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THE-61 8

STATE UNI VE

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This is to certify that the

thesis entitled

Examination of a population of somclones for variation
in DNA content, isozyme banding patterns, and tissue
culture response in sugarbeet (Beta vulgaris L.)

 

presented by
Yong Rhee

 

has been accepted towards fulfillment
of the requirements for

M.S. Crop and Soil Sciences

degree in

 

 

 

  

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me mechanisms-n14

 

EXAMINATION OF A POPULATION OF SOMACLONES FOR VARIATION IN
DNA CONTENT, ISOZYME BANDING PATTERNS, AND TISSUE CULTURE
RESPONSE IN SUGARBEET (Beta vulgaris L.)

By

Yong Rhee

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Sciences

1998

ABSTRACT
EXAMINATION OF A POPULATION OF SOMACLONES FOR VARIATION IN
DNA CONTENT, ISOZYME BANDING PATTERNS, AND TISSUE CULTURE
RESPONSE IN SUGARBEET (Reta vulgaris L.) ‘
By

Yong Rhee

Somaclonal variation is genetic change occurring in plants regenerated from
callus or other in vitro culture. Nineteen sugarbeet somaclones (regenerated plants) were
evaluated for somaclonal variation for three parameters Firstly, for tissue culture
response, intensity of leaf disc callus induction and of shoot regeneration on media with
and without cytokinin were not significantly different from the source genotype; shoot
regeneration occurred only on the medium with exogenous cytokinin. Secondly, isozyme
banding patterns with starch gel electrophoresis of 6-phosphogluconic acid, malate
dehydrogenase, Shikimate dehydrogenase, and glutamate dehydrogenase from leaves, as
well as alcohol dehydrogenase from callus showed no somaclonal variation. Thirdly, no
significant difference in DNA content in nuclei of leaf cells as determined by flow
cytometry was found. Additionally, a narrow leaf variant with altered lateral stalk
organization did not differ from the source genotype in isozyme banding patterns and
DNA content. The narrow leaf variation was not recovered in F , progeny. In an initial
direction of research, haploids were evaluated for their callus induction and shoot
regeneration prior to intended use in recovery of recessive somaclonal variation. But, an

unexpectedly low response was found in haploids in comparison with the source diploids.

To my parents

iii

ACKNOWLEDGMENT

I dedicate this thesis to my beloved parents. They always encouraged and gave
endless support to me. They also showed me the importance of considering others before
taking care Of myself. I believe that this philosophy will be the very basis of my whole
career in my life.

I am extremely grateful to three scientists for their valuable advises. Dr. James F.
Hancock and Dr. Joanne H. Whallon gave me good advises for completing the thesis.
Most importantly, I would like to thank my advisor, Dr. Joseph W. Saunders, for teaching

scientific thinking and giving valuable advises to a awkward beginner in science.

TABLE OF CONTENTS

LIST OF TABLES .......................................................................................................... vii

LIST OF FIGURES ......................................................................................................... .ix

CHAPTER I

Examination of a population of somaclones for variation in DNA content, isozyme
banding patterns, and tissue culture response in sugarbeet (Beta vulgaris L.)

Literature review... ........................................................................................................... 1
Introduction .................................................................................................................... 10
Materials and methods ..................................................................................................... 17
Experiment 1. Tissue culture response of leaf discs ...................................... . 17
Plant maintenance ............................................................................. 17
Leaf sample preparation .................................................................... 17
Media preparation ............................................................................. 18
Culture establishment ........................................................................ 18
Experiment 2. Isozyme banding patterns with starch gel electrophoresis ....... 20
Sample preparation ........................................................................... 20
Gel electrophoresis ........................................................................... 20
Gel Staining ..................................................................................... 32
Experiment 3. NuclearDNA contents as determined by flow cytometry .......... 24
Solutions required .............................................................................. 24
Preparation of nuclei by chopping plant tissues ................................... 25
Estimation of nuclear DNA content .................................................... 25

Results ............................................................................................................................ .27
Experiment 1. Tissue culture response of leaf discs ........................................ . 27
Experiment 2. Isozyme banding patterns with starch gel electrophoresis...... .. 28
Experiment 3. Nuclear DNA contents as determined by flow cytometry........ 29

Discussion............ .................................................................................................... .42

Bibliography ............................................................................................................... . 45

CHAPTER II

In vitro response in sugarbeet (Beta vulagris L.) haploids

Introduction... ... . .. ......................................................................................................... .55
Materials and Methods. . . . . . . .. ........................................................................................... 60
Plant materials... ................................................................................................ 60

Ovule culture of 6822-08. .. ................................................................................ 60

Leaf sample preparation ..................................................................................... 63

Media preparation... ........................................................................................... 63

Culture establishment ........................................................................................ 63

Results and discussion...... .. ............................................................................................ 65

Bibliography... .. .............................................................................................................. 70

vi

CHAPTER I

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

CHAPTER II

Table 1.

LIST OF TABLES

Inorganic and organic constituents of MS media ............................... . 19
Summary of electrophoretic materials and methods ............................. 23

Callus formation from discs of smaller and larger
leaves from source genotype 8266—10 and derivative
somaclones on BI media 10 weeks after culture initiation ................... 30

Callus formation from leaf discs of smaller and larger
from source genotype 8266-10 and derivative somaclones
on MO media 14 weeks after culture initiation ................................... .31

Shoot formation from discs of smaller and larger leaves
from source genotype 8266-10 and derivative somaclone
on B1 media 10 weeks after culture initiation ..................................... 32

Shoot formation from discs of smaller and larger leaves
from source genotype 8266-10 and derivative somaclones
on MO media 14 weeks after culture initiation ................................... .33

DNA contents of leaf cells from source plant 8266-10,
nineteen derivative somaclones, and narrow leaf variant
(SC-NL) by flow cytometry ................................................................. 39

Analysis of variance (ANOVA) of source genotype
8266-10, nineteen derivative somaclones, and narrow leaf
variant (SC-NL) ................................................................................... 41

Callus induction and shoot regeneration from leaf discs of
the diploid source plant REL-1 and its haploid plants
OV-9 and CV 16 ................................................................................. .66

vii

Table 2. Preliminary test of callus induction and shoot regeneration
in two diploid genotypes 6822-10 and FC 701/5-4 ............................. 67

Table 3. Callus induction and shoot regeneration of the diploid source
6822-08 and its derivative haploids OV-68-1, OV—68-2, and
OV-68-3 ............................................................................................. . 68

viii

CHAPTER I

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

CHAPTER II

Figure 1.

LIST OF FIGURES

A morphologically aberrant somaclonal variant (SC-NL) with
narrow leaf (Fig 1.1) and abnormal lateral stalk organization
(Fig 1.2 and Fig 1.3) ....................................................................... .14-16

The banding patterns of 6-phosphogluconic acid dehydrogenase
(6-Pgdh) from source plant 8266-10 and nineteen derivative
somaclones ........................................................................................... 34

The banding patterns of malate dehydrogenase (Mdh) (Fig 3.1)
and shikimate dehydrogenase (Skdh) (Fig 3.2) from source plant
8266-10 and nineteen derivative somaclones ..................................... .35

The banding patterns of glutamate dehydrogenase (Gdh)
(Fig 4.1) and alcohol dehydrogenase (Adh) (Fig 4.2) from source
plant 8266-10 and nineteen derivative somaclones ........................... . 36

The banding patterns of 6-phosphogluconic acid (6-Pgdh)

(Fig 6.1), malate dehydrogenase (Mdh) (Fig 6.2), shikimate
dehydrogenase (Skdh) (Fig 6.3), and glutamate dehydrogenase

(Gdh) (Fig 6.4) of source plant 8266-10 and narrow leaf

somaclonal variant (SC-NL) .............................................................. .37

Flow cytometric analysis of DNA contents from source
clone 8266-10, the nineteen somaclones, and SC-NL ......................... .38

The emerging embryo from the cultured ovule of diploid
source plant 6822-08 ........................................................................... 6O

ix

CHAPTER I
EXAMINATION OF A POPULATION OF SOMACLONES FOR VARIATION IN
DNA CONTENT, ISOZYME PATTERNS, AND TISSUE CULTURE RESPONSE
IN SUGARBEET (Beta vulgaris L.)

LITERATURE REVIEW

Plant cell and tissue culture procedures has been considered as a potentially
important tool to produce clonal plants in vitro, to study metabolism and its inheritance,
and to serve in plant transformation protocols. Genetic fidelity among cells or plants is
usually desired and assumed. The cell and tissue culture procedure, however, does not
always produce clonal cells or plants, and frequently results in numerous genetic changes.
Larkin and Scowcrofi (1981) have argued that these genetic changes are a general
phenomenon of cell and tissue culture to which they gave the name ‘somaclonal
variation’. Controlling this variation is desirable and will provide a powerful source of
useful mutants, or for plant improvement (Orton, 1984; Karp, 1991; Phillips et a1.
1994). But, in contrast to its potentials for plant biotechnology, the underlying genetic
bases of somaclonal variation have been not yet elucidated completely.

From these viewpoints, this review is aimed at providing general information on
somaclonal variation, hoping that the information presented will show a good guideline
for further research for somaclonal variation with the recognition of the importance of
this variation. For this purpose, the emphasis will be on both the various causes of

somaclonal variation and the general approaches for measuring somaclonal variation.
1

It has been known that chromosomal abnormalities with changes in chromosome
number and structure occur at a high frequency in some types of regenerated plants.
Polyploidy and aneuploidy are found in callus and regenerated plants (Bayliss, 1980;
D’Amato, 1985). Two main factors have been considered to play an important role in
the chromosome number changes in callus and regenerated plants: firstly, the
composition of the medium, especially kinds and /or concentrations of the plant growth
regulators was considered. Some studies suggested that auxins and /or cytokinins
induced chromosomal abnormalities, probably leading to chromosome number variation
in callus or regnerated plants (Bayliss, 1973; Ogura, 1982). Secondly, the duration of
the culture of callus may cause chromosome aberrations with the function of time after
callus initiation. It has been reported that chromosomal variation increased in proportion
to the duration of the callus culture over time after callus initiation (Ogura, 1982).
Swedlund and Vasil (1985) also reported that the proportion of diploid cells decreased,
while that of tetraploid and aneuploid cells increased afier 6 months of callus culture in
pearl millet embryogenic callus.

Chromosome structural changes or karyotypic changes of different types
(deficiencies, translocations, duplications, inversions) are common in callus and
regenerated plants. Murata and Orton (1983,1984) observed chromosome fusions in
celery suspension culture. They reported that chromosome fusion seems to be a primary
source of chromosomal aberrations such as translocation, duplication, and deficiency,
which are probably involved in chromosome number variation within celery suspension

cultures. Callus and regenerated plants can display cytologically observable structural

changes in their chromosomes (D’Amato, 1985). The consequence can be production of
chimeric genes with promoter substitutions which alter expression conditions and genetic
transmission of specific genes perhaps by deletion or duplication of one copy of a gene.
In addition, recombination or chromosome breakage can occur in hot spots of particular
chromosomes, thereby affecting some regions of the genome. For example, Karp and
Maddock (1984) reported that 29 % of the 192 regenerants from four different wheat
cultivars showed chromosome interchanges. And Lee and Phillips (1988) have cited
evidence for chromosome translocations, deletions, inversions, and other minor
reciprocal and nonreciprocal rearrangements in other many plant species such as ryegrass,
potato, maize and oats, and argued that late-replicating heterochromatin may occasionally
replicate so late in mitosis of cultured cells that bridge formation and subsequent
chromosome breakage occurs at anaphase. This would be lead to a breakage-fusion-
bridge cycle with consequent genetic effects, such as deletions and chromosome
interchanges.

Transposable elements can be activated and alter gene expression in callus and
regenerated plants (Peschke et al. 1987). Cell and tissue culture procedures are highly
mutagenic to plant cells (Karp and Bright, 1985). And so, they can be a cause for
genome alterations or rearrangements (McClintock, 1984), stimulating transposable
element activities. Evidence has become more numerous for transposable elements’
activation in callus and regenerated plants, although this evidence is generally limited to
maize (Evola et al. 1985; James and Stadler, 1989; Peschke and Phillips, 1991). In

alfalfa, the involvement of transposable element activities was suggested in the unstable

mutation to white flower with high frequencies of reversion in color in vitro (Groose and
Bingham, 1986). Activation of uncharacterized silent elements has also been suggested
based on the recovery of unstable variants consisting of an extremely high frequency of
green and yellow sectors in tobacco in vitro (Lorz and Scowcroft, 1983).

An increasing number Of plant genes are proving to be normally encoded and
expressed in multigene families usually in clusters in one or more positions in the
genome. Somaclones have been found with altered patterns of products from such
families. The gliadin storage protein of wheat (Cooper et a1. 1986; Larkin et a1. 1984;
Maddock et a1. 1985), the hordeins of wild barley (Breiman et a1. 1987), the beta-
amylases of wheat (Ryan and Scowcroft, 1987), and the esterases of potato (Allichio et
al. 1987) are the examples for this case. Some of these mutants express proteins not
present in the donor plants. There may be heritably repressed genes in these gene
families that are exposed following callus initiation.

It has been reported that specific genes can amplify or deamplify themselves
during differentiation or in response to environmental pressures. Depending how gene
expression is regulated, this could mean that the production of mRN A and protein from
that gene is up or down regulated. If such amplification or deamplification of DNA
sequence copies also can occur in callus or regenerated plants, it may account at least in
part for somaclonal variation. Cullis and Cleary (1986) showed that the copy number of
repeated DNA in flax varied when exposed to various environmental stimuli.
Amplification and deamplification of DNA have been reported in both callus and

regenerated plants of flax. Another example is the amplification seen in alfalfa following

cell selection. In a cell line selected for resistance to a competitive inhibitor of glutamine
synthase, the tolerant cell line contained 4-11 times the number of glutamine synthase
gene sequences (Donn et a1. 1984). Examples of dramatic deamplification have been
found in potato (Landsmann and Uhrig, 1985) and barley (Breiman et a1. 1987).

Plant DNA has been known to be partially methylated and the role of methylation
in gene expression of plants has been reviewed (Hepburn et a1. 1987). DNA methylation
can affect plant gene regulation and expression at the level of transcription (Palmgren et
a1. 1991). It was proposed that every steps in callus cutlure and regeneration could alter
DNA methylation patterns in regenerated plants (Phillips et a1. 1990; Kaeppler and
Phillips, 1993). For instance, in carrot, genomic DNA methylation was shown to be
tissue specific and influenced by plant growth, culture age, genotype, and primary
cultures during the transition from linear to stationary growthin vitro (Arnholdt-Schmitt
et a1. 1995). The maize plants regenerated in vitro showed both increases and decreases
in methylation (Brown 1989; Muller et a1. 1990). It has been also suggested that DNA
methylation could be correlated with transposable element activities (Chandler and
Walbot, 1986; Banks et al. 1988) and chromatin structure (Klaas and Amasino, 1989)
in vitro.

The measurement of the extent of somaclonal variation is important for several
reasons. Firstly, as somaclonal variation has been found to depend on the genotype
(Puolimatka and Karp, 1993), stable genotypes may be selected for genetic engineering
and propagation via somatic embryos. Secondly, such a measurement will allow the

development Of protocols that minimize variations. Lastly, by identifying the factors that

have effect on generation of somaclonal variation, such a measurement will enable the
study of the mechanisms underlying somaclonal variation. Actually, such measurement
may be on the morphological, on the cytological or on the biochemical / molecular level.
On the gross morphological level, the extent of somaclonal variation is usually
determined as the percentage of plants which shows one or more qualitative variations,
for example, the percentage of plants with distinct morphological alterations such as
dwarfism, albinism and aberrant leaf shape (Lee and Phillips, 1987). Alternatively, De
Klerk et a1. (1990) proposed to measure the extent of variation in a somaclonal population
by determining the value of the Standard Deviation (SD) for a quantitative trait and have
used the leaf shape (expressed as the natural logarithm of the ratio of the longest rib and
its longest perpendicular rib) of regenerants of Begonia x hiemalis as such a parameter.
_ Wang and H011 (1988) found an increased value of the SD for the ratio of length-width of
the leaf shape in a somaclonal population of Trifolium pratense. An increased value of
the SD for various traits was also reported in regenerated rice plants (Zheng et a1. 1989).
On the cytological level, chromosome number or ploidy of somaclones is
determined by chromosome counts in mitotic cells from the regenerant’s root tips, by
measurement of the DNA content per cell either by a microspectrofluorophotometric
determination after F eulgen staining, or by flow cytometry. But chromosome counts can
only be made on dividing cells, and thus the sample is limited and potentially biased.
Furthermore, chromosome counts do not measure changes in chromosome volume and
DNA content, and therefore there may be significant changes that such chromosome

counts do not reveal (Karp and Bright, 1985). MicrospectrofluorO-photometric

techniques avoid these problems, as they measure cells in all phases of the cell cycle, and
flow cytometry is probably the most rapid method of these for measuring DNA content.
It has been found that flow cytometric analysis of DNA content can successfully be
accomplished measuring the relative fluorescence of stained nuclei (Galbraith et al.
1983). For example, the ploidy stability of somaclones was evaluated by nuclear DNA
contents with flow cytometry and it was found that ploidy of somaclones from short term
callus culture was significantly different from somaclones’ ploidy from long term callus
culture (Jacq et a1. 1992). But most of these methods do not permit individual cell
selection and are limited to fluorescence dyes. The detection of small alterations of the
chromosomal structure is very laborious. But karyotypic analyses of cultured cells have
clearly demonstrated the existence of translocations, filSiOIlS, and deletions (Orton,
1980). With differential staining of chromatin using Giemsa banding, Ashimore and
Gould (1981) showed specific structural changes in altered karyotypes of cultured crown
gall tissues of Crepis capillaris. Interestingly, it has been observed that in regenerated
plants with the correct chromosome number, many plants Show structural chromosomal
changes (Lee and Phillips, 1988). However, phenotypically aberrant plants may have the
same chromosome structure as the phenotypically normal plants and vice versa
(Armstrong and Phillips, 1988).

On the biochemical level, the genetic fidelity of callus or regenerated plants is
often examined at the isozyme level. This approach has been conducted with many plant
species such as barley (Orton, 1980), tomato (O’Connell et al. 1986), orange

(Kobayashi, 1987), and beet (Sabir et a1. 1992). Qualitative changes such as loss of

electromorph activity or altered mobilities can be seen directly and are probably
indicative of underlying mutation, and can also be tracked by transmission genetics. For
a measurement based on isozymes, enzyme systems should be chosen that are expressed
very stably during plant development and that are not affected much by environmental
factors. For this reason, peroxidase isozymes were frequently examined in these studies
(Maheswaran and Williams, 1987).

New capabilities in molecular biology have and will continue to provide precise,
unequivocal measurement of somaclonal variation. This is a consequence of the ability to
discern variations of DNA directly using sequence-specific restriction enzymes and
cloned probes. For example, band polymorphisms were observed from sugarbeet
somaclones by restriction enzyme digestions based RFLP (Sabir et a1. 1992) and RAPD
(Munthali et a1. 1996). As the nuclear genome is far more complex than cytoplasmic
organelle DNA (Vedel and Delseny, 1987) and as the chloroplast DNA is relatively
stable in callus or regenerated plants (Shirzadegan et a1. 1989), most molecular studies
for somaclonal variation have been carried out on mitochondrial DNA. Hartmann et a1.
(1989) found that the extent of change of mitochondrial DNA in wheat depends on the
length of the tissue culture period. Furthermore, genome rearrangements in sugarbeet
mitochondrial DNA were detected in callus and suspension-cultured cells (Dikalova et a1.
1993) as well as somaclones from callus culture (Brears et a1. 1989; Dikalova et al.
1993). Rivin et al. (1983) argued that new rapid molecular cloning that utilizes improved
vectors has made it possible to obtain comprehensive stable nuclear genomic libraries. If

taken from standard or reference genotypes, specific constituents of such libraries will be

useful as direct genetic or chromosome structural probes for detection of variation.

In vitro micropropagation is being used increasingly as a production process to
rapidly propagate elite genotypes and genetically engineered plants. But, somaclonal
variation could be a serious disadvantage to such operations if adventitious shoot
production is involved, because clonal uniformity may not be sustained. Furthermore,
somaclonal variation is a random process which produces both desirable and deleterious
variants (Evans and Sharp, 1986). It can be very important therefore that reliable
procedures are developed to control this unwanted source of variation.

Since its intial recognition, somaclonal variation has been also regarded as a new
source of genetic variability for plant improvement in Spite of its limitations (Larkin and
Scowcroft, 1981; Bingham and McCoy, 1986; Ryan et a1. 1987). One of the major
benefits of somaclonal variation is the creation of additional genetic variability in
coadapted, agronomically useful cultivars, without the need to resort to hybridization.
This takes on added appeal if efficient in vitro selection is possible or if rapid plant
screening procedures are available. Characteristics for which the recovery frequency of
somaclonal mutants can be enriched in vitro include resistance to herbicides (Saunders et
al. 1992; Donn et a1. 1984), and tolerance of unfavorable environmental or chemical
conditions (Levall and Bomman, 1993). Somaclonal variation for other characters for
which there is no adequately defined in vitro response, such as seed protein quality (Zeng
et a1. 1989), photosynthetic efficiency or yield, can be of benefit, provided efficient

whole plant screening protocols are available (Evans and Sharp, 1986).

INTRODUCTION -

There has been continual success in producing superior crop varieties by
conventional plant improvement. However, it is likely that there are still a lot of valuable
gene combinations or additions that can not be achieved by conventional methods. At the
present time, considerable advances have been made in the genetic engineering of crop
plants through the coupling of recombinant DNA techniques with shoot or embryo
regeneration from plant tissue cultures. Amongst the current problems facing the advance
of this technology is the occurrence of somaclonal variation, that is, genetic infidelity
arising due to cell proliferation in callus or suspension culture. The term was originally
coined by Larkin and Scowcroft (1981) to describe “the variations displayed among the
plants derived from any form of cell and tissue culture”. Its origins and causes (Gould,
1986; Sree Ramulu, 1987) have been discussed speculatively, but so far basic knowledge
is fragmentary and only a few general principles have emerged.

Somaclonal variation is of particular interest because it may provide hints as to
the basic mechanisms underlying genotypic and phenotypic stability in normal plant
development. It is also of considerable practical importance in the application of plant
tissue culture in systems for plant improvement. When tissue culture is used for plant
propagation or as a system for genetic manipulation, unwanted variation must be

minimized. On the other hand, spontaneous and induced genetic changes arising in

10

11

culture may provide novel forms of variation (Meins, 1983), particularly if efficiently
screened.

From this viewpoint, if somaclones were obtained from adventitious shoots, for
the further evaluation of somaclonal variation, it would be advisable to carry out a careful
and critical assessment of various parameters on the extent of somaclonal variation.
Assessments can be carried out with evaluations at the phenotypic level of plant
morphology and electrophoresis banding patterns of isozymes, as well as at the genotypic
level of chromosome number, structure, DNA content, and DNA restriction fragments
(De Klerk, 1990; Orton, 1983).

Sugarbeet (Beta Eggs L.), a root crop plant grown mainly in temperate
regions, is an economically important agricultural plant and supplies nearly 40% of the
world supply of sugar (Smith, 1987). The leading sugarbeet producing countries are
France, Germany, Russia, Poland, Ukraine, and the United States (in alphabetical order).

A member of the Chenopodiaceae, sugarbeet is an allogamous species with a
biennial life cycle. In the first season, the plant grows a foliar rosette and sucrose is stored
in a large fleshy taproot. Following an overwintering experience and exposure to long-
day conditions, a large bushy flower stalk emerges on which as many as 5000 seeds ripen
under favorable conditions. The fruit is a nutlet, and, depending on the number of
potential seeds per fruit, there are two kinds of fruits - monogerm and multigerm.
Sugarbeet sometimes produces a precocious flower stalk during the first season in its life
cycle. This phenomenon is called bolting and is highly undesirable in sugarbeet

production.

12

In sugarbeet, with reliable Shoot regeneration methods available fairly recently,
comprehensive reports on the scope of somaclonal variation are lacking. Nevertheless, in
several studies, variation derived from in vitro culture has been detected in sugarbeet
plants regenerated from callus, cell suspensions, and protoplasts. This variation includes
apparent halving of chromosome number (Steen et a1. 1986), changes in isozyme
patterns (Steen et a1. 1986), including an overproducer of glutamate dehydrogenase
(Sabir et a1. 1992), in band polymorphism based on RF LP (Sabir et a1. 1992) and RAPD
(Munthali et al. 1996), genome rearrangements in mitochondrial DNA (Brears et al.
1989; Dikalova et a1. 1993), altered in vitro response (Saunders and Doley, 1986; Detrez
et a1. 1989), recurrence of a novel inttunescence character in regenerants from different
donor plants (Yu et a1. 1991), three monogenic dominant herbicide resistances (Saunders
et a1. 1992; Wright and Penner, unpublished), UV-tolerant somaclonal variants (Levall
and Bornman, 1993), as well as a leaf necrosis trait and a heritable pigment variant of
possibly unstable nature (Saunders et al. 1990). Some of these variants have not yet been
well characterized. Furthermore, there appear to have been no reports of somaclonal
variation in sugarbeet for quantitative agronomic traits (Saunders et a1. 1990). So, more
extensive and detailed studies of somaclonal variation in sugarbeet remain to be
conducted.

At least nineteen sugarbeet somaclones were available that had been regenerated
from leaf disc callus of diploid cytoplasmic male sterile genotype 8266-10 on a medium
which contained 30 mM glutamine as the only nitrogen source. Glutamine was used as

selective regime in a selection experiment to seek sugar beet somaclones with the better

13

nitrogen assimilation, perhaps by recovering a glutamate dehydrogenase overproducer
similar to that found by Sabir et al. (1992). It was proposed that these somaclones
should be evaluated for possible phenotypic identification of somaclonal variants, some
of which would be available for incorporation into sugarbeet breeding programs in the
future.

This research was aimed at detecting any possible somaclonal variation in these
nineteen somaclones for several parameters such as tissue culture response, DNA content
examination, and isozyme banding patterns. Additionally, a somaclonal variant with a
distinct morphological aberration - a narrow leaf (Fig 1.1) and abnormal lateral stalk
organization (Fig. 1.2 and Fig 1.3) - was obtained separately from the source clone 8266-
10 and named as SC-NL. The narrow leaf variant was examined at the biochemical and
cytogenetical level, namely, for isozyme banding patterns and DNA content. Molecular
approaches were not performed. Information on transmission genetics might be available

from seed harvested on the somaclones at a later date, but was not reported at this time.

 

Figure 1. A morphologically aberrant somaclonal variantion. One variation is narrow
leaf size in Fig 1.1 (right: source genotype 8266-10, lefi: SC-NL). Another variation is
abnormal lateral stalk organization in Fig 1.2 (right: source genotype 8266-10, lefi: SC-
NL) and Fig 1.3 (right: source genotype 8266-10, lefi: SC-NL). This variant was named

the narrow leaf somaclonal variant (SC-NL).

 

(Fig 1.2) The young abnormal lateral stalk from SC-NL (right: source genotype 8266-10,

lefi: SC-NL).

 

(Fig 1.3) The fully grown abnormal lateral stalk from SC-NL (right: source genotype

8266-10, left: SC-NL).

MATERIALS AND METHODS

EXPERIMENT 1. TISSUE CULTURE RESPONSE OF LEAF DISCS
Plant maintenance

Sugarbeet somaclones (regenerants) and source plants were used as donors of leaf
explants. Source plant genotype 8266-10 was a cytoplasmically male sterile BC3 (third
backcross generation) individual from the recurrent parent clone 436-3 (with
acknowledged shoot regeneration ability, Saunders and Daub, 1984), cytoplasmic male
sterility and biennial nature. The nineteen somaclones had been produced independently
by callus induction and shoot regeneration from control plant 8266-10. The somaclones
and control plants were grown in pots with Baccto high porosity professional planting
mix (55-65 % of horticultural sphagnum peat perlite, Michigan Peat Co.) in the
greenhouse without supplementary lighting and fertilized weekly with Peters 20-20-20
water soluble commercial nutrient mix (Peters Professional Co, Marysville OH).
Leaf sample preparation

Leaf discs were taken from one smaller and one larger leaf at 20-40 % of fully
expanded leaf size from source plants as well as each somaclone. Detached leaves were
surface sterilized with two twenty minute soakings in 15% commercial hypochlorite
bleach solution with 0.01% sodium laurylsulfate, followed by three rinses with sterile
distilled water. Leaf discs were excised from each leaf blade, excluding leaf veins, using

a #3 cork borer (0.7 mm in diameter).

17

18

Media preparation

The medium designated Bl was prepared from MS medium (Murashige and
Skoog, 1962) which consists of inorganic and organic mineral salts, 3% sucrose,
100 mg/L myo-inositol, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCI, 1.0 mg/L
thiamineHCl (Table l). 0.9% (w/v) Difco Bacto agar was included. 1.0 mg/L 6-
benzyladenine (6-BA) was added prior to autoclaving as the only growth regulator used
for B, medium. The final pH was adjusted to 5.95 with KOH prior to autoclaving for 30
minutes at 121 °C (15 lb/inz). Basal medium MO was identical but lacked 6-BA.
Medium was dispensed into 20 x 100 mm disposable plastic Petri dishes after
autoclaving, with 35 ml per dish.
Culture establishment

One leaf disc was used per dish. Dishes were sealed with two strips of parafilm
(American National Can Co, Neenah WI). Thirty six leaf discs were used for source
plant and each somaclone, respectively. The experimental system was a completely
randomized design. All cultures were maintained at 31 °C in the dark. Six weeks after
initiating the leaf disc cultures, observations of the responses began and were terminated
after 10 weeks with B] media and 14 weeks with M0 media (because callus initiation
took several weeks longer on MO media as reported by Doley and Saunders 1989). The
proportion of leaf discs for each entry producing callus, or shoots from that callus.
Statistical comparisons of each somaclone with source genotype 8266-10 were made by

the chi-square test with the Yates correction factor for one degree of freedom.

19

Table 1. Inorganic and organic constituents of MS“ media (Murashige and Skoog,

 

 

 

1962)

Constituents mg/L
INORGANIC
KNO3 1,900
NH,,NO3 1 ,650
CaC12-2HZO 440
MgSO4-7HZO 370
KH2P04 170
NaQEDTA 37.3
FeSO4-7H20 27.8
MnSO4-4H20 22.3
ZnSO4~7H20 8.60
H3BO3 6.20
KI 0.83
NazMoO4-2HZO 0.25
CoClz-6H20 0.025
CuSO4-5HZO 0.025
ORGANIC
Sucrose 30,000
Myo-inositol 1 00
Thiamine-HCI 1 .0
Nicotinic acid 0.5
Pyridoxine-HCI 0.5

 

* For B, media, 1.0 mg/L of BA was added to MS media.

For MO media, no BA was added to MS media.

20

EXPERIMENT 2. ISOZYME BANDING PATTERNS WITH STARCH GEL
ELECTROPHORESIS

Sample preparation

Young healthy leaves, approximately 2-4 cm long, were collected in the
greenhouse from control 8266-10, each somaclone from SC 1 to SC 19, and SC-NL
immediately before they were used. The genotype 80-111 (Aicher and Saunders, 1990)
was used as a reference of banding mobility for 8266-10. Three 1 cm diameter leaf discs
(= 0.03 g) were excised from each leaf blade, excluding leaf vein, using a #3 cork borer
(0.7 mm in diameter). The discs were homogenized using a glass pestle in porcelain spot
plates containing 200 uL of extraction buffer. Fresh calli (= 0.6 g) of each somaclone
from SC 1 to SC 19 was used for Adh. Callus was not available for SC-NL. The calli
were also homogenized in 250 uL of extraction buffer. The extraction buffer consisted of
0.1 M Tris-HCI, pH 7.0 buffer, containing 18% glycerol, 5% (w/v) soluble
polyvinylpyrrolidone (PVP-40T), 0.5% Triton X-100 and 1.0% 2-mercaptoethanol which
was added just before use. The spot plates were placed on blocks of ice in trays to
maintain temperatures near 0 °C throughout sample preparation.
Systems and gel electrophoresis

The apparatus was a slight modification of that described by O’Malley, Wheeler
and Guries (1980). Three buffer systems were used to resolve all of the enzyme systems
examined. System I consisted of a electrode buffer with 0.065 M L-histidine, 0.02 M
citric acid monohydrate, pH 5.7 and a gel buffer that was a 1:6 dilution of the electrode

buffer (Cardy, Stuber and Goodman, 1980). System Il consisted of a 0.125 M Tris-citric

21

acid, pH 7.0 electrode buffer modified from Cheliak and Pitel (1984). The gel buffer was
a 0.02 M DL- histidine-HCI, 0.56 mM Na2EDTA buffer adjusted to pH 7.0 with Tris
(Trizma base). System 111 consisted of the same electrode buffer as System 11. The gel
buffer was a 0.05 M DL-histidine-HCI, 1.4mM NaZEDTA buffer adjusted to pH 7.0

with Tris (Trizma base). Gels were prepared with 600 ml gel buffer, 75 g potato starch
(Sigma Chemical Company) and for system I gels only, 21 g sucrose. The heated and
aspirated gel solution was poured into a plexiglass frame with the dimensions: 21.7 cm
long x 14.0 cm wide x 1.5 cm deep. The gels were allowed to cool for 2 hours, and were
then covered with 2 layers of plastic wrap and kept in the room temperature overnight.
Before loading the samples, the gel was kept in a refrigerator at 4 °C for 30 minutes.
Twenty one paper wicks (Whatman Electrophoresis Paper No. 3MM), 4 x 15 mm in size,
were soaked in leaf extract, blotted on paper toweling, and inserted into a slice in the
starch gel 6 cm from the cathodal side of the gel, for system I, II, and 111, respectively.
After loading the samples, the gel was placed in a refrigerator at 4 °C, and the gels were
run at 20 watts constant power. After 30 minutes the wicks were removed and ice bags
were placed on top of the gels, and then electrophoresis was resumed for a total of 5
hours for system I, 6 hours for system 11, and 6 hours for system 111. The enzyme which
was examined using system I was 6-phosphogluconic acid dehydrogenase (6-Pgdh). The
enzymes which were examined using system II were malate dehydrogenase (Mdh) and
shikimate dehydrogenase (Skdh). The enzymes which were examined using system 111

were glutamate dehydrogenase (Gdh) and alcohol dehydrogenase (Adh).

22

Gel staining

After electrophoresis, gels were sliced into 1/16" thick slices using plastic spacer
and a fine steel guitar string. Although 5 or 6 thin slices were obtained from a single gel,
the top and bottom slices were discarded because they gave poor band resolution. The
third and fourth slices were harvested and immersed in stain solutions for 1 hour at 37 °C.
Adh was stained for 2 hours in room temperature. The 6-Pgdh, Skdh, and Mdh enzyme
stains were adapted from Cheliak and Pitel (1984). The 6-Pgdh was stained with 50 mg
MgClz, 20 mg 6-phosphogluconic acid, 10 mg NADP, 10 mg nitro blue tetrazolium
(NBT) and 1 mg phenazine methasulphate (PMS) in 50 ml 0.1 M Tris-HCl pH 8.0
buffer. Skdh was stained with 40 mg shikimic acid, 10 mg NADP (IS-nicotinamide
adenine dinucleotide phosphate), 10 mg NBT and 1 mg PMS in 50 ml 0.1 M Tris-HCl pH
8.0 buffer. Mdh was stained using 25 ml 0.5 M DL-malate pH 7.0 buffer and 12.5 mg [3 -
nicotinamide adenine dinucleotide (NAD), 10 mg NBT, and 1 mg PMS in 25 ml 0.1 M
Tris-HCI pH 8.0 buffer. Adh was stained with 10 mg NAD, 10 mg NBT and 2 mg PMS
in 49 ml 0.1 M Tris-HCl pH 8.5 buffer. 1 ml of 95% ethyl alcohol was added just before
staining. Gdh was stained with 4 g L-glutamic acid, 20 mg NAD, 20 mg NBT and 1 mg
PMS in 50 ml 0.1 M Tris-HCl pH 8.0 buffer. Gels were kept in the dark during staining
to prevent excess background staining and to protect light sensitive reagents. After
staining, gels were rinsed with distilled water two or three times and fixed by 6 (distilled
water) : 4 (95% ethyl alcohol) solution. Zymograms of source genotype 8266-10 and
nineteen somaclones were scored and examined for banding patterns. All methods for

system I, II and III are summarized in Table 2.

Table 2. Summary of electrophoretic materials and methods

 

 

 

 

 

 

 

 

System I System 11 System 111
pH 5.7 7.0 7.0
0.065 M L-histidine 0.125 M Tris 0.125 M Tris
Electrode buffer 0.020 M citric acid adjusted to pH 7.0 adjusted to pH 7.0
(EB) adjust pH with citrate with Tris acid with Tris acid
anhydrous anhydrous
0.02 M DL- 0.05 M DL-
Gel Buffer 1:6 dilution of EB histidineoHCl histidine-HCI
(GB) 0.56 mM EDTA to 1.4 mM EDTA to pH
pH 7.0 with Tris 7.0 with Tris
75.0 g potato starch 75.0 g potato starch 75.0 g potato starch
Gel and 21.0 g sucrose in in 600 mL GB and 21.0 g sucrose in
600 mL GB 600 mL GB
Supplied 20 watts 20 watts 18 watts
Power (50 mAmps, 400 V) (75 mAmps, 270 V) (70 mAmps, 250 V)
Duration 5.5 hours 6 hours 6.5 hours
Resolved 6-Pgdh Skdh Gdh
Enzyme Mdh Adh*

 

* The source of Adh in this study is only from callus.

24

EXPERIMENT 3. NUCLEAR DNA CONTENTS AS DETERMINED BY FLOW
CYTOMETRY

Solutions required

The following stock solutions were prepared before sample preparation. MgSO4
buffer (pH = 8.0; 10 mM MgSO,, 50 mM KCl and 5 mM Hepes) (van den Engh et al.
1984), 10 % (w/v) Triton X—100, propidiurn iodide (PI) stock solution (5 mg/mL, covered
with aluminum foil and stored at 2-4 °C), and Alsever’s solution (pH = 6.1; 0.55 mg/mL
citric acid, 20.5 mg/mL glucose, 4.2 mg/mL sodium chloride, 8 mg/mL sodium citrate).
Chicken red blood cells (CRBC) was used as an internal standard. 1:100 dilution of the
delivered fresh CRBC (Colorado Serum Co, catalog no. es 1151) in Alsever’s solution
gives approximately 107 CRBC/mL. This concentration was recommended for a good
DNA peak for chicken red blood cells as the internal standard (Arumuganathan and Earle,
1991). The diluted solution was stored at 2-4 °C. RNAase (DNAase free) was
purchased from Boehringer Mannheim Biochemicals (catalog no. 1119915) and stored
below 0 °C.

The following solutions were freshly prepared using the stock solutions,
protected from light with aluminum foil, and kept on ice. Solution A (15 mL enough for
up to 12 samples) consisted of 14.3 mL MgSO, buffer (ice-cold), 15 mg DL-dithiothreitol,
300 uL propidium iodide (PI) stock, 375 uL Triton X-100 stock. Solution B (3 mL
enough for up to 12 samples) was prepared by 3 mL solution A, 7.5 uL RNAase

(DNAase free), 3.0 uL chicken red blood cell suspension (CRBC).

25

Preparation of nuclei by chopping plant tissues

Young leaves, approximately 2-4 cm in length (Akinerdem, 1991) from healthy
source genotype 8266-10, nineteen somaclones, SC-NL in the green house were used.
The excised leaves were washed by distilled water to remove debris and chemical
residues. The weighed tissues (approximately 100 mg) were placed in plastic Petri dishes
(35 mm x 10 mm) on ice. 2 mL of the prepared solution A was added and the tissues
were finely chopped using a sharp scalpel. After chopping, the tissue homogenate was
filtered through a nylon filter (41 um pore size) into a microcentrifuge tube, and the
resultant solution was centrifuged at high speed (x 14,000 rpm) for 1 minute followed by
discarding of the supernatant. The pellet was resuspended in 400 uL solution B and
incubated for at least 15 minutes at 37 °C. After this incubation, the sample was ready
for the flow cytometer. Every procedure was performed on ice and in the dark.
Estimation of nuclear DNA content

Nuclear DNA content was analyzed on an Ortho Diagnostics Cyto-fluorograph

SO-H / Intel 80386 computer system using Acqcyte data acquisition software (Phoenix
Flow Systems, San Diego, Calif, USA) with an argon-ion laser to 488 nm. Each
measurement was based on propidium iodide fluorescence from 400 - 4000 plant nuclei.
The amount of DNA was estimated using MultiCycle version 2.5 (Phoenix Flow
Systems) as the fluorescence of the nuclei stained with propidium iodide relative to that
of chicken red blood cells (CRBC). The results were obtained in the form of a DNA
histogram (Figure 6). The DNA amount of CRBC (2C = 2.33 pg, Galbraith et al. 1983)

was used to convert the DNA content estimates for the source plant, the nineteen

26

somaclones, and SC-NL into absolute amounts (pg), using the following equation
(Arumuganathan and Earle, 1991a):
Plant nuclear DNA amount (pg / 2C) =

I mean position of plant nuclear peak x 2.33|
[mean position of CRBC nuclear peak]

Data of DNA contents were taken based on three observations and analysis of variance
(ANOVA) was made from the source genotype 8266-10, the nineteen somaclones, and

SC-NL.

RESULTS
EXPERIMENT 1. TISSUE CULTURE RESPONSE OF LEAF DISCS
The goal of this experiment was to determine if the in vitro responses of any of
the nineteen somaclones differed significantly from those Of the source genotype 8266-
10.

The parameters examined for in vitro response were leaf disc callusing proportion
and subsequent shoot regeneration proportion of the source genotype 8266-10 and the
nineteen somaclones on both B, and MO media. Two kinds of media (B, and M0) were
used because leaf discs respond to each somewhat differently for callus induction: B,
initiates callus and shoots after one month with some genotypes, while MO initiates
callus more slowly and produces somatic embryos with some genotypes (Doley and
Saunders, 1989). The leaf discs were taken from smaller and larger leaves from each
somaclone because optimal leaf size for somaclone leaf disc callus induction had not
been established, even though standard practice otherwise is to use leaves of 20-40 % of
final size.

Overall, no somaclone differed significantly from the source clone as to in vitro
leaf disc response for any of the four parameters. For callus induction, on E, medium,
72-89 % of leaf discs of somaclones from both smaller and larger leaves produced calli
compared to 92 % of callus induction in leaf discs of the source genotype 8266-10 (Table

3). The trends were the similar on M0 medium, although the range of callus induction

27

28

(61 -83 %) from both smaller and larger leaf of somaclones was lower on M0 medium
than on B, medium. All somaclones showed statistically insignificant callusing response
on M0 medium in comparison with the source genotype 8266-10 (83 %) (Table 4). For
shoot formation, 61-83 % of leaf discs from both smaller and larger leaves of somaclones
produced shoots on B, medium with no significant deviation of shooting proportions
from the source genotype 8266-10 (80 %) as in Table 5. On M0 medium, no shoot or
somatic embryos (0 %) were regenerated from either the source genotype 8266-10 or any
of the nineteen somaclones (Table 6).

EXPERIMENT 2. ISOZYME BANDING PATTERNS WITH STARCH GEL
ELECTROPHORESIS

The isozyme evaluation was performed to detect any variation in banding pattern
for the somaclones in comparison with the source genotype 8266-10 for five enzymes
6-phosphogluconic acid dehydrogenase (6-Pgdh), malate dehydrogenase (Mdh),
shikimate dehydrogenase (Skdh), glutamate dehydrogenase (Gdh), and alcohol
dehydrogenase (Adh). At best, despite considerable effort and troubleshooting, the
banding patterns were not clear and were often overstained. SO, it was hard to clearly
translate the banding patterns.

The banding patterns of the nineteen somaclones seemed to display no somaclonal
variation for the enzymes 6-Pgdh (Fig 2), Skdh (Fig 3.2), Gdh (Fig 4.1), and Adh (Fig
4.2). With Mdh (Fig 3.1 and Fig 5), all somaclones exhibited the absence of a fast band
in the Mdh-3 region sometimes seen in the source genotype. This is the position of
coordinate bands reported by Aicher and Saunders (1990) that have inconsistent

occurrence and appear to be physiologically influenced. No other differences were seen

29

for Mdh (Fig 3.1). In zymograms of Mdh and Gdh, bands of the source genotype 8266-
10 were first compared with those of standard genotype 80-111 as the reference to
identify 8266-10’s band mobilities. The banding patterns were identical between these
two genotypes. Thus the zymotype of 8266-10 was Mdh—l, FF; Mdh-3, SS; Gdh, SS as
in Aicher and Saunders (1990). The bands of the nineteen somaclones and SC-NL (Fig
5) were similar to 8266-10’s bands for Mdh and Gdh. For 6-Pgdh, Skdh, and Adh (this
enzyme was not examined in SC-NL because callus was not available in SC-NL), even
though 80-111’s bands were not available from Aicher and Saunders, (1990), no banding
patterns from the nineteen somaclones and SC-NL were seen as different from 8266-10
and 80-111.

EXPERIMENT 3. NUCLEAR DNA CONTENTS AS DETERMINED BY FLOW
CYTOMETRY

The purpose of the experiment was to examine DNA content variation of source
genotype 8266-10, nineteen somaclones, and SC-NL and all DNA histograms obtained
were similar to that described in Figure 6. The DNA content of source diploid 8266-10
was 1.73 pg / 2C. The mean DNA contents (pg / 2C) of the nineteen somaclones ranged
from 1.49 to 1.78 and SC-NL was 1.59 (Table 7). Analysis of variance showed no
significant variation of DNA content among source genotype 8266-10, the nineteen

somaclones, and SC-NL (Table 8).

30

Table 3. Callus formation from leaf discs of smaller and larger leaves from source

genotype 8266-10 and derivative somaclones on B, medium 10 weeks after culture

initiation.

 

Callusing proportion (%)

x2 value‘

 

Leaf disc origin

Smaller leaf Larger leaf Smaller leaf Larger leaf

 

 

8266-10 16/18 (88.9) 17/18 (94.4)
sc 1 14/18(77.8) 16/18(88.9) 1.516 2.632
sc 2 15/18(83.3) 16/18(88.9) 1.391 2.632
sc 3 15/18(83.3) 15/18(83.3) 1.391 2.618
sc 4 15/18(83.3) 16/18(88.9) 1.391 2.632
sc 5 15/18(83.3) 15/18(83.3) 1.391 2.618
sc 6 14/18(77.8) 16/18(88.9) 1.516 2.632
sc 7 13/18(72.2) 15/18(83.3) 3.891 2.618
sc 8 15/18(83.3) 15/18(83.3) 1.391 2.618
sc 9 15/18(83.3) 16/18(88.9) 1.391 2.632
sc 10 14/18 (77.8) 15/18 (83.3) 1.516 2.618
sc 11 15/18 (83.3) 16/18 (88.9) 1.391 2.632
sc 12 15/18 (83.3) 15/18 (83.3) 1.391 2.618
sc 13 14/18 (77.8) 16/18 (88.9) 1.516 2.632
sc 14 14/18 (77.8) 16/18 (88.9) 1.516 2.632
sc 15 13/18 (72.2) 16/18 (88.9) 3.891 2.632
sc 16 14/18 (77.8) 15/18 (83.3) 1.516 2.618
sc 17 14/18 (77.8) 15/18 (83.3) 1.516 2.618
sc 18 15/18 (83.3) 15/18 (83.3) 1.391 2.618
sc 19 13/18 (72.2) 15/18 (83.3) 3.891 2.618

 

‘ Comparisons of each somaclone were made with the source genotype 8266-10 using the

chi-square test; values of )6 were calculated with the Yates correction factor with one

degree of freedom; somaclones showed no significant deviation from 8266-10 at 99 %

confidence level.

31

Table 4. Callus formation from discs of smaller and larger leaves from source genotype

8266-10 and derivative somaclones on M0 medium 14 weeks after culture initiation.

 

Callusing proportion (%) )6 value‘

 

 

 

Leaf disc origin Smaller leaf Larger leaf Smaller leaf Larger leaf
8266-10 14/18 (77.8) 16/ 18 (88.9) --- ---
SC 1 13/18 (72.2) 14/18 (77.8) 1.071 1.516
SC 2 13/18 (72.2) 15/18 (83.3) 1.071 1.391
SC 3 12/18 (66.7) 15/18 (83.3) 1.009 1.391
SC 4 12/18 (66.7) 14/18 (77.8) 1.009 1.516
SC 5 13/18 (72.2) 15/18 (83.3) 1.071 1.391
SC 6 12/18 (66.7) 15/18 (83.3) 1.009 1.391
SC 7 13/18 (72.2) 14/18 (77.8) 1.071 1.516
SC 8 11/18 (61.1) 13/18 (72.2) 2.438 3.891
SC 9 11/18 (61.1) 14/18 (77.8) 2.438 1.516
SC 10 11/18 (61.1) 13/18 (72.2) 2.438 3.891
SC 11 13/18 (72.2) 14/18 (77.8) 1.071 1.516
SC 12 11/18(61.1) 15/18(83.3) 2.438 1.391
SC 13 13/18 (72.2) 13/18 (72.2) 1.071 3.891
SC 14 12/18 (66.7) 13/18 (72.2) 1.009 3.891
SC 15 13/18 (72.2) 15/18 (83.3) 1.071 1.391
SC 16 11/18(61.1) 15/18(83.3) 2.438 1.391
SC 17 11/18 (61.1) 14/18 (77.8) 2.438 1.516
SC 18 13/18 (72.2) 15/18 (83.3) 1.071 1.391
SC 19 13/18 (72.2) 15/18 (83.3) 1.071 1.391

 

‘ Comparisons of each somaclone were made with the source genotype 8266-10 using the
chi-square test; values of )6 were calculated with the Yates correction factor with one

degree of freedom; somaclones showed no significantly deviant proportions of callusing

from 8266-10 at 99 % confidence level.

32

Table 5. Shoot formation from discs of smaller and larger leaves from source genotype

8266-10 and derivative somaclones B, medium 10 weeks after culture initiation.

 

Shoot proportion (%)

)8 value‘

 

 

 

Leaf disc origin Smaller leaf Larger leaf Smaller leaf Larger leaf
8266-10 16/ 18 (88.9) 15/ 18 (72.2) --- ---
SC 1 15/18 (83.3) 14/18 (77.8) 1.391 0.983
SC 2 14/18 (77.8) 14/18 (77.8) 1.516 0.983
SC 3 14/18(77.8) 11/18(61.1) 1.516 5.433
SC 4 15/18 (83.3) 13/18 (72.2) 1.391 1.167
SC 5 13/18 (72.2) 11/18 (61.1) 3.891 5.433
SC 6 14/18 (77.8) 11/18 (61.1) 1.516 5.433
SC 7 15/18 (83.3) 13/18 (72.2) 1.391 1.167
SC 8 15/18 (83.3) 12/18 (66.7) 1.391 2.900
SC 9 13/18 (72.2) 14/18 (77.8) 3.891 0.983
SC 10 14/18 (77.8) 13/18 (72.2) 1.516 1.167
SC 11 13/18 (72.2) 12/18 (66.7) 3.891 2.900
SC 12 13/ 18 (72.2) 14/ 18 (77.8) 3.891 0.983
SC 13 13/18 (72.2) 11/18 (61.1) 3.891 5.433
SC 14 15/18 (83.3) 14/18 (77.8) 1.391 0.983
SC 15 15/18 (83.3) 13/18 (72.2) 1.391 1.167
SC 16 14/18 (77.8) 14/18 (77.8) 1.516 0.983
SC 17 13/18 (72.2) 12/18 (66.7) 3.891 2.900
SC 18 14/18(77.8) 13/18(72.2) 1.516 1.167
SC 19 14/18(77.8) 13/18(72.2) 1.516 1.167

 

‘ Comparisons of each somaclone with the source genotype 8266-10 using the chi-square

test; values of )8 were calculated with the Yates correction factor with one degree of

freedom; somaclones showed no significant deviation from 8266-10 at 99 % confidence

level

33

Table 6. Shoot fromation from discs of smaller and larger leaves from source genotype

8266-10 and derivative somaclones on M0 medium 14 weeks after culture initiation.

 

 

 

 

Shoot proportion (%)‘
Leaf disc origin Smaller leaf Larger leaf
8266-10 0/ 18 (0.0) 0/ 18 (0.0)
SC 1 0/18 (0.0) 0/ 18 (0.0)
SC 2 0/18 (0.0) 0/18 (0.0)
SC 3 0/ 18 (0.0) 0/18 (0.0)
SC 4 0/ 18 (0.0) 0/ 18 (0.0)
SC 5 0/ 18 (0.0) 0/ 18 (0.0)
SC 6 0/ 18 (0.0) 0/18 (0.0)
SC 7 0/18 (0.0) 0/18 (0.0)
SC 8 0/18 (0.0) 0/ 18 (0.0)
SC 9 0/18 (0.0) 0/ 18 (0.0)
SC 10 0/18 (0.0) 0/ 18 (0.0)
SC 11 0/ 18 (0.0) 0/18 (0.0)
SC 12 0/ 18 (0.0) 0/18 (0.0)
SC 13 0/18 (0.0) 0/18 (0.0)
SC 14 0/ 18 (0.0) 0/18 (0.0)
SC 15 0/18 (0.0) 0/18 (0.0)
SC 16 0/18 (0.0) 0/ 18 (0.0)
SC 17 0/18 (0.0) 0/18 (0.0)
SC 18 0/18 (0.0) 0/18 (0.0)
SC 19 0/18 (0.0) 0/18 (0.0)

 

‘ No shoots or somatic embryos were not formed from leaf discs of smaller and larger

leaves on M0 medium.

 

Figure 2. The banding patterns of 6-Pgdh from control (source) plant 8266-10 and
nineteen derivative somaclones. Lane A through S were not labeled but they correspond

from SC 1 to SC 19, T: 8266-10, U: 80-111.

35

 

Fig3.1 F1932

Figure 3. The banding patterns of Mdh (Fig 3.1) and Skdh (Fig 3.2) from control
(source) plant 8266-10 and nineteen derivative somaclones. Lane A through S were not

labeled but they correspond from SC 1 to SC 19, T: 8266-10, U: 80-111.

36

U)
>
(”-1
>

 

 

Fig 4.1 Fig4.2

Figure 4. The banding patterns of Gdh (Fig 4.1) and Adh (Fig 4.2) from control (source)
plant 8266-10 and nineteen derivative somaclones. Lane A through S were not labeled

but they correspond from SC 1 to SC 19, T: 8266-10, U: 80-111.

37

 

 

 

 

 

 

 

 

Skdh Gdh

Figure 5. The banding patterns of 6-Pgdh, Mdh, Skdh, and Gdh of control (source) plant
8266-10 and narrow leaf somaclonal variant (SC-NL). A: SC -NL, B: 8266-10, C: 80-

111.

38

 

Jr

150a * t t t

1300.
H
(I)
"i
:3 son.
.4
F1
0
C) 400.

 

 

 

Figure 6. Flow cytometric analysis of DNA content in the leaf cell nuclei from source
clone 8266-10, the nineteen somaclones, and SC-NL . Histogram shows the number of
nuclei as a function of relative DNA content. Nuclei were isolated from freshly excised
young leaves. The two peaks A and A* represent G,and G2 phase nuclei with 2C and 4C
complements Of sugarbeet plant DNA, respectively. B and B* are for G, and G2 of

chicken red blood cells (C.R.B.C) as the internal standard.

39

Table 7. DNA contents of leaf cells from control (source) plant 8266-10 and nineteen

derivative somaclones by flow cytometry.

 

Genotype

Replication

DNA content (pg)

Mean

t-value

 

8266-10

1.63
1.89
1.68

1.73

 

SCI

1.62
1.71
1.62

1.65 (nsb)

0.980

 

SC2

1.65
1.73
1.68

1.69 (ns)

0.209

 

SC3

1.59
1.62
1.71

1.64 (ns)

1.006

 

SC4

1.52
1.62
1.66

1.60 (ns)

1.453

 

SC5

1.56
1.31
1.61

1.49 (ns)

2.028

 

SC6

1.52
1.61
2.05

1.72 (ns)

0.054

 

SC7

1.59
1.62
1.69

1.63 (ns)

1.414

 

SC8

1.66
2.15
1.62

1.81 (ns)

0.428

 

SC9

1.56
1.74
1.91

1.74 (ns)

0.077

 

SC 10

1.63
1.58
1.73

1.65 (ns)

0.843

 

SCll

LAN—tLAND-tWNI—iWNHWN—‘WNIHUJN—PWN—‘WNI—‘WN—‘WNHWNfl

1.59
1.67
1.62

1.63 (ns)

1.195

 

40

Table 7. (Cont’d)

 

Genotype Replication DNA content (pg) Mean t-value

 

1.56
2.14 1.78 (nsb) 0.250
1.63

SC 12

 

1.59
1.62 1.59 (ns) 1.673
1.57

SC 13

 

1.57
1.67 1.63 (ns) 1.195
1.66

SC 14

 

1.65
1.87 1.74 (ns) 0.067
1.68

SC15

 

1.07
1.72 1.49 (ns) 1.084
1.67

SC 16

 

1.48
1.73 1.67 (ns) 0.474
1.81

SC 17

 

1.69

1.54 1.64 (ns) 1.006
1.69

1.67

1.68 ' 1.64 (ns) 1.006
1.58

1.61

1.57 l.59(ns) 1.673
1.59

a The comparisons of means were made between each somaclone and control 8266-10 by

SC18

SC 19

SC NL

wN—wwwwN—wm~ww~ww—wm—-wm~—ww—-

t-test.
b ns = non significance at 99 % at confidence level ; from each calculated t-value, each

mean is not significantly different from the mean of control 8266-10.

41

Table 8. Analysis of variance of source genotype 8266-10, the nineteen somaclones, and

the narrow leaf variant (SC-NL).

 

 

 

Source of Degree of Sum of squares Mean square F value (Fs)‘

variation freedom

Genotype 20 0.2523 0.0189 0.738
Error 42 1.0752 0.0256

 

 

. Fs < F 0.01 (20, 42) = 2370; PS < F 0.05 (20.42) = 1-840

DISCUSSION

In all the experiments, the nineteen somaclones and narrow leaf somaclonal
variant (SC-NL) showed no significant variation from source genotype 8266-10.

Delbreil and Jullien (1994) have reported somaclonal variation for in vitro
response, i.e. improved somatic embryogenesis in Asparagus ofi‘icinalis, and indicated
that in vitro response could be a good parameter for examining somaclonal variation.
Additionally, Saunders and Doley (1986) reported a more intensely shoot regenerating
somaclone in sugarbeet, one which could have been selected by the regeneration medium.
So, in vitro response for callus induction and shoot regeneration was chosen as one set of
parameters to be examined in this study. But, originally, when the experiments started,
the available population size of somaclones including narrow leaf somaclonal variant
(SC-NL) was small and thus it was realized that the probability for detecting any
variation from somaclones was relatively low for in vitro tissue culture response.

Sabir et al. (1992) identified somaclonal variations for eight isozyme banding
patterns, using 764 sugarbeet regenerants. Steen et al. (1986) also found isozyme variants
in somaclones of sugarbeet. Thus, precedents were on record for isozyme variants,
including the most interesting over-expression of Gdh (Sabir et al. 1992). With the
isozyme parameter, it is possible that some cryptic variation in somaclones and the

narrow leaf variant could be underestimated or not detected. This may be quite possible

42

43

due to several reasons. Firstly, the genetic variations in the non-coding portions of the
DNA (intron) will not be detected by electrophoresis because only transcription and
translation products of structural genes by coding portion (exon) are accessible to
electrophoretic analysis. Secondly, some mutations in coding sequence for an enzyme
may result in a slight size alteration for tertiary structure of an enzyme or in a substitution
of amino acids that does not alter the charge or activity of the gene product (enzyme).

So, these cryptic mutations will not be detected as a change in migration mobility of an
isozyme band. Thirdly, the redundancy of genetic triple code will make some changes in
base sequence of code not expressed as amino acid sequence of the encoded gene product
(isozyme). Lastly, from the zymograms with simple, single bands in the source genotype
8266-10, nineteen somaclones, and SC-NL, it was concluded that the alleles for the
isozymes examined may be at homozygous loci. So, if the mutations in null alleles for
the enzymes examined occur, they would not be detected at homozygous loci. For these
reasons, even though no genetic variation was found among the nineteen somaclones and
SC-NL for the banding patterns of isozymes, the possibility for undetected genetic
variation should not be excluded. More reliable molecular approaches such as DNA
sequencing might be necessary to detect any mutations.

Especially, there was no relationship between the isozyme patterns examined and
the morphological variations in the narrow leaf variant (SC-NL). A similar study in
maize also showed that the extent of morphological differences between five lines was
not correlated with qualitative differences between two-dimensional enzyme banding

patterns, but a variation was found when isozyme dosages were examined (Damerval et

44

al. 1987). It may be suggested that the patterns showed may be compared not only for
the absence or presence of specific bands, but for the amount of enzyme per band (band
intensity). However, no difference in either band number, position or intensity was noted
between SC-NL and source genotype 8266-10.

For examination of DNA content in somaclones and SC-NL, the precise
measurement of DNA content should be considered for detecting the variation of DNA
content with several possible explanations. Firstly, the condition of the leaf samples,
especially, health of source plants may be considered. In fact, it was difficult to maintain
uniform plant health over fifteen months in which leaves were sampled. Physiology of
donor plants may affect the synthesis of nuclear DNA or cell cycle. Therefore,
nonuniformity of conditions of donor plants may contribute to variability for the
measurement of DNA in leaf cells of the somaclones. Secondly, variability in execution
of the procedure for nuclei preparation from plant materials may occurs. Therefore, if
possible, procedure variations were to be more carefully controlled by the experimenter,
more precise data for DNA content might be obtained and significant differences found in
a population of somaclones and SC-NL. So, considerable effort should be made to more
tightly standardize conditions of sampling in terms of physiological conditions of leaves
and conditions of preparation of nuclei. Lastly, the debris of cell organelles may interrupt
the precise measurement of DNA content in somaclones and SC-NL. Especially,
chlorophyll from the incompletely destroyed chloroplast may emit the exogenous
fluorescence with the fluorescence of the stained DNA of isolated nuclei in flow

cytometry. This is also a possible cause for somewhat higher DNA content in normal

45

diploid 8266-10 (1.73 pg / 2C). The established DNA content in diploid sugarbeet is 1.57

pg / 2C (Arumuganathan and Earle, 1991b).

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Puolimatka, M.,and A. Karp. 1993. Effect of genotype on chromosome variation in
tissue culture of inbred and outbred rye. Heredity 71 :138-144.

Rivin, C.J., E.A. Zirnmer, C.A. Cullis, V. Walbot, T. Huynh, and R.W. Davis. 1983.
Evaluation of genomic variability at the nucleic acid level. Plant Mol. Biol. Rep.
1:9-16.

Ryan, S.A., P.J. Larkin, and F .W. Ellison. 1987. Somaclonal variation in some
agronomic and quality parameters in wheat. Theor. Appl. Genet. 74:77-82.

Ryan, SA. and W.R. Scowcroft. 1987. A somaclonal variant of wheat with
additional beta-amylase isozymes. Theor. Appl. Genet. 73:459-464.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

51

Sabir, A., H.J. Newbury, G. Todd, J. Catty, and B.V. Ford-Lloyd. 1992.
Determination of genetic stability using isozymes and RFLPs in beet plants
regenerated in vitro. Theor. Appl. Genet. 84:113-117.

Saunders, J .W., G. Acquaah, K.A. Renner, and WP. Doley. 1992. Monogenic
dominant sulfonylurea resistance in sugar beet from somatic cell selection. Crop Sci.
32:1357-1360.

Saunders, J.W. and ME. Daub. 1984. Shoot regeneration from hormone
autonomous callus from shoot cultures of several sugarbeet (Beta vulgaris L.)
genotypes. Plant Sci. Lett. 34:219-223.

Saunders, J .W. and WP. Doley. 1986. One step shoot regeneration from callus of
whole plant leaf explants of sugar beet lines and a somaclonal variant for in vitro
behavior. J. Plant Physiol. 124:473-479.

Saunders, J .W., W.P. Doley, J .C. Theurer, and M.H. Yu. 1990. Somaclonal variation
in sugar beet. Pages 465-90 in Y.P.S. Bajaj, ed. Biotechnology in Agriculture and
Forestry, vol 11: Somaclonal Variation in Crop Improvement 1. Springer Verlag,
Berlin.

Saunders, J .W., C.J. Tsai, and E. Samper. 1997. Evaluation of alternative nitrogen
and carbon sources for sugar beet suspension culture platings in development of cell
selection schemes. In Vitro Cell. Dev. Biol. 33:56-61.

Shirzadegan, M., M. Christey, E.D. Earle, and J .D. Palmer. 1989. Rearrangement,
amplification, and assortment of mitochondrial DNA molecules in cultured cells of
Brassica campestris. Theor. Appl. Genet. 77:17-25.

Steen, P., B. Keimer, K. D’Halluin, and HG Pedersen. 1986. Variability in plants of
sugar beet regenerated from callus, cell suspension and protoplasts. Pages 633-34 in
W. Horn, C.J. Jensen, W. Odenbach, O. Schieder, eds. Genetic Manipulation in Plant
Breeding. De Gruyter, Berlin.

Swedlund, B. and I.K. Vasil. 1985. Cytogenetic characterization of embryogenic
callus and regenerated plants of Pennisetum americanum (L.) K. Schum. Theor.
Appl. Genet. 69:575-581.

Van den Engh, G.B., S. Trask, and M. Bartholdi. 1984. Preparation of chromosome
suspensions for flow cytometry. Cytometry 52108-117.

Vedel, F. and M. Delseny. 1987. Repetitivity and variability of higher plant
genomes. Plant Physiol. Biochem. 25: 191-210.

52

85. Wang, H. and F .8. H01]. 1988. In vitro culture and the incidence of somaclonal
variation in regenerated plants of Trifolium pratense L. Plant Sci. 55:159-167.

86. Yu, M.H., L.M. Pakish, and J .W. Saunders. 1991. Association of a nematode
resistance bearing addition chromosome with a recurring leaf intumescence
somaclonal variation in sugar beet. Genome 34:477-485.

87. Zheng, K.L., Z.M. Shou, G.L. Wang, Y.K. Luo, and Z.M. Xiong. 1989. Somatic cell
culture of rice cultivars with different grain types: somaclonal variation in some grain
and quality characters. Plant Cell Tiss. Org. Cult. 18:201-208.

CHAPTER II
IN VITRO RESPONSE OF SUGARBEET (Beta vulgaris L.) HAPLOIDS

INTRODUCTION

Haploidy refers to the condition of any organism, tissue or cell having the
chromosome constitution Of the normal gametes of the species involved (Chase, 1952).
Since the first in vitro induction of haploids by androgenesis in Datura (Guha and
Maheshwari, 1964), haploids from various plant species have been produced by in vitro
culture techniques such as anther and microspore culture, ovary and ovule culture .

Haploids have been of value in plant biotechnology for two main reasons. Firstly,
doubled haploids (DH) can be obtained by diploidization of haploid shoots or cells by
chromosome doubling. The resultant doubled haploid is a homozygous diploid, and by
selfing gives rise to a pure line, otherwise obtained only after generations of selfing.
Usually, diploidization of haploid cells is possible by the use of colchicine. For example,
haploid cells of Atropa were diploidized by adding colchicine to exponentially growing
suspension cultures (Rashid and Street, 1974).

Secondly, haploid cells of diploid species are useful in finding somatic cell
mutants (Bajaj, 1983). By plating suspension cultures from haploid explants, they can be
handled like microbes, and, with the availability of large populations, it is possible to

select haploid recessive mutants arising from somaclonal variation or chemical

53

54

mutagenesis. If diploid cells are used, an arising dominant mutation creates a
heterozygote at the locus, and is expressed. Recessive mutations also create
heterozygotes but the mutant phenotype is not expressed, and the mutation is unavailable
for the selection process in that cell population. In contrast to that, the advantage of a
haploid cell containing only one copy of each gene is that all mutations arising in that cell
are expressed, since any recessive alleles are not masked by the presence of dominant
alleles. SO, only a system using haploid cells can effectively recover useful recessive
mutants. Mutants have been obtained from haploid cell cultures from various plant
species. For example, somatic cell mutants selected for herbicide resistance (Chaleff and
Parsons, 1978) and for antibiotic resistance (Maliga et al., 1973) have been reported for
haploid tobacco, and brought to the whole plant level.

However, for optimal haploid cell use in somatic cell genetics, it is essential
that the cultures remain at least predominantly haploid during the stages when mutants
are being selected. Haploidy in cell cultures is known to be unstable and within a matter
of weeks the ploidy of many cells may revert to the diploid state, or higher levels
(Sacristan, 1971; D’Amato, 1977, 1985; Novak and Vsykot, 1975). So, spontaneous
increase in ploidy level can be a major obstacle to the use of haploid cell lines in mutant
selection. The advantage of haploid cells for mutant selection is lost when spontaneous
increase in ploidy occurs in cultures, especially when diploidization, for example, occurs
relatively early. Bajaj (1972) regenerated both haploid and diploid plants from haploid
mesophyll protoplasts of Nicotiana tabacum and put forward various possible

explanations for this phenomenon: (1) some of the leaf vein cells that gave raise to the

55

protoplasts were diploid to begin with, (2) spontaneous fusion during protoplast isolation,
and (3) repeated endomitosis (endoreduplication) or nuclear fusion in callus cultures.
Observations favored the third mechanism for the differences seen in ploidy levels.
Likewise, mixtures of haploids and diploid plants were regenerated from mesophyll
protoplasts of haploid Petunia hybrida (Binding, 1974). Also, Bajaj et al. (1978)
observed a wide range of ploidy differences in the plants regenerated from mesophyll
protoplasts of haploid Atropa belladonna. They obtained haploid, diploid, triploid, and
hexaploid plants , along with various types of aneuploids.

Since the first in vitro induction of androgenetic haploid plants by anther culture
(Guha and Maheshwari, 1964), induction of haploids by tissue and cell culture has
attracted considerable attention. It is evident that in vitro induction of haploidy can
speed up the production of haploids for plant breeding programs (Reinert and Bajaj,
1977; Vasil, 1980; Maheshwari et al., 1982; Bajaj, 1983). Especially, ovule or ovary
culture has enabled haploid production in certain species for which anther culture is not
so effective, thus extending the number of species for which haploids can easily be
available.

Given the potential advantages of haploidy, sugarbeet breeding would also be
facilitated by the availability of haploids. Sugarbeet haploids have successfirlly been
obtained as a result of pollen irradiation before pollination (Bosemark, 1971; Seman,
1983), crosses between diploid and tetraploid plants of B. vulgaris (De Jong and De
Bock, 1978), between wild species and B. vulgaris (Bosemark, 1971), or between red

beet and B. vulgaris (Seman, 1983). But, these conventional productions of haploids are

56

time-consuming, laborious, and not very efficient.

Procedures for in vitro induction of haploidy have also been developed for sugar
beet. Although anther or microspore culture has not been successful for haploid
production in sugarbeet (Banba and Tanabe, 197 2; Atanasov, 1973; Rogozinska and
Goska, 1982; Van Geyt et al., 1985), gynogenesis, i.e., in vitro culture of unfertilized
ovules or ovaries, has proven to be more promising for the production of haploids than
the other techniques used in sugarbeet (Hosemans and Bossoutrot, 1983). Since the first
successful report of ovule culture of sugarbeet, the haploid production frequency has
increased dramatically (Bomman, 1985; D’Halluin and Keimer, 1986; Van Geyt et al.,
1987; Doctrinal et al., 1989, 1990).

In the experiment described below, on the basis of callusing and shoot
regeneration, a comparison was made on the in vitro response of leaf discs between the
source diploid REL-1 (with good callusing as well as regeneration ability as in Saunders
et al., 1992) and two sugarbeet haploids OV-9, OV-l6, respectively, which had been
produced from REL-1 by in vitro gynogenesis. A second comparison was made between
the source diploid 6822-08 and three haploids OV-68-1, OV-68-2, and OV-68-3 which
were also produced from the source diploid 6822-08 by in vitro gynogenesis. But their
ploidy had not been confirmed cytologically. However, all fit the previous description of
beet haploids as conspicuously reduced in vigor and size, and OV-9 and OV-16 had been
grown in replicate long enough to display complete sterility. The three presumptive
haploids from 6822-08 had been developed directly from their ovules, minimizing the

chance of somaclonal deviation from haploidy. Literatures of sugarbeet gynogenesis

57

report a preponderance of haploidy in the products of ovule culture (Bossoutrot and
Hosemans, 1985; Van Geyt et al., 1987). SO, there is a high probability of haploidy for
these five products of ovule culture. Furthermore, from observation point of view, all of
these five products of ovule culture showed high sensitivity to environment, delayed
development, slower growth rates. They had also a smaller mass and a greater number of
small leaves. All of these observed characteristics were similar to common characteristics
of sugarbeet haploid plants as in Seman’s report (1983).

In vitro response data of these five sugarbeet haploid plants provided basic
information about the suitability of these haploid plants for the experiment of
spontaneous diploidization in callus and regenerated plants and suspension cultures. The
haploids could ultimately be the source material for developing a somatic cell selection

system in sugarbeet.

MATERIALS AND METHODS

Plant materials

Sugarbeet haploid plants OV-9 and OV-l6 had been produced by in vitro
gynogenesis from the source diploid clone REL-1 which has good callus induction and
shoot regeneration ability (Saunders et al., 1992) and was available for leaf discing in the
greenhouse. Other haploids OV-68s (OV-68-1, OV-68-2, and OV-68-3) were produced
from the diploid 6822-08 by ovule culture in this experiment. The haploid plants and
diploid plants were grown in 6" plastic pots with Baccto high porosity professional
planting mix (55-65 % of horticultural sphagnum peat perlite, Michigan Peat Co.) in the
greenhouse without supplementary lighting in 1995 and fertilized with Peters 20-20-20
water soluble commercial nutrient mix (Peters Professional Co, Marysville OH) at the
intervals of two weeks.

Ovule culture of 6822-08

Donor plant material. The induction of another haploid with a different genetic
background from diploid REL-1 derived haploids OV-9 and OV-16 was attempted. As
the ovule donor genotype, a diploid 8266-10 was used. Flower buds of 8266-10 were
collected from the incompletely expanded secondary or tertiary branches of the
inflorescences in the greenhouse without supplementary lighting as in Doctrinal et al.

(1989).

58

59

The isolation of ovules and culture establishment. After washing the excised
flower buds with distilled water,.the flower buds were sterilized twice in a 15%
commercial hypochlorite bleach solution with 0.01% sodium lauryl-sulfate for 20
minutes. The explants were rinsed three times in sterile distilled water and were dried
between sterile papers. Dissection of floral buds was done with a sharp dissecting needle
under a dissecting microscope under aseptic conditions. The isolated ovules were put on
the induction medium M20 which is MS medium (Murashige and Skoog, 1962) with 0.25
mg/L BA (6-benzyladenine) as the only growth regulator, Five ovules were put on each
plate. A total of 700 ovules were cultured. The plates with cultured ovules were kept in a
growth chamber at 24 °C i 1 °C under cOOI-white fluorescent light (about 25 umole-m‘z-
s').

The response of cultured ovules. During the next 60 days, regular observations
were made once a week to recover any prospective haploid embryos from the ovules
(Figure 1). Three embryos were recovered independently from three ovules and labeled
as OV-68-1, OV-68-2, and OV-68-3. The recovered haploid embryos were transferred to
new M20 medium for further development and shoot growth. The shoots were propagated
in M20 medium and kept under the same culture conditions as ovule culture.

The recovery of whole plants. Haploid shoots were transferred to N3 medium
for rooting. N3 medium is MS medium with 3.0 mg/L NAA (or -naphthaleneacetic acid)
as the only growth regulator and kept under the same culture conditions as ovule culture.
The plantlets with roots were then potted into soil and grown in the greenhouse without

supplementary lighting.

60

 

Figure 1. The emerging haploid embryo from the cultured ovule of diploid source clone

6822-08 after about 80 days since culture initiation.

61

Leaf sample preparation

Leaf discs were taken from incompletely expanded (20-40 % of final leaf size)
young leaves of diploid plants REL-l and 6822-08 as well as haploid plants OV-9, OV-
16, and OV-68s. Detached leaves were surface sterilized with two twenty minutes
soakings in 15% commercial hypochlorite bleach solution with 0.01% sodium
laurylsulfate, followed by three rinses with sterile distilled water. Leaf discs were
excised from each leaf blade, excluding leaf veins, using a #3 cork borer (0.7 mm in
diameter).
Media preparation

B, medium was prepared from MS medium (Murashige and Skoog, 1962) which
consists of inorganic and organic mineral salts, 3% sucrose, 100 mg/L myo-inositol, 0.5
mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCI, 1.0 mg/L thiamine-HCI. Final
concentration of agar was 0.9% (w/v) Difco Bacto agar. The final pH was adjusted to
5.95 with KOH prior to autoclaving for 30 mins at 121 °C (15 lb/inz). 1.0 mg/L BA
(6-benzyladenine) was added prior to autoclaving as the only growth regulator used for
B, medium. After autoclaving, media were dispensed into 20 x 100 mm disposable
plastic Petri dishes with 35 ml per dish.
Culture establishment

One leaf disc was put in each plate. Dishes were sealed with two straps of
parafilm (American National Can CO, Neenah W1). All cultures were maintained at 31
°C in the dark. Eight weeks after initiating the cultures, the data for callusing and shoot

regeneration were collected from the dishes of haploids OV-9, OV-16, OV-68s (CV-68-

62

1, OV-68-2, and OV-68-3) and the source diploids REL-1, 6822-08, on the basis of the
proportion of discs callusing and regenerating shoots. The data were analyzed by chi-

square test with the Yates correction factor for one degree of freedom.

RESULTS AND DISCUSSION

The goal of this experiment was to obtain information about callus induction and
shoot regeneration for five haploids (OV-9, OV-l6, OV-68-1, OV-68-2, and OV-68-3).
OV-9 and OV-16 showed extremely low proportions of leaf disc callusing and shoot
regeneration in comparison with source genotype REL-1; REL-1 had high percentage of
callus induction (95 %) and shoot regeneration (70 %). However, OV-9 produced callus
only from 7 % of leaf discs and shoots from 5 %. OV-16 showed about 6 % of callusing
proportion and 4 % of shooting proportion (Table 1).

On the other hand, in order to obtain other haploids with a different genetic
background from REL-l, two diploid genotype 6822-08 and F C 701/5-4 were identified
as the candidates for ovule donor genotypes from the populations of SP 6822 and FC
701/5 (these two populations had earlier shown good callus induction and shoot
regeneration ability as in Saunders and Shin, 1986), respectively, and were tested for
callus induction and shoot regeneration.

These two diploid genotypes 6822-08 and F C 701/5-4 showed high proportions of
leaf disc callus induction and shoot regeneration. Callus and shoots were produced from
about 90 % and 80 % of leaf discs of 6822-08, respectively (Table 2). FC 701/5-4
showed 80 % of callusing proportion and 70 % of shoot regeneration (Table 2).

However, in this experiment, 6822-08 was chosen as the ovule donor genotype because it

63

64

Table l. Callus induction and shoot formation from leaf discs of the diploid source plant

REL-1 and its haploid plants OV-9 and OV-16

 

Callusing proportion (%) Shooting proportion (%)

 

 

 

Genotype
)8 value‘ x2 value‘
133/140 (95.0) 101/140 (72.1)
REL-l
10/140 (7.1) 7/140 (5.0)
OV-9
2258.43** 312.58**
8/140 (5.7) 5/140 (3.6)
OV-l6
2332.74’" 326.05**

 

‘ Values of )8 were calculated with Yates correction factor with one degree of freedom;

** = significantly different from the diploid source clone REL-1 at 99 % confidence

level.

 

65

was available first as a flowering plant in the greenhouse.

Table 2. Preliminary test of callus induction and shoot regeneration in two diploid

genotypes 6822-08 and FC 701/5-4.

 

 

 

Genotype Callus induction (%)‘ Shoot regeneration (%)
6822-08 52/60 (87.0) 49/60 (81.7)
F C 701/5-4 47/60 (78.3) 39/60 (65.0)

 

‘ one leaf disc (total 60 leaf discs) was put on each plate for 6822-08 and FC 701/5-4,

respectively.

From 6822-08, three ovules each responded and directly produced (without
callus) a presumptive haploid shoot, each of which was multiplied and rooted as OV-68-
1, OV-68-2, and OV-68-3. When the callus induction and shoot regeneration abilities of
three haploid genotypes were tested, they showed extremely low proportions of leaf disc
callusing and shooting in comparison with their source diploid 6822-08 (Table 3). In
fact, none of the three haploid genotypes regenerated shoots. From these results, it was
concluded that none of the five haploid genotypes were adequate as materials for somatic
cell selection or studies of spontaneous diploidization.

Several possible explanations for this low in vitro response of five haploid
individuals in two different genetic backgrounds may be given. Firstly, unconscious

selection of eggs/ovules that produced haploid plants with low intensity of callus and

66

Table 3. Callus induction and shoot regeneration of the diploid source 6822-08 and its

derivative haploids OV-68-1, OV-68-2, and OV-68-3

 

Callusing proportion (%) Shooting proportion (%)

 

 

 

 

Genotype

)6 value‘ )6 value‘
2880 (93.3) 24/30 (80.0)

6822-08
“30 (3.3) 0/30 (0.0)

OV-68-l
378.14’” 117.05'”
1/30 (3.3) 0/30 (0.0)

OV-68-2
378.l4** 117.05'MI
3/30 (10.0) 0/30 (0.0)

OV-68-3
323.35** 117.05“I

 

‘ x2 values were obtained with Yates correction factor with one degree of freedom;
** = significantly different from the diploid source clone 6822-08 at 99 % confidence

level.

67

shoot production may be made when ovules were taken for culture. Secondly, haploid
plants may be intrinsically less responsive than diploids. Thirdly, haploid leaf samples
may be sensitive to surface sterilization reagents. Before making leaf discs, leaf samples
were surface-sterilized by 15 % commercial hypochlorite bleach solution with 0.01 %
sodium laurylsulfate. The leaves of haploid plants are thinner than leaves of diploid
plants. So, during surface sterilization, the sterilization reagents may have damaged the
thin leaves of haploid plants and injured leaf cells. Lastly, the choice of appropriate leaf
size from the haploid plants for leaf disc culture may be not made. Actually, leaves of
haploid plants are smaller than the leaf of a diploid. So, in a haploid plant, it is more
difficult to measure the degree of expansion of a leaf. Standard practice is to use 20-40 %
of final leaf size. So, in this experiment, there is a possibility that fully expanded small
leaves of haploid plants were taken as samples. If fully expanded leaves were used for
explants, the in vitro responses of the explants would be quite different from young
incompletely expanded leaves. Therefore, the choice of leaf sample size may be critical

for normal in vitro responses of haploid genotypes.

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10.

11

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