue age and source ------------------------ 22

F. Senescence retardents ------------------------ 23

G. Temperature and humidity --------------------- 23
MATERIALS AND METHODS --------------------------------- 24
I. Protoplast isolation and culture ---------------- 24

A. Source Material ------------------------------- 24

B. Seedling Preparation -------------------------- 24

1. CEC grown seedlings ------------------------ 24

2. In vitro grown seedlings ------------------- 24

3. Cold and dark pretreatment of donor tissue — 25

ii

C. Protoplast Isolation -------------------------- 25

1. CEC grown seedlings ------------------------ 25

2. In vitro grown seedlings ------------------- 27

D. Protoplast Culture and Shoot morphogenesis -—-— 27

II. Experimental design and data analysis ———————— ~—— 28
RESULTS ----------------------------------------------- 29
I. Yield ------------------------------------------- 29

A. Test of interaction effect -------------------- 29

B. Goodness of fit ------------------------------- 29

C. Contrasts ------------------------------------- 30

II. Viability --------------------------------------- 33
A. Test of interaction effect -------------------- 33

B. Goodness of fit ------------------------------- 33

C. Contrasts ------------------------------------- 33

III. Microcalli Diameter ------------------------------ 36
A. Test of interaction effect -------------------- 36

B. Goodness of fit ------------------------------- 36

C. Contrasts ------------------------------------- 36

IV. Shoot Morphogenesis ------------------------------ 39
A. Test of interaction effect -------------------- 39

B. Goodness of fit ------------------------------- 39

C. Contrasts ------------------------------------- 40
DISCUSSION -------------------------------------------- 44
I. Protoplast Yield ------------------------------- 44
II. Protoplast Viability --------------------------- 45
III. Microcalli Diameter ---------------------------- 47
IV. Shoot Morphogenesis ---------------------------- 49
SUMMARY AND CONCLUSIONS -------------------------------- 51
APPENDIX A Summary Statistics and Data Analysis ------- 53
APPENDIX B Least Squares Approach --------------------- 72
1. General fixed model ------------------------- 73

2. Estimation ---------------------------------- 76

3. The normal equations ------------------------- 77

4. Solving for b ------------------------------- 80

5. Estimable functions ------------------------- 81

6. Other calculations -------------------------- 85

111

7. MEthOd of calculation ———————————————————————

LIST OF REFERENCES

iv

Table

A1.
A2.
A3.

A4.

A5.
A6.
A7.
A8.
A9.
A10.
A11.
A12.
A13.
A14.
A15.
A16.
A17.

A18.
A19.
Bl.

82.
83.
84.

LIST OF TABLES

Page
Reductions in sum of squares for yield ------------ 53
Reductions in sum of squares for viability -------- 54
Reductions in sum of squares for microcalli
diameter ------------------------------------------ 55
Reductions in sum of squares for shoot
morphogenesis ------------------------------------- 56
Single factor evaluation -------------------------- 57
Goodness of fit for 2-way interaction model ——————— 58
Goodness of fit for 3-way interaction model ------- 59
Smallest subclass means for yield ----------------- 60
Contrasts for yield ------------------------------- 61
Smallest subclass means for viability ------------- 62
Contrasts for viability --------------------------- 63
Smallest subclass means for microcalli diameter --- 64
Interaction effects ------------------------------- 65
Contrasts for microcalli diameter ----------------- 66
Smallest subclass means for shoot morphogenesis --- 67
Contrasts for shoot morphogenesis ----------------- 68
Pre-conditioning treatments with the highest means
for protoplast yield, viability, microcalli
diameter and.shoot morphogenesis --------------- 69
Tomato protoplast media --------------------------- 70
Purification of agar ------------------------------ 71
Protoplast yield (x106) of dark pre-conditioned
seedlings ----------------------------------------- 74
x matrix as a 2-way table ------------------------- 78
Four solutions for b0 ----------------------------- 82
Estimates of four linear functions ---------------- 84

LIST OF FIGURES

Figure

1.

2.

Effect of cold and dark pre-conditioning on

protoplast yield. (a) CEC grown seedlings. (b)

IV grown seedlings. -----------------------------

Effect of cold and dark pre-conditioning on

protoplast viability. (a) CEC grown seedlings.
(b) IV grown seedlingS. _________________________

Effect of cold and dark pre-conditioning on
microcalli diameter. (a) CEC grown seedlings.
(b) IV grown seedlings. -------------------------

Effect of cold and dark pre-conditioning on shoot

morphogenesis. (a) CEC grown seedlings. (b) IV
grown seedlings. --------------------------------

vi

Page

31

34

37

42

ABBREVIATIONS

BAP — 6—benzylaminopurine
NAA - alpha-naphthalenacetic acid
2,4-D - 2,4-dichlorophenoxyacetic acid

CEC - controlled environment chamber
IV - in vitro

C — factor cold

D - factor dark

8 - factor source

CD - cold x dark interaction

CS - cold x source interaction

DS - dark x source interaction

CDS - cold x dark x source interaction

uEm"28-1 - Micro Einstein per square meter and second

vii

INTRODUCTION

Most plant breeding programs follow a process of
recombination and selection within an elite germplasm pool to
produce superior cultivars. Techniques such as induced

polyploidy, wide hybridization, mutant induction and i gitro

 

methods supplement primary breeding operations by providing
geneth: variability difficult and/or impossible to obtain
otherwise. ;_ yitrg methods can be classified into three
general catagories; 1) the micropropagation of shoot tips to
produce viruS*free stock, multiply hard to propagate species
and expensive F1 hybrids, 2) the culture and manipulation of
protoplasts, cells and tissue to produce genetically useful
individuals and, 3) vector introduced genes.

Plant regeneration from protoplasts has been
demonstrated in over 70 species, half of which are
represented by the Solanaceae. Successful application of in
yitgg techniques such as exogenous DNA and chromosome uptake
by protoplasts, somatic cell fusion and cell selection to
improve a species economically, requires a base technology
of efficient plant regeneration from protoplasts. Factors
important in successfully regenerating plants from cultured
protoplasts include genotype, growth conditions of the donor

plant, pre-conditioning of donor tissue, types and

1

2

concentrations of isolation enzymes, composition of the
culture medium and the environmental conditions during
isolation and culture. In tomato and potato the
physiological condition of the donor plant is of critical
importance in achieving successful isolations of viable
protoplasts capable of sustained divisions (160,164,168).
Tabaeizadeh et al. (175) found that a cold/dark treatment of
whole plants and detached leaves significantly increased the
number of protoplast-derived calli though the effect was not
as great with detached leaves. Shahin (160) reported the
necessity of dark and low temperature preconditioning of
donor tomato tissue in isolating viable protoplasts.
Likewise, Shepard and Totten (165) used a short photoperiod
and low'temperature preconditioning of donor plants. Without
such preconditioning, protoplasts failed to undergo division
regardless of the culture medium used. How these factors
influence the various stages of growth from initial cell
viability to shoot formation from.protoplast-derived calli is
little understood as much of the research to date is
empirical and/or anecdotal.

This study attempts to identify those stages of
protoplast development most influenced by dark and cold
preconditioning of the donor plant by preconditioning donor
tissue and measuring protoplast yield, viability, rate of
cell colony growth and shoot morphogenesis. The extent that
such treatments are necessary in achieving shoot regeneration

can then be examined by determining the contribution of cold

1"

 

 

3

and dark on each measured variable. The tomato, Lycqpersicon

 

esculentum, is used in this study as it is the worldws
leading vegetable crop and is also a prominent species for

somatic cell genetic manipulations.

LITERATURE REVIEW

I. History of the Tomato

The cultivated tomato, Lyggpersicon escglentgm,

 

originated in western South America and is a member of the

family Solanaceae. All ten species in the genus Lycqpersicon

 

are 2g-2g-24 (146). Domestication is thought to have first
occurred in Mexico. When the tomato was first taken to
Europe in the 16th century it was thought to be poisonous due
to deadly glycoalkaloids present in the other members of the
nightshade family such as belladonna and henbane.

The tomato currently ranks second to potato in economic
importance among vegetables, with a combined value for
processing and fresh market fruit exceeding 800 million
dollars per year in the United States (111). In addition to
its culinary appeal the tomato is one of the best known crop
plants in terms of its established genetics and cytogenetics
(147). A favorable biology such as a short life cycle,
ecological versatility, high seed yields (up to 20,000 seeds
per plant), diploid genome, self—pollinating and amenability
to hybridization are responsible for this species appeal to
researchers. Plant breeders have made significant
improvements during the past 50 years as follows:

1. Increased yields - larger and greater numbers of
fruit per plant, improved flower set and a more
concentrated fruit set

2. Plant habit changed to accomodate cultural and
harvesting operations - the gp gene for determinate

growth is mainly responsible for such improvements

4

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3. Improved handling and storage

4. Pest resistance.

II. In Vitro Culture

 

A. Tissue culture

The first successful 1 yitro culture of tomato was

 

reported by White in 1934 (199). He succeeded in culturing
isolated root tips in liquid suspension culture. The roots
grew indefinitely for over a year. This indicated to White
that root growtthas not dependent on the top of the plant.
Twenty years later root cultures were again utilized, but
from the wild.species Lycgpersicon peruvianum (127). Some of
the cultured roots formed shoots that were transferred to
soil.

It was not until 1973 that routine shoot morphogenesis

was first reported using cultured internodes of’;u_esculentum

 

(42). Numerous reports followed detailing rapid shoot
morphogeneous from a variety of tissues: leaves
(8,35,74,80,87,106,129,133,137,17B), stem internodes
(41,103), apical meristems (86), cotyledons (66,92,134,195)
and hypocotyls (66,71,103,131,210). The general methodology
consisted of placing explants from in gitgg or greenhouse
seedling explants onto a semisolid medium containing
inorganic salts, macronutrients and micronutrients, usually
those of Murashige and Skoog (120) , vitamins of the B group
(thiamine, pyridoxine and nicotinic acid), glycine, myo-

inositol, sucrose and an auxin and/or cytokinin.

6

Using 64 treatment combinations of indole—3-acetic acid
(IAA) and 6-furfurylaminopurine (kinetin), Padmanabhan (133)
classified tomato as one of the plant species with
morphogenetic expression regulated by the auxin/cytokinin
ratio as first discovered by Skoog and Miller (169). When an
auxin is used, IAA is reported to be optimum for achieving
shoot morphogenesis (80,87), with alpha-naphthaleneacetic
acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) almost
always inhibitory to shoot morphogenesis (7,131,195).
Moreover, 2,4-D is generally most useful when initiating or
maintaining callus and suspension cultures. The cytokinins
zeatin, 6-benzlyaminopurine (6-BAP), kinetin and 6-
(gamma,gamma-dimethylallylamino)-purine (21?), in increasing
efficiency, all seem to initiate shoots of tomato with or
without an accompanying auxin.

Although there are numerous reports on shoot
morphogenesis in the cultivated tomato, these experiments
achieved shoot formation from primary leaf, stem or hypocotyl
explants (8,80,87,131,133,178) and not from secondary or
older callus. DeLanghe and DeBruiJne (41) studied shoot
regeneration in L; esculentum and L. peruvianum. They
hypothesized that the lower shoot—forming capacity of L;
gggglggggg was due to a higher endogenous level of
gibberellic acid (CA), as addition of GA to the culture
medium resulted in a high production of callus and fewer
shoots. Adding 1000 ppm chlormequat (CCC), a GA inhibitor,

to the culture medium had no effect on the shoot-forming

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VI .

7
ability of the explants. However, if the donor seedlings
were sprayed daily with 2000 ppm CCC callus derived from
these seedlings maintained a undiminished shoot-forming
capacity after 2 years.

Meredith (109) studied shoot regeneration from 17—28
month old callus cultures of L2. eggulgntgm and LA
pimpinellifiglm. L_._ pimpinellifiglm callus did not
regenerate shoots, but callus from L; esculentum 'VFNT
Cherry' formed shoots, however the shoots were
morphologically'abnormal and could not be rooted. Plantlets
regenerated from younger, 4-month-old callus, were normal
and rooted easily.

Behki and Lesley (7) studied the response of secondary
leaf callus to the nitrogen source used in the culture medium
and the growth regulators used in the induction and
differentiation media. They found that decreasing the amount
of nitrogen (MS salts used) inhibited shoot regeneration. A
similar inhibition of shoot regeneration occurred when the
NR4+ concentration equalled or exceeded the N03‘

concentration.(NH4+:N03 is 1:2 in MS). Noa', however, could
be substituted for NH4+ without any deleterious effect on
shoot formation. They also found in both the callus
induction and the differentiation media the duration of the
induction period and the presence of light during the

induction period was important.

 

I .
. , .7 . .'
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8
B. Genetic control of shoot morphogenesis
In addition to media components, a strong genetic effect

on shoot morphogenesis is found among L; esculentug genotypes

 

(56,64,71,96,97,131,134,186,210). The wild Lycopersicon

 

species L; peruvianum is in general superior to LL<escu1entum

 

in shoot forming-ability (103,118,178,209).

Ohki et al. (131) was the first to study the genetic
transmission of shoot-forming capacity in tomato. The F1
hybrid whose seed parent had a higher shoot-forming capacity
was superior to the reciprocal hybrid in shoot formation
indicating a maternal effect. Heterosis was also observed in
this particular hybrid. The other hybrid whose seed parent
was the poorer of the two parents possessed an intermediate
regeneration capacity.

In contrast to Ohki et al. (131), Frankenberger et al.
(57) found no reciprocal cross differences or heterosis.
Regressing shoot formation of the hybrids onto the mid—parent
values gave a heritability estimate of 0.98 indicating
additive gene action. A highly significant general combining
ability indicated that shoot—forming ability could be
predicted from the general combining ability estimates of the
parents. A possible explanation for some of the
discrepancies between these two experiments.(ine. reciprocal
cross differences and heterosis) might be the culture media
used. Frankenberger et al. (57) used one culture medium
whereas Ohki et al. (131) used three media chosen from a

screening test of 35 IAA and 2iP combinations. In only 2 of

9

the 3 media were reciprocal cross differences and heterosis
observed suggesting that the capacity of the hybrids in
comparison to their parents depends also upon the growth
regulators in the medium. With only one medium, different
from any that Ohki et al. (131) used, lrankenberger et al.
(57) could have missed observing these effects. Behki and
Lesley (8) evaluated different tomato genotypes on media with
a range of growth regulator concentrations and demonstrated
that the type and concentration of auxins and cytokinins
which stimulated shoot formation from leaf tissue differed
among genotypes.

Because of the high totipotency of L_._ peruvianum, some
workers have hypothesized the presence of regeneration
controlling genes in L; pegggigngm and have proposed
interspecific hybridization as a means to transfer those
genes into the cultivated tomato (32,183). The ability to
form shoots appears to be a dominant trait in reports to date
(95,97,128,183). Thomas and Pratt (183) developed a tomato

genotype L2, with an ancestry of 75% L; peruvianum and 25% L;

 

esculentum. L2 was selected for self-fertility, high rate of
callus growth, and high efficiency of regeneration of shoots
from callus. Kut and Evans (97) found the P1 hybrids L;
esculentgm x Solanum lycopersicoides and L; esculentgm x L;
hirsutum to regenerate»as well as the more responsive*wild
parent. Koornneef et al. (95) developed a tomato genotype
MsK93 for superior shoot regeneration from cell cultures.

Like L2, MsK93 contains the favorable shoot regeneration

 

10
genes from L; peruvianum but its genetic backround is greater

than 50% L; esculentum.

 

One strategy not yet employed in tomato is the selection
for regeneration potential as successfully pursued by Bingham
et al. (16) in alfalfa. Bingham and his co-workers
identified five fertile regenerable genotypes. Regenerants
from these were intercrossed and the progeny screened for
regeneration. Shoot morphogenesis was increased from 12 to
67% after two cycles of recurrent selection.

Why L; esculentwm is more recalcitrant than L;

 

pggggl_ggm and L; ggilgggg is not known. There are some
observations that may contribute to its morphogenetic
response. First, chromosome changes are negatively
correlated with the capacity of cultured tissues to
regenerate shoots (203). Plant cells in culture from a wide
range of species exhibit a high incidence of polyploidy,
aneuploidy and chromosomal rearrangements
(6,55,38,98,173,204). Bayliss (6) reported that of 55
species studied only 11 retained the original chromosome
number while in culture. Murata and Orton (121) showed that
even when the chromosome number remains unchanged there may
be a high incidence of chromosomal rearrangement. The extent
of chromosomal variation is influenced by several factors.
First, differences of cultivars within a species, for
example, celery (19), corn (4) and oats (107), indicate a

genetic effect. Ogura (130) reported a dominant Mendelian

trait inducing chromosome instability in tobacco~ Second,

11
culture conditions such as the concentration and type of
growth regulators used in the culture medium can also
influence chromosomal stability (89,132,203). Third,
Cassells (25) suggested that high endogenous auxin levels in
tomato may suppress shoot formation from callus cultures.
Comparative assessments of auxin levels in non-regenerating

genotypes, regenerating genotypes and wild Lycopersgcon

 

species have yet to be done.
C. Protoplast culture

Protoplasts were first isolated from tomato roots by
Cooking (33) followed by cotyledon tips (32) and placental
tissue from immature fruits (63). These were also the first
successful protoplast isolations using enzymatic digestion
rather than mechanical methods (31). One recent exception to
the prevalent use of cell wall degrading enzymes is the
mechanical isolation of protoplasts from auxin conditioned
callus of Saintpaulia ionantha (African violet) (13). Though
these early attempts demonstrated the feasibility of
recovering large numbers of protoplasts using fungal derived
enzymes, the protoplasts rapidly degenerated. Pojnar et al.
(139) reported the first observation of cell wall synthesis
using protoplasts isolated from tomato fruits. The first
report of sustained cell division was by Nagata and Takebe
(123) for tobacco leaf mesophyll protoplasts. The following
year they reported the regeneration of plants from
protoplast-derived calli (122).

L; peruvianum with its high morphogenetic capacity was

12
the first Lycgpersicon species regenerated from protoplasts
(118,149,207,209). Plants of L; geruvianum var. dentatum and
L: chilense also are easily regenerated from callus and cell
suspension-derived protoplasts (73,138). Numerous early
attempts to regenerate plants from protoplast-derived calli
of L; esculentum failed (27,45,117,118,166,177,209). Morgan
and Cooking (115) screened fourteen cultivars of L;
esculentum for their morphogenetic potential using a petiole—
derived callus assay. The cultivar 'Lukul lus' produced many
green meristematic regions which readily developed into
shoots. When protoplasts were isolated from leaf tissue of
Lukullus they divided and formed calli that regenerated
shoots when placed onto MS salts and vitamins (120), 2%
sucrose, 0.8% agar and 1 mg/L zeatin. However, of the nine
cultivars from which protoplasts were isolated, plants were
only recovered from 'Lukullus'. Protoplasts were isolated
from cell suspension cultures derived from two-year old
callus cultures of 'Lukullus' (94). Shoots were regenerated
from callus derived from protoplasts but they were abnormal
and failed to root. However, using cotyledon protoplasts
from the cultivar 'Nadja' morphologically normal plants were
recovered (93). Shahin (160) used pre—conditioned Lg gigs;
grown seedlings and was able to regenerate plants from
fourteen tomato cultivars. .However, he could not isolate
protoplasts from greenhouse or growth chamber grown
seedlings. In our laboratory we have succeeded in

regenerating fertile plants from both in vitro and growth

13
chamber grown seedlings of seven diverse genotypes (124,125)
without pre—conditioning.
D. Anther Culture

Most attempts to regenerate haploid tomato plantlets via
anther cur microspore culture have failed
(21,30,39,44,65,101,161,162,205,206) except for L; peruvianum
(144). In tomato, haploid plantlets have only been obtained
from cultured anthers (23,64,212,213) but not in large
numbers. .Although, haploid embryoids were obtained culturing
uninucleate microspores in liquid medium (192). Gresshoff
and Day (64) reported the recovery of abnormal haploid
plantlets from one of forty—three tomato lines studied. The
factors identified.as essential for haploid plantlet recovery
were an amenable genotype, anthers cultured when the pollen
mother cells were still in metaphase I and the use of light
during’plantlet development.

Given a responsive genotype, one of the principle
factors affecting haploid induction is the stage of
microsporogenesis at which the anthers are cultured (76,172).
The correct stage must be determined experimentally for each
species. Several workers (3,44,150) reported on the
beneficial effect of a cold pretreatment on anthers which
disrupts microsporogenesis. This finding led Zamir et al.
(206) to use fifteen male-sterile mutants representing four
stages when microsporogenesis was blocked - pre-meiotic,
meiotic, tetrad and microspore. Mg 1035, a male-sterile

mutant characterized by an arrest in the development of

l4
meiocytes at the tetrad stage, formed haploid callus;
however, no plants were regenerated. Ziv et al. (212,213)
using mg 1035 and the culture system of Zamir et al. (206)
succeeded in recovering haploid plants. Anthers of
heterozygous plants (tf/+) carrying the allele for trifoliate
were placed into culture (212). .All plants regenerated were
trifoliate; thus, they were believed to be doubled haploids
of sporogenic origin. It was unknown why normal plants were
not recovered.
E. Uses

I gitro culture systems have been used to study a wide

 

variety of physiological, genetic, developmental and breeding
problems.

Warren and Routley (197) used callus cultures of
resistant and susceptible cultivars to Phytophthora infestans
to determine if the disease response in culture correlated
with the reaction of the whole plant. Some differences were
noted but they were not as delineated as at the whole plant
level. A somaclonal variant resistant to race 2 of Fusarium
ggysporum f. sp. lycopersici was selected from the progeny of
plants regenerated from callus cultures of 00828 (50,112).
No selective agents were used and resistance was determined
to be governed by a single, dominant gene. Scala et al.
(69,156) were able to select cell lines from cotyledon-
derived cultures showing higher partial resistance to L;
oxysporum f. sp. lycopersici race 1 filtrate than unselected

controls. No plants were regenerated. Of 370 plants

15

regenerated from leaf discs of a fully TMV susceptible line
six were selected as putatively resistant (5). Progeny
testing revealed varying degrees of resistance. Screening
for tobacco mosaic virus (TMV) resistance at the callus and
protoplast level, Toyoda et al. (184) found significant
differences between callus from resistant and susceptible
genotypes. These differences were not observed at the
protoplast level. Zhuk et al. (211) used cell suspension
cultures to show that TMV infection increases the number of
metaphases with concomitant disturbances of the spindle and
in the frequency of chromosome aberrations at anaphase and
telophase. No ploidy changes were observed.

Somatic hybrids have been recovered between potato +
tomato (108,148,163), §9_Lap_u_m Lycopersgcoiggg + tomato (70),
Solanum nigrum + tomato (68,79), tomato + §; rickii (128) and
L; peruvianum + Petunia hybrida (174). Binding (14) achieved
cell division between petunia + tomato heterokaryons but no
plants were recovered.

Meredith (110) selected variant cell lines tolerant of
aluminum. ‘Variant cell lines were stable but no plants were
regenerated making it difficult to rule out epigenetic
effects. Suspension cultures of L; peruvianum were exposed to
a step-wise increase of the concentration of cadmium sulphate
(9). Exposure of tolerant and sensitive genotypes to Cd, Cu
or Zn led to the intracellular accumulation of a low
molecular weight cystein-rich cadmium—binding protein. This

protein was increased 5-fold in selected cell lines.

16

Epigenetic effects could not be ruled out as plants were not
regenerated for genetic analysis. Tolerance to NaCl was
observed between cultivars using cultured cotyledon explants
(54). Differences were noted on the basis of explant growth
and callus proliferation. Ellis (48) was unable to isolate
cell lines resistant to the herbicide metribuzin using cell
suspension cultures. However, Harrison et al. (72) detected
differences in response of tomato cultivars to metribuzin
using callus cultures initiated from the hypocotyl. The
sensitivity of this assay was decreased due to a high level
of variabililty. Paraquat-tolerant tomato mutants isolated
from cell cultures had a 30—fold increase in whole plant
tolerance (181). A detailed genetic analysis was not
performed though some of the progeny of tolerant individuals
retained the trait. When cell suspension cultures of a L;
35;:ng x L_. 2232215193.! hybrid were irradiated with 294
rads/min for a total of 11,760 rads glyphosate tolerant cell
lines were selected (170).

Tomato protoplasts were able to take up laboratory made
positively charged lipid vesicles without any fusion
treatment (28) indicating a potential vehicle for delivering
compounds directly into the cell.

Root cultures were compared to stem-derived callus for
sugar uptake (29). Roots grew better with sucrose than
glucose and fructose while callus grew well in sucrose and
glucose and slightly less well on fructose. Coleman and

Greyson (34) studied root formation using leaf discs and

17
found that GA inhibited rooting when IAA was present but
stimulated rooting in the dark without IAA.

The L3 23539 response of ovaries from newly opened
flowers can be used in screening genotypes for parthenocarpic
progeny to be used in breeding the same season (69). Rastogi
and Sawhney (145) studied the nutritional and hormonal
factors controlling flower morphogenesis by culturing young
floral buds. Embryo culture has been used to obtain
interspecific hybrids between L; escuLentug and L; peruvianum
(18,95) and Léggggiggggg and L_._ minutum (187).

Utilizing tissue culture induced variation in tomato,
Evans and Sharp (51) recovered thirteen single gene mutations
including male-sterile, Jointless, tangerine fruit, lethal
albino, virescent, indeterminate, mottled and green base.
Buiatti et al. (20) found 17.04% of the progeny of tissue
culture-derived plants had chlorophyll mutations or other
morphological abnormalities. These mutants segregated 3:1.
Polyploids and chimeras were also found. Plants regenerated
from hypocotyl segments from a homozygous (LgLa) leafless
lanceolate mutant exhibited both leafy and leafless
phenotypes (157L. Progeny of leafy regenerates segregated
into homozygous mutants (LgLa), heterozygotes (hilt) and
normal plants (:11) indicating genetic reversion. These
studies suggest that tissue culture induced variation may be
of value to tomato breeders.

Tissue culture provides a convenient way'to transform

plants with Agrobacterium Lgmefaciens (106). Kanamycin

18
resistance was incorporated into 4 cultivars using stem
explants (11). Tomato protoplasts were used to study the

hormone relationships of a number of nggbactegitum

 

transformed cell lines (194L
III. Preconditioning of donor tissue

Protoplasts have now been isolated from every plant
tissue and organ including tissue derived callus and cell
suspension cultures. The physiological condition of the
donor plant markedly effects the yield, viability and
subsequent differentiation of the protoplasts.

A. Genotype

Given suitable culture medium and environmental
conditions, genotype is probably the single most important
factor determining the physiological response of plant
protoplasts (125,160). As mentioned earlier, profound
differences are observed even between cultivars within the
same species. Niedz et al. (125) regenerated plantlets from
six diverse tomato genotypes. The frequency of calli forming
shoots ranged from 2-22% with two genotypes not responding.
Likewise, Morgan and Cocking (115) found only one tomato
cultivar, 'Lukullus', of 14 tested, that was capable of shoot
morphogenesis from protoplast-derived calli.

Arguably it might be stated that all genotypes are
capable of shoot morphogenesis from protoplasts if only the
proper cultural conditions are found. However, such
conditions have not always been found even with a

considerable search. For instance, many investigators have

19
attempted to regenerate plantlets from cereal protoplasts but
only in a few cases, sugar cane (193), pearl millet (193) and
rice (37,202), has success been reported. In Lg; ggyg.
Potrykus (140) tested tens of thousands of culture media to
no avail. To date, only 3 lines of maize have been reported
(113) which are capable of cell wall formation and cell
division from protoplasts - inbred line A188, Black Mexican
Sweet and B73.
B. Greenhouse, CEC, Ln giggg

Greenhouse grown plants have been used to isolate and
regenerate plants from protoplasts (26,114,115,175L.
However, because of varying light and temperature regimes
year-round repeatability is difficult (198). Controlled
environmental chambers (CEC) reduce some of the variability
associated with greenhouse grown plants
(1,2,12,24,67,126,152,155,165,168). For instance, leaves
taken from pea plants in a CEC yielded protoplasts that
divided and formed callus, while leaf-derived protoplasts
from greenhouse grown plants died and only rarely divided
(2).

Aseptically grown plant material provides a number of
advantages over greenhouse and CEC grown plants. First,
light, temperature and nutrient supply are uniformly
controlled and, second, tissue sterilization with toxic
substances is avoided (81,171). In one case it was the only
successful method to isolate and culture tomato leaf

mesophyll protoplasts (160).

20
C. Light

Little work has been done on the effects of duration,
intensity and quality of light on protoplast source plants.
Bhatt and Fassuliotis (12) found a long photoperiod (16 hr)
and high light intensity (7500 lux) beneficial for
consistently high yields of eggplant protoplasts capable of
division. When plants were grown under a 10 hour photoperiod
and high light or lower light (4300 lux) and 12 or 16 hour
photoperiod, protoplasts failed to divide.

In potato, protoplast division was only observed when
source plants were previously conditioned for 4—10 days under
a 6 hour photoperiod of 7000 lux with fluorescent light
(164). Binding (15) found protoplast yield of La giggg grown
haploid and diploid Petunia hybrida dependent on several
factors including an optimal light intensity of 7000 lux,
similar to both previous studies.

Watts et al. (198) discovered that illumination in
excess of 25,000 lux of greenhouse grown tobacco plants
decreased protoplast yield. Tal and Watts (177) reported
that greater numbers of viable tomato protoplasts, as
measured by their plating efficiency, were obtained from
plants preconditioned under low temperature (15°C) and high
relative humidity (82%). Modification of membrane
characteristics was suggested for the increased stability of
protoplasts under these conditions.

The production of viable protoplasts from.greenhouse—

grown plants depended on a dark pre—conditioning treatment of

 
 

21
shoot cuttings 30-162 hours prior to isolation (36). Shahin
(160) recommended transferring donor plants 48 hours before
isolation to the dark to reduce starch accumulation and to
favor the osmotic adjustment of the cells after isolation.

Cell divisions and colony formation were high when
SgLanum penneLLLL plants were transferred 6 days before
isolation in: short—day conditions (8/16 hour
photoperiod)(49).

Protoplasts isolated from freshly removed leaves of
greenhouse or CEC grown ELQLQ narbonenng plants did not
divide (46). A 2-4 day dark treatment was not beneficial.
However, when the leaves were first pre-cultured on basal
medium plus BAP and p-chlorophenoxyacetic acid (CPA) for 7—8
days, the isolated protoplasts divided.

D. Nutrition

Cassells and Barlass (27) reported that protoplast
viability from leaves of greenhouse grown tomatos was
increased by feeding with calcium nitrate (0.1 M twice
weekly) and low light intensities (2.52-10.8 MJ m-zday'l, 16
hr photoperiod). Protoplast stability seemed dependent on a
relatively high calcium content (4-4.7% of dry matter) of the
leaves. Supplemental feeding of donor plants with calcium
also helped increase protoplast stability of tobacco (197%
The authors believe feeding is important as it increases
membrane calcium and may have other beneficial effects
connected with increased nitrate and chloride uptake and with

anion/cation interactions. One difference between tomato and

22
tobacco in these two previous studies is the influence of the
flowering state. Poor quality protoplasts were obtained once
a tobacco plant entered the flowering state but not in
tomato. It is suggested that endogenous changes are
occurring that are incompatible with the isolation of stable
protoplasts.
E. Tissue age and source

The age of the tissue affects protoplast isolation and
viability (102,119,191). David et al. (40) report that yield
and plating efficiency are increased in expanding cotyledons
compared to fully expanded cotyledons of LLnus pinaster.
Likewise, isolation of protoplasts from cotyledons of cotton
led to an increase in the number of damaged protoplasts
partly due to the increase in incubation time (91). Kao and
Michayluk (84) found protoplasts from the youngest, not fully
expanded leaves divided sooner then those from older leaves.
Wallin et al. (196) found the use of very young leaves (3-5
mm) in buds from Lg yLng cultured shoots of apple increased
protoplast yield.

Berry et al. (10) obtained the highest yields and
plating efficiencies from protoplasts derived from cotyledons

of Lg gitro seedlings of Lactuca sativa. Using Lg gitro

 

seedlings of sunflower, Lenee and Chupeau (100) found that
only protoplasts isolated from hypocotyls divided with

repeatibly high plating efficiencies (60%).

23
F. Senescence retardents
Sawhney et al. (153,154) found that pretreating detached
oat leaves for 18 hours in a solution containing a senescence
retardant (eg. cycloheximide, kinetin, L-lysine, L-arginine)
increased protoplast stability. An associated decrease in
nuclease activity and an improvement in the incorporation of
protein and nucleic acid precursors was also observed.
Conversely, the addition of L-serine, a senescence promoter,
to the conditioning solution decreased protoplast release and
did not improve stability. High RNase activity, low rates of
amino acid and nucleoside incorporation are phenomena
associated with senescent leaves.
G. Temperature and humidity
Mesophyll protoplasts of rape plants grown at 19—21°C
and 40-45% relative humidity were capable of sustained
division and subsequent plant regeneration. ProtOplasts
derived from plants grown at a higher temperature and

humidity, 26°C and 70% respectively, failed to survive (88).

24
MATERIALS AND METHODS

I. Protoplast isolation and culture
A. Source Material

Seeds of P.I. 367942 (L157) were obtained from the North
Central Regional Plant Introduction Station, Ames, Iowa. A
single plant selection was made and selfed seed collected
from this individual. The process was repeated for a total
of 3 generations to reduce any heterogeneity that may have
existed in the original seed sample.

B. Seedling Preparation

1. CEC grown seedlings

Seeds of L157 were sown on soil—less planting medium
(VSP, Bay Towing Co., Houston, TX) in plastic containers and
germinated in a controlled environment chamber (CEC). Total
light intensity'of 150'uEm'2s'1 was provided by cool-white
fluorescent lamps and 6 incandescent bulbs providing 20 uEm'
2s'l, both on a 14 hour photoperiod. The temperature was
27°C/25°C day and night, respectively. Seedlings were
transplanted 10-12 days following sowing into VSP in plastic
4-cell (2"x2") pack containers and grown under the same
environment; Seedlings were‘watered daily with a 20-20—20
fertilizer at 150 ppm N, with the pH adjusted to 6.2 with 50%
H3P04.

2. Lg yLng grown seedlings

Seeds of L157 were surface sterilized with 70% ethanol

for 3 minutes followed by 30 minutes in a solution containing

25

30% (v/v) Big Chief Bleach (Patterson Laboratories, Detroit,
MI) and 6 drops/l Tween-20 (Sigma Chemical Co., St. Louis,
MO), and subsequently washed 4 times with sterile distilled
water. Thirty to forty seeds were sown per container
(Magenta Corp., Chicago, IL) containing 50 mls of MS medium
plus 30 g/l sucrose. Ten to twelve days after sowing
seedlings were cut at the hypocotyl, and the apical portion
reinserted into fresh MS medium. Seed germination and
seedling growth were under a light intensity of 35 uEm'zs'l,
16 hr photoperiod provided by cool-white fluorescent lamps
and a 27°C/25°C day/night temperature regime.

3. Cold and dark pretreatment of donor tissue

I gitro and CEC grown donor plants were preconditioned

 

immediately prior to protoplast isolation, cold treatments
included 0, 12 and 24 hours at 10°C. Dark treatments
included 0. 24 and 43 hours dark at 26°C. All possible
combinations (9 cold/dark treatments) of cold and dark were
tested. The 9 cold/dark treatments were given to 2 donor
tissue sources, CEC and Lg gLng, for a total of 18
treatment combinations (3x3x2 factorial). All

preconditioning treatments were carried out in CEC.

C. Protoplast Isolation
1. CEC grown seedlings
The first fully expanded leaves of 3—week.old plants,
following any preconditioning, were excised and surface
sterilized for 20 minutes in a solution containing 15% (v/v)

Big Chief Bleach (Patterson Laboratories, Detroit, MI) and 6

26

drops/l Tween-20 (Sigma Chemical Co., St. Louis, MO) and
subsequently washed 4 times with sterile distilled water.
Leaflets were sliced transversely into 1 mm strips and
plasmolyzed for 1 hour in CPW salt solution (58) containing
13% (w/v) sorbitol at pH 5.7. This plasmoticum was removed
and replaced by a filter—sterilized enzyme solution
containing 3.0% (w/v) Meicelase-P (Meiji Seiki Kaisha Ltd.,
Tokyo, Japan),(L1% (w/v) Macerase (Calbiochem, San Diego,
CA), 9.0% (w/v) sorbitol and CPW salts at pH 5.7. Leaf
strips plus enzyme solution were incubated at 27°C in the
dark on a gyratory shaker (45 rev/min) for 4—6 hours.
Digested tissue was filtered through a 60 um sieve, the
filtrate was collected and placed into 16-ml screw-capped
tubes and centrifuged at 35 x g for 10 min. The supernatent
was discarded and the protoplast pellet resuspended in
protoplast washing medium (KM-T w/o growth regulators and 0L4
molar sorbitol as osmotic stabilizer). The protoplasts were
washed 2 times in protoplast washing medium and finally
resuspended in flotation medium and centrifuged at 100 x g
for 10 min. Flotation medium was prepared by mixing 1 part
Lymphoprep (Accurate Chemical and Scientific Corp., Westbury,
NY), 2 parts CPW salts plus OLSM sucrose, adjusting the pH to
5.8, and filter—sterilizing the solution. After
centrifugation, protoplasts at the surface were removed and
resuspended in culture medium, KM—T (Table A18). .Protoplasts
were counted and plated in 2 mls of KM-T over 2 mls of A4

(Table A18) in 60 x 15 polystyrene culture dishes (Falcon,

27

Becton, Dickinson and Co.) at a density of 3 x 104
protoplasts/ml. A4 was sterilized by autoclaving for 15

minutes at 15 p.s.i.

2. Lg y_L_t_r_g grown seedlings

All leaf tissue of 3-week old transferred shoot tips,
following any preconditioning, were excised and sliced into
1-2 mm sections. Leaf sections were placed into a filter-
sterilized enzyme solution containing 1% (w/v) Cellulysin
(Calbiochem, San Diego, CA), 0.1% (w/v) Macerase, 0.4 molar
sorbitol and CPW salts at pH 5.7. Incubation of Lg gLng
tissue was the same as described for CEC tissues except the
time was 4-5 hours. Purification was as previously described

for CEC tissues. Plating density was 6 x 104 protoplasts/ml

in 2 mls KM-T over 2 mls A4.

D. Protoplast Culture and Shoot Morphogenesis
The protoplasts were initially cultured at 27°C in the
dark for 7-10 days and then transferred to low light (3—4

’29-1 from cool-white fluorescent lamps). Two to three

uEm
weeks after isolation, depending upon the cell division rate,
the liquid medium containing the cells and cell colonies was
removed and placed onto fresh medium, KM—T with 0.3 molar
sucrose, the 2,4-D deleted and 0.6% purified agar added
(Table A19). Thirty day-old calli were removed by pipette
and placed onto Whatman No. 1 filter paper overlaid on MS + 2
mg/L zeatin (MS2Z) with 0.15 M sucrose. Filter paper with

attached calli was transferred to MS2Z with 0.087 molar

 

28

sucrose in another 2 weeks. As individual calli reached 3-4
mm in diameter they were removed and placed directly onto
M822 medium. Calli showing shoot organogenesis were
transferred to regeneration medium containing 1 mg/L zeatin
(trans isomer) for further shoot development. Shoots were
excised and rooted on MS medium without growth regulators.
Rooted shoots were transplanted into VSP soilless mix and
kept under clear plastic for 2 weeks before transfer to the

greenhouse.

II. Experimental design and data analysis

The four variables measured were protoplast yield,
viability, cell colony diameter and shoot morphogenesis.
Yield was calculated as protoplasts released/two gram of
fresh weight of tissue in 25 ml enzyme solution. Percent
viable protoplasts twenty—four hours after isolation was
determined by fluorescein diacetate staining (200) and visual
observation. Microcalli diameter was measured thirty days
after plating with an ocular micrometer. Shoot morphogenesis
was the number of calli.that underwent shoot morphogenesis
during 3 months. The method of analysis used in these

experiments was the Least Squares Approach (158,159).

29
RESULTS

There were two empty subclasses for the variables
microcalli diameter and shoot morphogenesis from CEC grown
seedlings — 12 hr cold/0 hr dark (CD12'0) and 24 hr cold/0 hr
dark cold (CD2"O). Protoplasts isolated from CEC grown
seedlings pre—conditioned in either manner died within 24-48
hours.

I. Yield
A. Test of interaction effects

Using the sums of squares for the models R(u,CDS) and
R(u,C,D,S,CD,CS,DS)(Table A1) the cold x dark x source (3-
way) interaction is significant at the 1% level (Table A5).
Testing seedling source separately, the cold x dark (2—way)
interaction is significant at the 5% level for both CEC grown
seedlings and Lg gLng (IV) grown seedlings (Table A5).

B. Goodness of fit

The 3-way interaction model accounts for 90.9% of the
observed variation in yield and the model is significant at
the 1% level (Table A7). The 2-way interaction model for CEC
and Lg gLng grown seedlings accounts for 88.7% and 92.3%
(Table A1) of the variation in protoplast yield,

respectively. Both 2-way interaction models are significant

at the 1% level (Table A6).

   

 

 

 

30

C. Contrasts

The smallest subclass means for the variable yield are
simply subclass averages u + CD13 (Fig. 2 a Table A8).
Because of a significant interaction, comparisons between
classes within a factor (the most interesting kind) cannot be
made but are limited to comparisons between subclasses or
groups of subclasses. For CEC grown seedlings, the contrast
to compare all treatments receiving 0 hours of cold to all
treatments receiving 12 and 24 hours cold, 2(°D0,o + CD0,24 +
CD0,48) ‘ “3012.0 + CD12,24 + CD12,43 + CD24,0 '* CD24.24 +
CD2"‘8)(Table A9), is significant at the 1% level, and
indicates that cold preconditioning decreases protoplast
yield from CEC grown seedlings. However, the treatment 12
hours cold and 48 hours dark is not significantly different
from the 3 treatments of 0 hours cold over the three dark
levels (Table A9). For IV grown seedlings the contrast to
compare all treatments with 48 hours dark to all treatments
receiving less than 48 hours dark, 2(CD0'48 + CD12'48
CD24,48) ‘ (CDo,o + CD0,24 + CD12,0 + °912,24 * CD24,0 +
CD24'2‘HTable A9), is significant at the 1% level indicating
a 48 hour dark treatment decreases yield over all levels of
cold. Some other contrasts are listed in Table A9. Four
preconditioning treatments, CDO,0' CDO,24' CD0,48 and
CD12'48, resulted in the highest yields from CEC seedlings
(Table A17). Three preconditioning treatments, 090,24'
CD21.” and CD12'0 resulted in the highest protoplast yield

from IV seedlings (Table A17).

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31

Figure 1. Effect of cold and dark pre-conditioning on
protoplast yield. (a) CEC grown seedlings. (b)
IV grown seedlings.

Yield (CEC)

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33

II. Viability

A. Test of interaction effect

Using the sums of squares for the models R(u,CDS) and
R(u,C,D,S,CD,CS,DS)(Table A2) the cold x dark x source
interaction is significant at the 1% level (Table A13).
Testing seedling source separately, the cold x dark
interaction is highly significant for CEC grown seedlings and
significant for IV grown seedlings (Table A5).

B. Goodness of fit

The 3—way interaction model is significant at the 1%
level (Table A7) and accounts for 96.4% of the observed
variation in protOplast viabilityu The 2-way interaction
models are significant at the 1% level (Table A6) and account
for 98.0% and 22.7% in the variation of protoplast viability
for CEC and IV derived protoplasts, respectively.

C. Contrasts

The smallest subclass means for the variable viability
are subclass averages (Fig. 4 & Table A10). As for
protoplast yield comparisons between classes within a factor
are not possible. The contrast CD0,48 - CD0'24 (Table A11)
indicates CD0'48 gave the highest viability for CEC grown
seedlings. However, the contrasts CD12'O — CD2"48 and
CD12,0 - CD12'24 (Table A11) indicate the highest viability
for IV grown seedlings was obtained from the treatments
CD12'O, CD12'24 and CD24'48. For CEC seedlings the
preconditioning treatment CD0,48 had the highest viability

while for IV seedlings CD12'0 had the highest viability

34

Figure 2. Effect of cold and dark pre-conditioning on
protoplast viabiltiy. (a) CEC grown seedlings.
(b) IV grown seedlings.

Viability (CEC)

EEE

 

 

 

 

 

 

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Viability (IV)

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36

(Table A17).
IV. Microcalli diameter

A. Test of interaction effect

Using the sums of squares for the models R(u,CDS) and
R(u,C,D,S,CD,CS,DS)(Table A3) the cold x dark x source
interaction is significant at the 1% level (Table A5).
Testing seedling source separately, the cold x dark
interaction is not significant for CEC grown seedlings and
highly significant for IV grown seedlings (Table A5).

B. Goodness of fit

The 3-way interaction model accounts for 73.6% of the
variation in microcalli diameter and is significant at the 1%
level (Table A7). The 2—way interaction model accounts for
29.3% of the observed variation in microcalli diameter from
IV grown seedlings and is significant at the 1% level (Table
A6). Whereas, the 2-way interaction model is not significant
for accounting for microcalli diameter from CEC grown
seedlings (Table A5). The full model R(u,C,D) accounts for
37.6% (Table A3) of the observed variation and is significant
at the 1% level.

C. Contrasts

The smallest subclass means for microcalli diameter of
IV and CEC grown seedlings are listed in Table A12 and Fig.
6. Referring to Table A17, single factor evaluation of CEC
source seedlings shows the factors cold and dark together
accounting for 37.6% of the variation, and considered

separately accounting for 35.2% and 2.7% of the variation.

37

Figure 3. Effect of cold and dark pre-conditioning on
microcalli diameter. (a) CEC grown seedlings.
(b) IV grown seedings.

38

a)

Microcalli diameter (CEC)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Microcalli diameter (IV)

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39
respectively. The factor dark is not significant.

Factors cannot be examined individuallyrfor'IV source
seedlings due to interaction. The contrast 2CD12'O - (CDO,0
+ CD0'24)(Table A14) shows the treatment C012,0 having the
highest microcalli diameter for IV source seedlings. The
largest microcalli diameter from CEC seedlings were observed
from the two treatments Co + D24 and C0 + D43, while for IV
seedlings the largest microcalli was observed from the
treatment C012,0 (Table A17).

IV. Shoot morphogenesis
A. Test of interaction effect

Using the sums of squares for the models R(u,CDS) and
R(u,C,D,S,CD,CS,DS)(Table A4) the cold x dark x source
interaction is not significant (Table A5). Testing seedling
source separately, the cold x dark interaction is not
significant for both CEC grown seedlings and IV grown
seedlings (Table A5).

B. Goodness of fit

The 3—way interaction model accounts for 41.4% of the
variation in shoot morphogenesis and the model is significant
at the 1% level (Table A7). The full model with no
interaction accounts for 25.4% of the variation in shoot
morphogenesis from CEC grown seedlings and 38.9% from IV

grown seedlings.

 

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40
C. Contrasts

The smallest subclass means for shoot morphogenesis of
CEC and IV grown seedlings are u + Ci + Dj (Table A14)

As CEC and IV grown seedlings had no significant
interaction effect (Table A5) between cold and dark pre-
conditioning single factor effects were assessed (Table A17).
The two factors, cold and dark, together describe 39.61% and
25u38% of the variation in shoot morphogenesis from CEC and
IV source seedlings, respectively. Cold accounts for <1% of
the variation in shoot morphogenesis and dark accounts for
38.40% for CEC source seedlings. For IV source seedlings,
cold and dark describe 13.77% and 23,34%, respectively; Cold
and dark together or individually are significant at the 1%
level from both sources (Tables A5).

For CEC grown seedlings the contrasts CO = 012 = C24 and
D0 = D24 2 D48 are both significant at the 1% level
indicating differences exist among the classes within both
the factors cold and dark. These two contrasts are
equivalent to the models R(Clu,D) and R(Dlu,C), respectively;
Contrasting the highest class within each factor with the
other two classes, 2C0 - (C12 + C24) for the factor cold and
2°48 — (D0 +.D24) for the factor dark, shows both contrasts
significant at the 1% level. Contrasting the two lower
classes of each factor for CEC calli, 012 - C24 and Do - D24
for the factors cold and dark respectively; results in the
cold contrast being not significant and the dark contrast

being significant at the 1% level. It seems the cold

41

preconditioning treatments, 12 or 24 hours, decreases
regeneration and that a dark preconditioning treatment
progressively increases regeneration with a 48 hour treatment
being the most effective. From CEC seedlings C0 + D48
resulted in the highest frequency of shoot morphogenesis,
while for IV seedlings six preconditioning treatments were
equally'high - Co + 90,24 & 48 and.C12 + D0'24 & 48 (Table
A17).

For CEC seedlings CD0’48 (and C0 + D48)(Table A17) ranks
as the "best" (highest treatment mean) preconditioning
treatment under all 4 measured variables, while for IV

seedlings, CD12’0 (and C12 + D0) ranks as the "best".

42

Figure 4. Effect of cold and dark pre-conditioning on shoot
morphogenesis. (a) CEC grown seedlings. (b) IV
grown seedlings.

Shoot morphogenesis (CEC)

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44
DISCUSSION
P.I. 367942 (L157) was chosen for this study as it
had the lowest frequency (2%) of shoot morphogenesis of the
seven cultivars previously regenerated.in our laboratory.
The morphogenetic response of the other cultivars using the
protocols developed for this research remains to be

investigated.

I. Protoplast Yield

Protoplast yield from CEC and IV grown seedlings was
differentially affected by cold and dark pre-conditioning.
The decrease in yield from CEC seedlings under all cold
treatments except CD12'48 might be due to stress as the
treatment times were too short to:significantly alter cell
wall composition. After 12 or 24 hours of cold, CEC
seedlings were wilted. Other experiments with wilted seedling
tissue always resulted in a decrease in protoplast yield
(unpublished data). Perhaps the low yields associated with
water stress are due to mechanical rather than physiological
reasons. A wilted leaf does not cut as cleanly as a turgid
one thereby resulting in damaged cells along the cut edge.
This would limit enzyme ability to penetrate the
intercellular tissue resulting in decreased yields.
Accordingly, the two treatments with the lowest yield, 12
cold/ 0 dark and 24 cold/ 0 dark were also the most severly
wilted. IV grown seedlings were not as affected by the cold
treatments as CEC grown seedlings. As IV seedlings were

grown under 100% relative humidity they would be less prone

45

to wilting and water deficits. It is difficult to compare
protoplast yields from CEC and IV seedlings as the isolation
enzymes used for each source were different. Varying
toxicity, specificity and activity of the enzymes plus the
possibility of differences in cell wall composition between
the two sources makes contrasts between sources almost
impossible. Valid contrasts are therefore limited to within
source comparisons.

It is clear that cold pre-conditioning is of no
beneficial value for increasing protoplast yield from either
CEC or IV grown seedling tissue. A 24 hour dark pre-
treatment did increase»protoplast yield from IV seedlings.
The presence of high tissue starch levels has been shown to
correlate with a decrease in protoplast yield due to a
decrease in the protoplasts ability to withstand mechanical
handling'(198L. By’dark treating tissue prior to isolation
starch levels are reduced and protoplast stability under
handling increases, and more protoplasts are recovered.
Perhaps differences in cellular starch levels exist between
CEC and IV grown seedlings to explain the beneficial effect

of dark on IV seedlings—derived protoplast yield.

II. Protoplast Viability
There are a number of short-term methods of assessing
protoplast viability:

1.Intact membrane exclusion of Evans blue dye
(60,62,83) or phenolsafranine (53,77,85,105,185,200).

46

2. Intracellular accumulation of fluorescein
(99,167,200)cu'neutral red (43).

3. Intracellular reduction of methylene blue (75).
4. Cyclosis (136,143).

5. Respiratory metabolism measured by oxygen uptake
(176).

6. Photosynthetic activity (83).

7. Ethylene release (52).

Visual assessment of spherical protOplasts with the
chloroplasts evenly distributed around the cell proved the
most conservative estimate of predicting protoplast
viability; Cyclosis is difficult to observe in mesophyll
cells. Oxygen, photosynthetic measurements and ethylene
release are only applicable to whole cell populations and
dyes are more liberal viability indicators. For instance,
protoplasts that were clearly of unacceptable quality (e4L
chloroplasts aggregated on one side and the plasmalemma
membrane bulging outwards) would stain using fluorescein
diacetate (FDAL AJso, protoplasts will vary quantitatively
in the amount of stain excluded or taken up. Estimates using
FDA were consistently 15-20% higher than estimates obtained
by visual observation (unpublished data).

Viability is between 50-60% for both CEC and IV derived
protoplasts and though statistical differences were noted,
the variances were so small that dark and cold pre—
conditioning are of little practical consequence except in
two classes - when CEC seedlings were exposed to light and

cold viability dropped to zero.

47

The two empty subclasses caused by protoplast death from
CEC grown seedlings preconditioned with a combination of
light and cold were most likely due to photooxidation, a
light and oxygen-mediated chlorosis (141) in cold sensitive
species (190). A number of reports have distingushed between
the response of plants to chilling in the dark and to
chilling in the light (90,142,179,180,188). Chilling in the
dark has little effect on photosynthesis (particularly PSII),
but: in the light chlorosis, photosystem damage
(90,142,141,180,188,190) altered chloroplast ultrastructure
(179,201) and cellular lipid degradation (141) are observed.
These results suggest the potential use of protoplasts in
tomato to assay for cold tolerant genotypes.

Other factors might also contribute to the differences
in response to cold and dark preconditioning between CEC and
IV seedlings. For instance, CEC seedlings were grown under
higher light (150 uEm-zs'l) than IV seedlings (35 uEm-zs-IL
Perhaps a difference in the cellular milieu such as osmotic
pressure and membrane composition would contribute to the
observed response. Also, the available carbon was supplied
to each source differently, atmospheric C02 for the CEC
seedlings and sucrose in the culture medium for the IV
seedlings. Carbon source has previously been shown to

profoundly effect cold tolerance of tomato cells (117).

III. Microcalli Diameter
Microcalli diameter at thirty days was indicative of the

rate of cell colony growth. Only colonies composed of small

48

cytoplasmically—dense cells were measured, as colonies with
large filamentous cell with large vacuoles are poor
morphogenetically and would have distorted the data. For
example, a single large filamentous cell could easily be the
size of a entire colony of smaller cytoplasmically—dense
cells. Large vacuolated cells constituted a large proportion
of the cell types in CEC-derived cell colonies pre-treated
with cold. The fewer cytoplasmically-dense cell colonies
grow slower in these cultures than in cultures where the
vacuolated cells were less numerous.

In spite of differences between the pre-conditioning
treatments all treatments, except the two empty subclasses,
yield cell colonies of acceptable quality and quantity.
Colony size differed considerably between CEC and IV
protoplast-derived colonies. Comparing the subclass means
from CEC and IV sources in even the poorest CEC treatment, 48
dark/0,12,48 cold had a larger mean colony diameter than the
best IV treatment, 0 dark/12 cold. Most probably the
difference is due to the protoplasts from IV source seedlings
dividing later (2-3 days) than protoplasts derived from CEC
source seedlings. Also, the initial average protoplast size
of the two source tissues differs considerably. CEC and IV
derived protoplasts average about 35 uM and 10 uM,
respectively. Initial protoplast size probably does not
contribute greatly to thirty day colony diameter as the cells

are virtually indistingushable after ten days.

49
IV. Shoot morphogenesis

With the exception of the two empty subclasses shoot
morphogenesis was acceptable from all treatment combinations.

Within 7—10 days after p-calli were placed onto
regeneration media darker green areas appeared in the callus
from which shoots would eventually arise. Chlorophyll
synthesis was generally observed to precede shoot
morphogenesis. Calli varied from seven days to four weeks
(experiment was terminated at this time) before the first
green meristematic area appeared.

Interaction effects disappeared in both CEO and IV
protoplast derived calli assayed for shoot morphogenesis.
Analysis of single factor effects showed that only dark pre—
conditioning effects were still present in CEC—derived calli,
but both cold and dark effects were present in IV-derived
calli. Cold treatments in all cases exerted no beneficial
effect on shoot morphogenesis. Cold treatments were not
significant for CEC-derived calli and accounted for a lesser
proportion of the variation observed among IV-derived calli.

The effect of cold and dark on shoot morphogenesis is in
contrast to their effect on microcalli diameter. For
microcalli diameter of dark preconditioned CEC-derived calli
was not significant. As previously explained for microcalli
diameter a large proportion of the CEC-derived cells from
cold preconditioned tissue were vacuolated resulting in
slower growth of the calli. Also, an increase in the degree

of browning was observed in these cultures. In all

50
treatments only calli of a compact and spherical morphology
were chosen for transfer to shoot regeneration medium (M822).
As calli from all treatments grew rapidly and underwent
chlorophyll synthesis it appears the variation due to cold

was removed when morphologically desirable calli were

selected.

51

SUMMARY AND CONCLUSIONS

Leaf mesophyll protoplasts of tomato were enzymatically
isolated from CEC and IV grown seedlings previously subjected
to cold (0, 12 and 24 hours at 10°C) and dark (0. 12 and 43
hours) in a 3x3 factorial experiment. Protoplasts were
cultured in KM-T over A4 solidified with 0.5% purified agar.
Media were supplemented with 6—benzylaminopurine, alpha-
naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid.
Shoot regeneration was achieved on M822.

Four variables were measured; protoplast yield,
viability, microcalli diameter and shoot regeneration~.As
data was unbalanced and included two empty subclasses for 12
and 24 hours cold/O hours dark/CEC the Least Squares Approach
was used for data analysis.

CEC and IV grown seedlings responded differentially to
cold and dark preconditioning. Cold did not benefit CEC
protoplasts at any stage, but did increase viability and
microcalli diameter for IV protoplasts. CEC grown seedlings
were severly affected when subjected to cold and light as
protoplasts died within 48 hours. This may have been due to
several factors including photooxidation, lipid degradation
and chloroplast ultrastructural changes. The use of tomato
protoplasts to one, assay for cold tolerance and, two, study
chilling injury so as to separate the primary causes of
chilling injury from the secondary whole plant effects may be
beneficial.

Further work on the effects of preconditioning could

 

52

include environmental preconditioning of detached tissue.
The composition of the incubation medium during
preconditioning could markedly influence the subsequent
response. Also, preconditioning of freshly isolated
or day old protoplasts might provide a means, as with the
study of chilling injury, of seperating the secondary effects
of preconditioning on the whole plant from the primary
effects. The two best preconditioning treatments for all.44
variables were CD0,48 and CD12'0 (or Co + D48 and C12 + Do)
for CEO and IV seedlings, respectively. This suggests a
fundamental difference between CEO and IV seedlings regarding
their response to cold and darkg Where CEC seedlings benefit
from.dark IV seedlings benefit from cold" Determining the
difference would require growing CEC seedlings under IV
conditions and vice versa. For instance, growing CEC
seedlings enclosed in plastic bags to raise the relative
humidity to 100% as for IV seedlings, or decreasing the light
intensity from 150 uEm'zs'1 to 35 uEm-28'1 for CEC seedlings
and conversely increasing the light for IV seedlings. Also,
the nutrients used could be more closely matched.

In spite of the statistical differences noted for cold,
dark and donor plant on shoot regeneration, whole plant
preconditioning is clearly unnecessary for successful culture

and shoot regeneration from protoplast-derived tomato calli.

r4

APPENDIX A

53

Summary statistics and analysis

Table A1. Reductions in sum of squares for yield.

 

Sum of Degrees
Model Squares of freedom R2
y'y 41.919 47 100.0 %
R(u,CDS) 41.676 18 90.9 %
R(u,C,D,S,CD,CS,DS) 41.386 12 80.1 %
R(u,C,D) CEC 18.281 5 72.7 %
IV 23.098 5 84.4 %
R(u,CD) CEC 18.454 9 88.7 %
IV 23.222 9 92.3 %
R(u) 39.244 1 0.0 %

 

54

Table A2. Reductions in sum of squares for viability.

 

Sum of Degrees
Model Squares of freedom R2
y'y 82.495 283 100.0 %
R(u,CDS) 82.183 18 96.4 %
R(u,C,D,S,CD,CS,DS) 80.553 12 77.5 %
R(u,C,D) CEC 31.339 5 71.2 %
IV 49.007 5 16.4 %
R(u,CD) CEC 33.162 9 98.0 %
IV 49.021 9 22.8 %
R(u) 73.871 1 0.0 %

 

55

Table A3. Reductions in sum of squares for microcalli

 

diameter.
Sum of Degrees

Model Squares of freedom R2
y'y 286,196.04 255 100.0 %
R(u,CDS) 281,397.67 16 73.6 %
R(u,C,D,S,CD,CS,DS) 281,030.18 12 71.6 %
R(u,C,D) CEC 178,843.62 5 37.6 %

IV 102,161.33 5 20.0 %
R(u,CD) CEC 178,888.8 7 38.9 %

IV 102,508.92 9 29.3 %
R(u) 268,016.79 1 0.0 %

 

56

Table A4. Reductions in sum of squares for shoot
morphogenesis.

 

Sum of Degrees
Model Squares of freedom R2
y'y 319.587.3 276 100.0 %
R(u,CDS) 315,924.5 16 40.6 %
R(u,C,D,S,CD,CS,DS) 315,850.9 12 39.4 %
R(u,C,D,8) 314,949.9 6 24.8 %
R(u,C,D) CEC 156,891.2 5 25.4 %
IV 158.874.6 5 38.9 %
R(u,CD) CEC 156,894.2 7 21.5 %
IV 159,030.3 9 43 5 %
R(u) 313,420.1 1 0.0 %

 

Table A5. Single factor evaluation.

 

 

 

partial

Variable Source Factor(s) F sign. sz

Microcalli

diameter CEC C,D 16.123 1% 37.61%
C 29.132 1% 35.26%
D 1.531 n.s. 2.78%

Shoot

morphogenesis CEC C,D 22.846 1% 39.61%
C <1 n.s. <1%
D 44.885 1% 38.40%

IV C,D 10.372 1% 25.38%

C 9.743 1% 13.77%
D 18.572 1% 23.34%

 

58

Table A6. Goodness of fit

for

2-way interaction model.

 

Variable Source R2 Significance
Yield CEC 88.7 1%
IV 92.3 1%
Viability CEC 98.0 1%
IV 22.7 1%
Microcalli CEC - —
diameter IV 29.3 1%
Shoot CEC — -
morphogenesis IV - -

 

t\

59

Table A7. Goodness of fit for 3-way interaction model.

 

Variable R2 Significance
Yield 90.9% 1%
Viability 96.4% 1%
Microcalli

diameter 73.6% 1%
Shoot

morphogenesis - -

 

60

Table A8. Smallest subclass means for Yield

 

 

Means
Smallest Subclass CEC IV
u + 000,0 1.022 1 0.121 1.029 1 0.033
u + 000,24 1.031 1 0.121 1.230 1 0.107
u + 000.43 1.140 1 0.121 0.612 1 0.107
u + 0012 0 0.563 1 0.143 1.077 1 0.131
u + 0012,24 0.794 1 0.143 0.953 1 0.107
u + 0912.43 1.146 1 0.143 0.636 1 0.107
n + 0024 0 0.533 1 0.143 1.237 1 0.131
u + 0024'24 0.797 1 0.121 1.063 1 0.131
u + 3924,43 0.734 1 0.143 0.714 1 0.131

 

61

Table A9. Contrasts for Yield

 

 

Source ho F value Sign.
CEC CD - CD0 = 0 0.0046 n.s.
12 48 48
000, 24 — 0012, 24 = 0 7.171 5%
CD12 48 - CD0, 24 3 0 1.688 n.s.
2CD0: ‘8 - (CDO o + CD0,24)
= 0 2.741 n.s.
CD12,48 - CD0 0 = 0 1.963 n.s.
CD12,48 - CD12 24 f 0 13.181 1%
CD12 ‘8 - CD2‘: 2‘ - 0 15.550 1%
CD24: 24 - 24 0 a O 8.564 5%
2CDO: o - (CD12:O + CD24'0)
3 0 40.543 1%
ch0.24 ‘ (C912,24 + C924.24l
= 0 10.894 1%
(090.43 + 0912.43) ‘ ch24.43
= 0 19.356 1%
CD12,48 ‘ CD12,0 a 0 36.158 1%
(CD + CD ) - 2CD
24.48 24,248 0 24,0 9.575 1%
IV CD24'0 ’ CD0,24 = 0 <1 n.s.
CD2"0 - CD12,O . O 3.368 n.s.
0024'0 - CD24'2‘ 3 0 3.758 n.s
CD24,O - CD0,0 3 0 8.132 5%
CD0,24 ‘ CD24'2‘ = 0 ‘.1‘5 n.s.
000,24 - 0012'24 = 0 15.144 1%
CD24,48 - CD0,48 u 0 1.643 n.8
- B 0 9.967 1%
2000, 224 - 0313,24 + 0024,24)
= 0 11.704 1%
CDO,O - CDO,48 = 0 42.90 1%
n.s

CDS24,0,Iv ‘ CDS12,48,030 = 0 ‘1

 

62

Table A10. Smallest subclass means for viability

 

 

Means

Smallest Subclass CEC IV

u + CD0,0 0.525 1 0.018 0.559 1 0.013
u + 000.24 0.547 1 0.016 0.582 1 0.014
u + 000,48 0.689 1 0.021 0.566 1 0.018
u + 9912.0 0.000 0.622 t 0.018
u + 01112,24 0.647 1 0.021 0.696 1 0.019
u + CD12,48 0.646 1 0.021 0.679 1 0.021
u + 0024'24 0.624 1 0.024 0.691 t 0.015

 

\t.

63

 

 

Table A11. Contrasts for viability
Source ho F value Sign.
CEC CDO,48 ’ CD0,24 3 0 11.786 1%
CD0'24 - CD12,48 = 0 <1 n.s.
CD0,24 - CD0,0 3 0 6.702 1%
CDO,24 ' CD24'24 3 0 2.599 n.s.
(9912,24 + CD12,48) ‘
(CD24'24 + CD24,48) 3 O 4.202 5%
IV CD12'0 - C024'48 g 0 2.966 n.s.
CD12’O “ CD12'24 - 0 3.143 n.s.
CDIZ'O “ CD2"2‘ a 0 7.042 1%
CD12,O ‘ CD24,O = O 4.636 5%
$38'2‘ “ ED8D48 .)O 20D 1.468 n.s.
, 4 24, 4 — ,
12 2 2= O 0 21.455 n.s.
CD24'24 - CD0,24 : 0 <1 n.s.
CD24,48 - CDO,48 - O 6.818 1%
CD24,48 - CD24'24 z 0 <1 n.8
2CD0,48 ' (CD12,48 + CD24,48)
B 0 16.067 1%
CDS12.0.Iv ‘ CDS0.48.020 = 0 5-345 5*

 

64

 

 

Table A12. Smallest subclass means for microcalli diameter.
Means

Smallest Subclass CEC IV
u + CD0,0 26.845 : 1.416
u + CDO'24 25.720 1 1.606
u + CD0,48 23.912 1 2.041
u + 0012.0 31.187 3 1.967
u + CD12'24 30.062 1 2.125
u + CD12,48 28.254 1 2.327
u + CD24'0 26.348 1 1.688
u + 0024,24 25.223 1 2.041
u + CD24,48 23.415 1 2.041
u + C0 + no 41.389 1 1.769
u + C0 + 024 43.686 1 1.274
u + CO + D48 43.420 i 1.573
u + 012 + Do -
u + 012 + 024 36.594 1 1.626
u + 012 + D48 36.328 1 1.628
u + 024 + no -
u + 024 + 024 36.068 1 1.793
u + 024 + D48 35.802 1 1.671

 

 

 

 

 

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66

Table A14. Contrasts for microcalli diameter.

 

Source ho P value Sign.
CEC 012 ' C24 = 0 <1 n.s.
024 ’ D‘s = 0 <1 n.s.
Co ‘ C24 3 C48 58.528 1%
Do 3 024 3 D48 2.329 n.s.
IV 2CD12.0 - (CD0,o + CDo,24)
= 0 13.182 1%
(090,24 + CD12,24’
- ZCD24'24 = 0 4.231 n.s.

CD12,‘8 ’ CD0,48 a O 5.407 5%

 

67

Table A15. Smallest subclass means for shoot morphogenesis.

 

 

Smallest Subclass Means
CEC IV

u + co + ”0 33.460 1 1.271 33.436 1 0.966
u + 00 + 024 36.041 1 1.169 33.757 1 1.040
u + co + 048 39.576 1 1.316 34.091 1 1.176
n + C12 + no - 34.450 1 1.180
u + 012 + 024 32.133 1 1.332 34.771 1 1.115
u + 012 + D48 35.670 1 1.254 35.105 i 1.138
u + 02‘ + Do - 27.206 1 1.211
u + 024 + 024 32.915 1 1.259 27.526 1 1.226
u + 024 + 048 36.453 1 1.239 27.661 1 1.310

 

68

 

Table A16. Contrasts for shoot morphogenesis.
Source ho F value Sign.
CEC co=012=c24 7.665 1%
130=024==D48 14.991 1%
C24 — 012 = 0 2.782 n.s
20o - (012 + 024) a 0 15.334 1%
C12 - C24 = 0 <1 n.s.
2048 - (00 + 024) = 0 29.963 1%
no - 02‘ a 0 4.679 1%
IV (012 + Co) - 2024 = 0 >1000 1%
c0 - 012 = 0 17.660 1%
012 - 024 = 0 701.365 1%
0o - 024 = 0 1.749 n.s
024 - 048 = 0 1.654 n.s

 

69

Table A17. Pre—conditioning treatments with highest means
for protoplast yield, viability, microcalli
diameter and shoot morphogenesis.

 

 

 

Source

Variable CEC IV
Yield 090.0' CD0.24 CD0.24 CD24.0

CD0.46 CD12,46 CD12.0
Viability CD0,43 CD12,0
Microcalli Co + 924 CD12,0
diameter 00 + D48
Shoot Co + D43 Co + D0,24 & 48
morphogenesis C12 + D0,24 & 48

 

70

Table A18. Tomato protoplast culture media

 

 

Components KM-T A4
KN03 1875 1875
MS salts w/o

NH4N03

myo-Inositol 2000 2000
Thiamine HCl 10 10
Nicotinic acid 1 0.5
Pyridoxine HCl 1 0.5
L—glutamine 100 100
D-Ca Pantothenate 1 -
Folic acid 0.4 -
p—ABA 0.02 -
1/2 V-KM

organic acids2 +

Biotin 0.01 —
Choline Chloride 1 -
Ascorbic acid 2 -

V-KM sugars
and sugar

alcohols2 + -
V-KM

supplements2 + —
NAA 1 1
6-BAP 0.5 0.5
Sucrose 17100 17100
Mannitol 63700 31850
Glucose — 31500
Purified agar1 - 6000
pH 5.8 5.8

 

1 See Table 2.
2 ref. 17.

71

Table A19. Purification of agar

 

5.

Suspend 1/2 pound of Bacto Agar in ddH20 and wash by
drawing of the supernatent.

Resusgend agar in ddeo and allow it to stand overnight
at 4 C.

The resultant cake is resuspended in acetone and
filtered through 2 ply cheesecloth.

The resultant cake is resuspended in 100% ethanol and
filtered through 2 ply cheesecloth.

The cake is dried.

 

APPENDIX B

72

Least Squares Approach

In cell and tissue culture experiments the data
collected is commonly unbalanced. In other words, there are
unequal numbers of observations in the individual treatment
classes. The problem with unbalanced data is the sums of
squares due to the individual factors do not sum to the
total sum of squares due to all of the factors. Hence, the
factors are not independent and so must be considered
simultaniously. The calculations involved in this type of
analysis are usually more complicated than those of
traditional analysis of variance for balanced data. Prior
to the era of computers only limited demand existed for
analyzing unbalanced data. Now, however, because of the
availability of computers, analysis of unbalanced data is
increasingly more common. Analysis of unbalanced data
cannot be made Just by means of some minor adjustments to
the traditional analysis of variance of balanced data.
Unbalanced data analyses have their own analysis of variance
techniques, and those for balanced data are merely special
cases of the techniques for unbalanced data. Unbalanced
data analyses use matrix expressions, many of which do not
simplify in terms of summation formulas. When the data is
balanced or, in other words, the number of observations are
all the same, these matrix expressions simplify
considerably. They reduce, in fact, to the well-known
summation formula of traditional analysis of variance of

designed experiments. It is easy then to think of balanced

 

 

 

 

 

y-

 

73

designed experiments as special cases of the more basic
analysis of variance for unbalanced data. The method of
analysis used in these experiments is the Least Squares
Approach, also known as the method of fitting constants of

the linear model.

1. General fixed model
A simplified example will best introduce the idea of
the general fixed model used in these experiments and the
matrices necessary to manipulate and solve them.

Example. Numbers of protoplasts isolated per gram of
fresh tissue was obtained for 47 individual protoplast
isolation experiments. Protoplasts were isolated from
seedlings preconditioned by 0, 24 or 48 hours in the dark
prior to protoplast isolation. I will consider just 9
isolation experiments for illustrative purposes, 4 from 0
hours, 3 from 24 hours and 2 from 48 hours dark
preconditioning. For the entries in TableBi let Yij
represent the protoplast yield of the 3th plant of the ith
dark treatment, i having a value of 1, 2 or 3 for 0, 24 and
48 hours respectivelyu and j=1,2,. ..,n1, where n1 is the
number of observations in the ith dark treatment. The
problem is to estimate the effect of dark preconditioning on
protoplast yield. To do this the observation Yij is

considered as the sum of three parts

Yij 3 u + d1 + e13, (1)

C}

l
a O

LIE"

fit

.. r. . .
x *O r u
.. .. . \
r.
. L ... ,2 1. .
n .
9 . .
I
. . .
‘ I m r.
_

74

Table Bl. Protoplast yield (x106) of dark precondition-
ed seedlings.

 

9 hrs. _1 hrs 18 hrs
1.073 1.002 0.884
0.992 1.202 0.973
1.053 1.136
1.023

Totals 4.106 3.393 1.857

 

 

 

 

 

 

 

 

75
where u represents the population mean or the constant
common to all observations, d1 is the effect of dark
preconditioning i on protoplast yield, and e13 is the random
error associated with each Yij' The eij's are assumed to be
independent and distributed around a mean of zero. Also, it
is assumed each e13 has the same variance,<32, so that the

variance-covariance matrix of the vector of eij's is €21.

r — F —
620....0 10...0
2 (1‘2...0 201...0
C I a = (T
2
00 q. 0 1
a a L A

 

 

 

 

The problem is to estimate u and the three d1 terms, and
also <72, the variance of the error terms. To provide an
estimation of these parameters each observation is written

in terms of the above a-part equation (1):

1.038 = Yll g u + ldl + ell
0.992 = Y12 3 u + 1d1 + 312
1.053 ' Y13 : u + ldl + 313
1.023 = Y14 = u + 1d1 + 814
1.055 = Y21 = u + 1d2 + 921
1.202 a Y22 = u + 1d2 + 922
1.136 a Y23 a u + 1d2 + 823
0.88‘ B Y31 g u + 1d3 + 831
0.973 3 Y32 B u + Ida + 832

The equations are easily converted into matrix forms

76

 

 

 

 

 

 

 

 

 

1.038 VII 1 1 O 0 ell
0.992 y12 1 1 0 0 u e12
1.053 y13 1 1 0 0 d1 + e13
1.023 = Y14 = 1 1 O 0 d2 614
1.055 Y21 1 0 1 0 daj 821
1.202 Y22 1 O 1 0 _ 822
1.136 Y23 1 0 1 O 823
0.884 Y31 1 0 O 1 331
0.973 Y32 1 O O 1 832
L a h d L J b a

 

representing the general form of the fixed model
y = Xb + e

where y is the vector of individual observations, the X is
termed the incidence matrix, because the presence of 0's and
1's describes the incidence of the terms in the model (u,
d1, d2 and d3), and b is the vector of parameters to be

estimated,

 

 

and e is the vector of error terms.

2. Estimation

The problem is to estimate b as all computational
procedures for‘estimation and hypothesis testing requires
estimates of b. Applying the method of "Least Squares"
results in an estimator of b that minimizes the squared

errors (i.e. sum of squared deviations)

Differentiating e'e with respect to b minimizes e'e. The
resultant equations are termed the normal equations.
A
3'!!!) - X'y
A

A
where the "A" represents the solutions of b. Therefore, b

is solved for algebraically from:

A
b - (x'X)'1x'x

where (X'X)"1 is the inverse of the coefficient matrix (X'X).

3. The normal equations

The coefficient matrix, mat, in the normal equations is
obtained from the incidence matrix x in the model, y - Xb +
e. The incidence matrix, 1:, can be thought of as a 2-way
table with the parameters as headings to the columns and the
observations as headings to the rows (Table 82). From the

example,

,“A‘

78

Table 82. x matrix as a 2-way table.

 

 

Parameters
Observations u d1 d2 d3
y12 1 1 0 0
y14 1 1 0 0
23 1 O 1 0

31 1 0 0 1

 

O

79

— — — -

1 1 1 1 1 1 1 1 1 1 1 0 0 9 4 3 2

1 1 1 1 O 0 0 O O 1 1 O 0 4

0 0 0 O O O O 1 1 1 O 1 O 2

L... .1 ._ _J

 

 

 

 

l

|
000011100 1010 3.030

I

I

1 O O 1
L. .3

 

 

The top left-hand element, 9, is the total number of
observations with the next three elements, 4, 3 and 2 being
the number of observations in those particular classes, 0,
24 and 48 hours dark preconditioning, repectively. The
submatrix is composed of the number of observations in each
subclass. So, the intersection of 24 hrs. and 48 hrs. is 0
as the seedlings were preconditioned in one of the three
treatments. Likewise, the 1!"! matrix is obtained from the
incidence matrix X and the y observation vector from the
model y - X'b + e. In the )1"! 9.356 is the top element and
is the sum of all nine observations, and the following
elements are the corresponding sums of the individual
classes 0, 24 and 48 hrs dark ( d1, d2 and d3 ),

respectively.

80

1.038
0.992
1 1 1 1 1 1 1 0 0 1.053 9.356
1 1 1 1 0 0 0 0 0 1.023 4.106
0 0 0 0 1 1 1 0 0 1.055 3.393

000000011 1.202 1.857

 

 

 

 

1.136

0.884

0.973

 

 

Writing the normal equations from the example out in full is

the fol lowing:

 

9 I 4 3 2 u 9.356

4 : 4 0 0 d1 4.106

3 | 0 3 0 d2 3 393
I

2 | 0 0 2 d3 1.657

 

 

 

 

 

The normal equations are constructed using numbers of

observations in the 3': and their sums in the X'y.

4. Solving for b

Unlike the regression model the fixed classification
model is a non—full rank model. The result is the inverse
of X'X, (X'X)'1, does not exist and, estimates cannot

therefore be calculated by b - (X'X)"1X'y. For the fixed

81
classification model a generalized inverse, (XUU') is used.
One of the properties of a generalized inverse is that
solutions can be found for b but those solutions are not
unique as with a regression model. In fact there are an
infinite number of solutions for b. Therefore, the normal
equations are now written as

(x'X)b° - 1m:

with b° signifying these equations have no single solution
for b°. None of these solutions estimates b, the solutions
only solve the normal equations. So, for the example Table

83 lists four of the many solutions for b.

5. Estimable functions

As they stand none of the solutions for b° in Table 83
estimate b, but certain linear functions of the elements of
b° can be used. Suitable linear functions yield the same
estimate regardless of the solution for b°. This set of
linear functions is called "estimable”. Table B4 lists four
linear functions and their respective solutions using the
solutions for b° from Table 83. Linear functions 1 and 2
give a different solution for each b° solution, but linear
functions 3 and 4 are invariant to the solutions for b0.
Hence, linear functions 3 and 4 are estimable linear
functions and can be used to estimate the parameters of the
linear model.

To determine the estimability of a given linear function

it is not necessary to test its invariance with several bo

u
0 hrs (d1)
24 hrs (d2)

‘8 hrs ((13)

82

 

Table 83. Four solutions for b°.
b1 b2 b3 b‘
0 .929 1.131 1.027
1.027 .098 -.105 0
1.131 .203 0 .1045
.929 0 -.203 -.098

 

83
solutions. There are two methods to determine the
estimability of a function.

1) The smallest subclass means are estimable as are any
linear combination of them. In the example, the smallest
subclass means are (u + d1), (u + d2) and (u + d3). So,
linear function 3 in Table 84 is estimable as it is a linear

combination of the smallest subclass means (u + d1) and (u +

d2).

(u+d1)-(u+d3)
=u+d1-u—d3

“d1’d3

2) A linear function k'b is estimable if it satisfies

the following equation:
k'(X'X)'X'x a k'

where k is a vector containing the values of the interested
contrast. So, linear function 3, (d1 - d3), would be

written as follows:

k'b=[0101]u

 

 

and,

84

Table 84. Estimates of four linear functions.

 

 

Solutions
linear function b1 b2 b3 b4
1) 1/2 (d1 + d3) .978 .049 -.154 -.049
2) (u + d1 + d2 + d3)/3 1.029 .410 .274 1.034
3) d1 — da .098 .098 .098 .098
4) (d1 ‘1" d2) "' 2d3 .3 .3 .3 .3

 

k(X'X)-X'X = [ 0

BIG

=k'

1

1

85

0 1 ] 0 0

0'1/4
0'0
0:0

1 1
1 0
1 0

 

0 1 ]

hence, d1 - d3 is estimable.

6. Other calculations

With solutions for b° and estimable functions suitable
to answer the research questions of interest sums of squares

are calculated and hypotheses tested.

equations used in this study:

0 0
0 0
1/3 0
0 1/2
0
0
0
1

 

 

 

9 4 3 2
4 '4 .0—0-
3 :0 3 0
2 :0 0 2

L _.

The following are the

1) Sums of squares (SS) of the full model

a R(full)

_ bOsxlx

2) Sums of squares minus the mean

= R(factorslu)

= bOlex _ ny2

 

3)

4)

5)

6)

7)

8)

9)

10)

11)

86
Residual SS

,3 YIY _ bOIXIx

= residual SS/(n-r)

where, n is the number of observations and

r is the rank of the X'X.
Proportion of variation explained by the model
- 32
- R(factors|u)/(y'y - ny2)
Contribution of individual factors
3 32:
- R(factorlother)/[y'y - R(others)]
Confidence intervals for an estimable function, k'b
k'b° 16th,“ “/2 k'(X'X)"k
Test for goodness of model
. r -= R(factorslu)/(r-1)3'2

Test for partial contribution of factors

a F a R(factors|other)/(r-rothers)2

Numerator sum of squares (Q-value) for specific

hypotheses.
0 = (k'b° - m)[k'(X'X)’k]’1(k'b° - m)
where, m is a vector of specified
constants of order s x 1 and s
is the number of simultaneous
hypotheses being considered.

Test for specific hypotheses (F-ratio).

‘“2
F(H) = Q/SQ’

 

87
7. Method of calculation
Matrix manipulations were preformed on an IBM Personal
Computer using software written in BASIC. Matrix inversion

was done by a modified Cause-Jordan Elimination Method.

8E3

(X'X) for colony diameter with dependencies removed.

C322
0532

0322
0332

CD32
CD33

 

19
13

29
27

104

52

S2

12
19

24
21

42
36

10
13

28
24

52
42

112
0

10
14

36
46

52

36

143

36
46

52
36

19
13

CS
22

36
0

12
10

CS
32

0
46

19
13

19
13

19
13

DS
22

12
19

DS
32

10
13

CD
32

 

(X'XY for oolmy dimer.

C0

33

05 CO CD CD C0 C0 C0 C0 C0

6 6666 6

32

23

11 12 13 21 22 32 13

32

11 21 31 12 22

11 21 31 12 22

6666 6

S
2

U

8833!!!

iii!

‘6'.

89

 

 

33g? 3&5: 33. 333333: 3333313. 3333333ig-ifis-23:1
I I II
23 a: 8 8° $8 53
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33°3 333 33 333333 333333 333333333
3393 333 33 333333 333333 333333333
3363 '333 33 333333 333333 333333333
3333 333 33 333333 333333 333333333
3393 333 33 333333 333333 333333333
3393 333 33 333333 333333 333333333
°°3° 333 33 333333 333333 333333333
3 ‘5" $3 3 '- 8°
33g'3: 333 83 333383. 3333;; 333333333.
‘ II I II II
3388 383 3_ 333338 3333—.v-_ 333333338
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3393 333 33 -333333 333333 333333333
3393 333 33 333333 333333 333333333
°°3° 333 33 333333 333333 333333333
°°3° 333 33 333333 333333 333333333
.-l I II
8 2 '- 3 g 31’
33%3. 333 32. 333333 33333§ 33333338.;
,°I I II
3303 333 33 333333 333333 333333333
3393 333 33 333333 333333 333333333
3363 333 33 333333 333333 333333333
°°3° 333 33 333333 333333 333333333
8 9'- 5; 83 853
3393. 3§§_ 33 33338.: 333333 333333333
I I II II
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3303 388 83 333333 3333 8 333333333
II
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oogg :33 3g 333333 999933 9999:9983
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3303 333 33 333333 333333 333333333
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33%,: 333 33 33333:. 333385. 33333333.?
' II I II II
3 '— 28 2
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3333 333 33 333333 333333 333333333
L3333 333 33 333333 333333 333333333]
an at use.» not.» Qflflflflih
o-Nm v-wn v-N 3:385:33 3:889:33 2532538338
3333 333 mm 888888 888888 888888888

(X'Y) for oo1cny diameter.

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d

C511
$21
C331
C812
C322
$32

$11
$21
$31
$12
$22
$32

CD11
CD12
CD13
0021
CD22
CD32
CD13
CD23
CD33

02.911"
4151.9
2100.8

2014.4

2275.3
3476.3
2514.5

4458.9
3808.2

2576.1
1020.9

861.9
1575.8
1079.9
1152.5

745.0
2096.2
1617.7
1530.3
1380.1

897.8

1438.9
1785.9
927.1
475.3
858.6
766.9
361.1
831.8

 

 

L_821.5_J

90

(KW-(X7) = b

91

 

.4

 

 

 

 

88 ‘38 “8 ° :3 2‘"
as a; §8 33 :8 3%
L99fi‘9 9 7 9a 999999 999990? 9999999 F4
II
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F783 2 a 2 gm 8M
9989. 933 93. 9999§8 999993 9999999293-
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99°9 999 99 999999 999999 999999999
99°9 999 99 999999 999999 999999999
9999 999 99 999999 999999 999999999
9999 999 99 999999 999999 999999999
99°9 999 99 999'999 999999 999999999
99°9 999 99 999999 999999 999999999
99°9 999 99 999999 999999 999999999
8: a" 32 ~ ~ 8°
993.3 98§ 33 99993.3 9999;; 999999933
II I II II
9
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9938 988 ES: 9999§8 9999§§ 999999933
II I II II
99°9 999 99 999999 999999 999999999
99°9 999 99 999999 999999 999999999
9999 999 99 999999 999999 999999999
9969 999 99 999999 999999 999999999
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9909 999 99 999999 999999 999999999
9999 999 99 999999 999999 999999999
9999 999 99 999999 999999 999999999
9999 999 99 999999 999999 999999999
8 __ '- 8“ 3
9998. 9§§_ 93 999933 99993§ 99999993§
I I II II
3
9999 933 §9 999999 9999§§ 999999999
II
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|'9 I 8| II
2 “2 S'— 251' 3
eggs 9&3 3§ 999933 eoeogg 9999999§§
I'I II II I
9999 999 99 999999 999999 999999999
II I II II
II II I
9999 999 99 999999 999999 999999999
[3999 999 99 999999 999999 999999999]

 

Far CEC data:

(X'X) ufith dependencies removed.

01 P50 0 0110 20
|

C2 020 0'014

03 0 0241010

0 1 18 D 0 : 18 0

 

 

02 30 14 10 I0 52
(m-(X'th
'0' 0 0 0
0 .0139 .015 .0125
0 .015 .041 .0094
0 .0125 .0094 .0495
0 -.0439 -.015 -.0125
0 -.0301 -.0225 -.0100
_0 0 0 0

 

-.0439

-.015

-.0125

.0994

.0301

-.0301

-.0226

-.0188

.0301

.0451

£312

°J

 

"0050.9”
2575.1
1020.9
861.9
745.0

2096.2

 

 

@171

_ 1

0

43.4204

36.3278

35.8018

-2.0315

.2658

 

 

 

 

0

-.0302

-.0274

-.0257

.0465

.0279

ForIVdata:

(X'X)I11thdepende101'a reroved.

c c c 0 0

1 2 3 1 2

01 ”6'1 0 0127 21‘

l

02 0 33 0.11 12

03 0 0 15:11 19

01 27 11 11:55 0

02 g 12 19: 0 52

(X'X)-(X'Y)=b

0 0 0 0
0 .0395 .0213 .021
0 .0213 .0173 .0193
0 .021 .0193 .0111
0 -.0302 -.0271 -.0251
0 -.0200 -.0235 -.0279
_0_ 0 0 0

 

0

5313

-.0286

-.0265

-.0279

.0279

.0471

 

1575.8

1079.9

1152.5

1530.3

1380.1

3800.27

 

897.8

 

_ q

23.9124

28.2542

23.4151

2.9326

1.8077

 

 

_ d

LIST OF REFERENCES

10.

LIST OF REFERENCES

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94

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95

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Bhatt, D.P., Fassuliotis, G. 1981. Plant
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Pflanzenphysiol. 105:285-288.

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E; PflanzenphysiolL 74:327-356.

 

Bingham, E.T., Hurley, L.V., Kaatz, D.M., Saunders,
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Bokelmann. G.S., Roest, S. 1983. Plant regeneration
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Bonito, R.D. 1985. Embryo culture in tomato: a
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Buiatti, M., Marcheschi, G., Tognoni, F., Lipucci, M.,
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Cappadocia, M., Ramulu, K.S. 1980. Plant
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Agomgsicon esculentum L. x Lycopersicon peruvianum
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Carlberg, 1., Glimelius, K., Eriksson, T. 1983.
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Caruso, J.L., Franco, P.I., Snider, J.A.. Pence, V.C.,
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Cassells, A.C., Cocker, F.M. 1982. Seasonal and
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Cassells, AJL, Barlass, M. 1976. Environmentally
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Cassells, A.C., Barlass, M. 1978. A method for the
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