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University of Wollongong Thesis Collections
1994
The relationship between ploidy, gene expression
and vigour in citrus
L. Slade Lee
University of Wollongong
Recommended Citation
Lee, L. Slade, The relationship between ploidy, gene expression and vigour in citrus, Doctor of Philosophy thesis, Department of
Biological Sciences, University of Wollongong, 1994. http://ro.uow.edu.au/theses/1062
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T H E RELATIONSHIP B E T W E E N PLOIDY, G E N E EXPRESSION
AND VIGOUR IN CITRUS.
A thesis submitted in fulfilment of the
requirements for the award of the degree
DOCTOR OF PHILOSOPHY
from
THE UNIVERSITY OF WOLLONGONG
by
\J
L. Slade LEE, B.App.Sc.(Hons.)
Department of Biological Sciences
1994
SUMMARY
Autotetraploid Citrus plants exhibit growth retardation compared to their diploid
counterparts. Agricultural scientists are attempting to exploit this characteristic to
produce dwarfed trees, which offer horticultural advantages over the normally large trees.
No conclusive data exist with regard to the dwarfing propensity of tetraploids of various
cultivars. Neither is there any information on underlying gene expression effects of
autotetraploidy in Citrus cultivars. Tetraploids of Citrus arise apomictically and are
therefore somatic and isogenic with their diploid progenitors. Therefore, they provide
a useful model for studying the effects of genome duplication. Furthermore, a better
understanding of gene expression characteristics in Citrus somatic autotetraploids will
result in improved efficiency in tetraploid rootstock breeding programs by aiding the
selection of superior germplasm.
In this thesis, differences in gene expression between eight diploid Citrus cultivars and
their somatic tetraploid counterparts were investigated, using quantitative methods
involving isozyme electrophoresis and laser densitometry. For this purpose, appropriate
methods of sample preparation, isozyme electrophoresis and quantification, and DNA
measurement were established and verified. Isozymes were electrophoresed on cellulose
acetate gels and individual band activities were quantified using a laser densitometer.
The effect of genome duplication on quantitative gene expression had not been previously
studied for a range of proteins in a number of related genotypes. To compare isozyme
activity between diploids and their isogenic tetraploid counterparts it was necessary to
compensate for the difference in cell sizes between ploidy levels. This was achieved by
expressing sample isozyme activities in terms of their DNA concentrations, which were
determined by densitometric and fluorometric methods.
In tetraploid plants, m a n y
isozyme bands exhibited differing levels of activity compared to diploids of the same
cultivar. This effect was repeatable but a very complex situation was apparent in the
relationships between individual isozyme bands and between cultivars. When all
diploid:tetraploid ratios of isozyme activity per unit DNA for each cultivar were
considered collectively, it was found that the majority of the values for each cultivar f
within one of three distinct activity groups. The effect of genome duplication on
quantitative gene expression in each cultivar exhibited a strong tendency to either
enhanced, reduced or unaffected activities. For any given cultivar, this tendency was
uniform across a diverse range of isozymes. For example, isozyme activity per unit
DNA was always greater in the tetraploid of the Troyer citrange cultivar compared with
its diploid counterpart (for 26 different isozyme bands the ratio of diploid:tetraploid
isozyme activity per unit DNA ranged between 0.562 and 0.805). Conversely, the
tetraploid of Volkameriana lemon always exhibited reduced isozyme activity, relative to
its diploid partner (the diploid:tetraploid ratios for 21 isozyme band activities ranged
between 1.412 and 2.034). On the other hand, two mandarin cultivars showed little
difference between their ploidy levels. This uniformity of the diploid:tetraploid isozyme
activity response of each cultivar suggested that gene expression was modulated by some
non-gene-specific or 'extra-genomic' (DeMaggio and Lambrukos, 1974) feature, such as
differences in cell morphology between ploidy levels. Very importantly, the assignment
of cultivars to the three groups coincided exactly with three distinct phylogenetic groups
This strongly suggested that the non-gene-specific feature above is genetically influence
A field trial showed that scions grafted on tetraploid rootstocks were smaller than
identical scions grafted on the diploid counterpart rootstocks. This result suggested tha
tetraploids are physiologically different from their isogenic diploid equivalents. Other
field studies compared tree growth characteristics of diploid and tetraploid counterparts
of the range of cultivars as examined in the isozyme studies. This work showed cultivardependent growth differences between diploids and their tetraploid counterparts. As was
the case for their isozyme activity characteristics, growth responses to genome duplicati
varied between groups of phylogenetically related genotypes. The findings suggested that
where there was a marked difference in quantitative gene expression between a diploid
and its isogenic tetraploid, vigour may be relatively impaired in the tetraploid. On the
other hand, no such vigour effect may occur in genotypes where both ploidy levels
exhibit similar quantitative gene expression characteristics.
PREFACE
This project made use of tetraploid Citrus accessions selected from the much larger
collection of Citrus polyploids at Queensland Department of Primary Industries,
Bundaberg Research Station (Queensland, Australia). I have developed these out of a
series of on-going Citrus polyploid breeding projects since 1984. That work aims to
exploit useful horticultural characteristics of Citrus triploids and tetraploids. There i
considerable body of knowledge concerning inheritance in polyploids. However, there
is limited understanding of the effects of polyploidy on gene expression. This is probabl
because the study of polyploidy became unfashionable before modern molecular methods
of genetics investigation were available. This PhD project arose from a desire to
understand how polyploidy affects gene expression - an important consideration in
aspiring to exploit the characteristics of polyploids in plant breeding. Citrus and relat
genera provide a convenient model for this purpose because they are the only plants
known to produce tetraploids through apomictic embryogenesis in diploid progenitors.
The result is perfectly isogenic tetraploid/diploid counterparts, free from the problems
of mixoploid chimeras and mutations which can arise from induction of such polyploids
by artificial means.
Acknowledgements
I wish to express my gratitude to the Queensland Department of Primary Industries and
in particular John Chapman and my superior officers and colleagues for their financial,
moral and professional support and encouragement, both in the PhD project and in my
Citrus polyploidy work in general. I particularly wish to thank m y supervisors at the
University of Wollongong, Prof. Helen Garnett and Dr. David Ayre, for their guidance
and counsel, and my friend and colleague Dr. Gonzalo Hortelano-Hap for his
encouragement and advice. I also thank Gary Blight, QDPI biometrician, for his
assistance with data analysis, and my technician David Gillespie for maintaining my
breeding programme while I was engaged in this project.
Contents 1
CONTENTS
SUMMARY
PREFACE
Acknowledgements
CONTENTS
section
page
Chapter 1
INTRODUCTION
1.1
Polyploidy
1.1
Classification and types
1.2 Citrus taxonomy and reproduction 1.5
1.2.1
1.2.2
Citrus taxonomy
Poly embry ony
1.5
1.6
1.3 Apomixis and polyploidy 1.7
1.4 Characteristics of polyploids 1.8
1.5 Effects of genome duplication on physiology
and gene expression
1.13
1.5.1
1.5.2
1.13
1.16
Physiological effects
Isozyme effects
1.6 Project objective 1.20
Chapter 2
METHODOLOGY
2.1
2.1.1
2.1.2
2.1.3
Introduction
Cultivar selection
Isozyme analysis procedures
Isozyme selection
a) Selection criteria
b) Isozymes
(i) Shikimate dehydrogenase
(ii) Isocitrate dehydrogenase
(iii) Cytosol aminopeptidase
2.1
2.4
2.5
2.6
2.6
2.8
2.8
2.11
2.14
Contents 2
4
5
6
(iv) Glucose-6-phosphate dehydrogenase
(v) 6-Phosphogluconate dehydrogenase
(vi) Malate dehydrogenase
(vii) Ribulose 1,5-biphosphate carboxylase
Effects of sample lyophilisation
a) Effects of lyophilisation on proteins
b) Effects of lyophilisation on isozyme activity
Densitometry
a) Comparisons of densitometry with other
quantitative methods
b) Different approaches to densitometry
(i) Internal standards
(ii) Subfraction apportionment
(iii) Comparative densitometry
c) Linearity of band density response
(i) Peak height vs. curve area
D N A quantification
Methods 2.39
1
Isozyme analysis procedures
a) Identification of cytosolic R B C isozyme
2
Investigation of the effect of lyophilisation on isozyme
band density
a) Investigation of the effect of lyophilization
on M D H band density
3
Assessment of densitometry procedures
a) A n experiment to assess the effects of a series
of concentrations of sample extract on band
densities of I D H , S k D H and M D H
b) A n experiment to test sample applicator reliability.
c) Isozyme activity calibration
4
D N A quantification methods
a) D N A quantification by densitometry of
electrophoretic bands
(i) Assessment of the relationship between D N A
concentration and electrophoretic band density
(ii) Quantification of D N A in Citrus crude extracts
by densitometry of electrophoretic bands.
b) Quantification of D N A in Citrus crude extracts
by fluorometry
2.15
2.17
2.18
2.22
2.25
2.26
2.27
2.28
2.31
2.32
2.32
2.33
2.33
2.33
2.34
2.36
2.39
2.41
2.42
2.43
2.44
2.45
2.46
2.46
2.48
2.48
2.50
2.51
2.52
Results and Discussion 2.54
1
2
Investigation of the effect of lyophilisation on isozyme
band density
a) Investigation of the effect of lyophilisation
on M D H band density
Assessment of densitometry procedures
2.54
2.54
2.55
Contents 3
3.3
a) A n experiment to assess the effects of a series
of concentrations of sample extract on band
densities of I D H , S k D H and M D H
2.55
b) A n experiment to test sample applicator reliability.
2.59
c) Isozyme activity calibration
2.62
D N A quantification
2.66
a) D N A quantification by densitometry of
electrophoretic bands
2.66
(i) Assessment of the relationship between D N A
concentration and electrophoretic band density
2.66
(ii) Quantification of D N A in Citrus crude extracts
2.71
by densitometry of electrophoretic bands.
4 Conclusion 2.74
Chapter 3
T H E RELATIONSHIP B E T W E E N PLOIDY A N D
G E N E EXPRESSION IN CITRUS
Introduction
Methods
a) Small scale study
b) Large scale study
(i) Sample collection and preparation
(ii) Isozyme and D N A analysis
(iii) Experiment design
Results and Discussion
a) Small scale study
(i) D N A concentrations
(ii) Isozyme band density per unit D N A
b) Large scale study
(i) D N A concentrations
(ii) Isozyme bands
(iii) Analysis of the data
(iv) Interpretation
4
Conclusion
3.1
3.4
3.4
3.5
3.5
3.6
3.6
3.7
3.7
3.7
3.8
3.11
3.11
3.13
3.20
3.24
3.28
Chapter 4
T H E RELATIONSHIP B E T W E E N PLOIDY A N D G R O W T H
IN CITRUS
1 Introduction
4.1
Contents 4
Methods
4.3
a) In vitro growth studies
b) In vivo nursery growth studies
c) A n experiment to investigate the effect of tetraploid
rootstocks on the growth of various diploid Citrus
scion cultivars
d) A field study of growth differences between diploid and
tetraploid counterparts of eight Citrus cultivars
4.3
4.4
4.5
4.6
Results and Discussion 4.9
a) In vitro growth study
4.9
b) A n experiment to investigate the effect of tetraploid
rootstocks on the growth of various diploid Citrus
scion cultivars
4.11
c) A field study of growth differences between diploid and
tetraploid counterparts of eight Citrus cultivars
4.14
(i) Flush duration
4.14
(ii) Flush length
4.15
(iii) N u m b e r of internodes
4.15
(iv) Internode length
4.15
(v) Dry matter
4.18
(vi) Interpretation
4.19
Chapter 5
CONCLUSION
REFERENCES
APPENDICES
Appendix 1
Extraction buffer formulae
Appendix 2
Isozyme stain protocols
Appendix 3
Peak height data for isozymes of eight citrus cultivars,
their diploid:tetraploid ratios and their ratios per unit D N A
Appendix 4
Origins of the accessions
Appendix 5
Estimation of Total D N A in Crude Extracts of Plant Leaf Tissue
Using 4',6-diamidino-2-phenylindole (DAPI) Fluorometry.
Introduction 1.1
section 1.1
Chapter 1
INTRODUCTION
The usual manner of propagating Citrus varieties is to graft or, more usually, bud the
desired fruit cultivar (the scion) onto an appropriated rootstock cultivar. The polyploid
Citrus breeding program I have conducted since 1984 has two broad objectives. One is
to produce seedless triploid mandarin scion varieties, the other is to develop dwarfing
tetraploid rootstock varieties (Lee, 1988a, 1988b). The prospect of the latter objective
arises from the observation that when used as rootstocks, tetraploids of Citrus cause the
scion to be dwarfed (Lee et ah, 1990). This characteristic offers several agronomic
benefits. Molecular techniques have not been applied comprehensively to investigate the
genetic differences between diploid and tetraploid counterparts of the same Citrus
cultivars. Such methods have the potential to reveal much about the genetics of genome
duplication and to provide an rapid screening procedure to identify the most potentially
useful plants in rootstock breeding programs.
1.1 Polyploidy
The exploitation of polyploidy in plant breeding was heralded as a potential revolution
in the 1930's, 1940's and 1950's when the use of colchicine became popular for artificial
induction of polyploidy. This technique did not live up to its promise (Dewey, 1980),
chiefly because breeders failed to appreciate the genetic complexities of polyploidy and
section l. l
Introduction 1.2
the attendant constraints to its application (Sanford, 1983). In describing these
complexities, detailed dissertations on polyploid origins, classification, genetics and
characteristics (Stebbins, 1947, 1950; Harlan and DeWet, 1975; Lewis, 1980a; Levin,
1983) revealed that the terminologies used have caused confusion.
In their pioneering work with tetraploid Datura, Blakeslee et al. (1923) recognized that
tetraploids exhibit tetrasomic inheritance. They realized that while diploids have only
type of heterozygote (Ad), in tetraploids there are three types, and they coined the ter
simplex (Aaaa), duplex (AAaa), and triplex (AAAa). Homozygotes in diploids (AA and
ad) become quadriplex (AAAA) and nulliplex (aaaa) in tetraploids. Furthermore, they
reasoned that plants should exist which have doubled chromosome numbers but
homologous pairs only, rather than homologous foursomes as found in their subject
plants. Such observations led to the initial notion (Kihara and Ono, 1926) that there we
two types of polyploids - autopolyploids, arising from doubling of the diploid genome,
and allopolyploids arising from hybridization between disparate species with doubling
resulting from nonhomology of the two genomes (DeWet, 1980). It soon became
apparent that some polyploids did not fit neatly into these categories (Haldane, 1930;
Miintzing, 1933; Lindstrom, 1936; Clausen et al., 1945). Following attempts by various
authorities in the late 1930's and early 1940's, Stebbins (1947) proposed four classes:
autopolyploids, which contain multiple genomes derived from within a single
species, and hence multiple homologous chromosomes which often produce polyvalents
during meiosis;
section l.l
Introduction 1.3
segmental allopolyploids, generally derived from interspecific hybridizations,
containing multiple genomes which possess considerable portions of homologous
chromosome segments, but sufficient nonhomology that homologous chromosomes pair
preferentially at meiosis resulting in a high proportion of bivalents;
true or genomic allopolyploids, which are derived from hybridizations between
distantly related species or across genera. Chromosome pairing only occurs between
homologues of the same genome, yielding exclusively bivalents, and;
autoallopolyploids, which are higher polyploids (hexaploids and higher) derived
from lower-order allopolyploids. They contain significant portions of multiple
homologous genomes.
In documenting examples of these four classes, Stebbins (1950) showed that there are
numerous forms within each. The amphiploid class is any polyploid containing whole
genomes from two or more separate species (Strickberger, 1985) and encompasses
segmental allopolyploids, true allopolyploids, autoallopolyploids and aneuploids
(Stebbins, 1950).
Whereas these classifications have served to emphasize the diversity amongst polyploids
DeWet (1980), Stebbins (1980), and Sanford (1983) explained that they have been used
in a taxonomic sense, and that this has been a major source of confusion as, due to the
largely hybrid origin, taxonomic variation in polyploid plants is continuous rather tha
discrete (Love, 1964). Ironically, Stebbins (1980) conceded that his terms should now
section l.i
Introduction 1.4
be abandoned. From a genetic standpoint, the major consideration is the relationship
between the genomes comprising the polyploid. Accordingly, Stebbins (1980), and also
Lewis (1980b), proposed the terms intraspecific and interspecific polyploidy. Sanford
(1983) suggested the terms polysomic and disomic polyploidy, which better define the
cytogenetic relationship between the constituent genomes. In polysomic polyploids the
genomes are homologous and exhibit random intergenomic meiotic pairing and hence
polysomic segregation. Conversely, a disomic polyploid contains differentiated sets of
homoeologous genomes in which pairing occurs primarily between homologous
chromosomes from the same genome (Jackson, 1991). The result is disomic segregation,
with the polyploid behaving as if diploid.
Lewis (1980b) made an important distinction between two types of polysomic polyploids.
Using the general term autoploidy, he described homozygous and heterozygous types.
Genomes of disomic polyploids are necessarily heterozygous. However, in polysomic
polyploids the constituent genomes may be either, (i) derived from two homologous but
heterozygous genomes through intraspecific fertilization, or alternatively, (ii) the gen
may derive from one chromosome set. This latter phenomenon, referred to as
homozygous autoploidy by Lewis (1980b), results in polyploids with genomes which are
entirely isogenic with their diploid progenitors. They are usually tetraploids. Love
(1964) coined the term panautoploid for such polyploids, but the term has not been
widely adopted. This situation applies to Citrus nucellar tetraploids which are the subj
of this thesis. Whereas each gene locus of heterozygous tetraploids may possess any
genotype, from mono-allelic to tetra-allelic, in homozygous tetraploids, loci can only b
nulliplex, quadriplex or duplex (Bingham, 1980). The presence of duplex loci, with their
section 1.2.1
Introduction 1.5
heterozygous alleles, in so-called homozygous tetraploid organisms, ostensibly contradi
the concept of homozygosity in diploids, and this term may therefore cause confusion.
A diversity of reproductive aberrations gives rise to the various types of homozygous
polyploidy (Den Nijs and Peloquin, 1977; Lewis, 1980b; Veilleux, 1985) of which
somatic chromosome doubling is a particularly rare type (Harlan and DeWet, 1975;
DeWet, 1980). These can be broadly categorized as (i) zygotic chromosome doubling,
(ii) meristematic chromosome doubling, and (iii) gametic chromosome non-reduction
(DeWet, 1980). I have previously described a number of such phenomena which occur
in Citrus (Lee, 1988b - attachment). Insofar as it dictates genetic structure, the mode
origin of Citrus tetraploidy must be considered.
1.2 Citrus taxonomy and reproduction
1.2.1 Citrus taxonomy.
Rootstock varieties for cultivated Citrus are derived from species within the genus Ci
from Poncirus trifoliata and intergeneric hybrids thereof. Agreement has not yet been
reached on a correct taxonomy for the genus Citrus (Cameron and Soost, 1969), however
recent investigations (Potvin et al., 1983; Green et al., 1986; Handa et al., 1986; Sc
1988; Vardi, 1988) support the three-species view first expressed by Scora (1975), and
Barrett and Rhodes (1976). The three basic species are the citrons (Citrus medico), the
pummelos (C. grandis (syn. C. maxima, Scora and Nicolson, 1986)), and the other
Eucitrus types (C. reticulata). The former two are included in the conventional Swingle
section 1.2.2
Introduction 1.6
(1967) classification of ten species which also defines the mandarins (C. reticulata),
oranges (C. sinensis), the lemons (C. limori), the sour oranges (C. aurantium), the lim
(C. aurantifolia), the grapefruit (C. parodist), the Indian wild orange (C. indica), an
Tachibana orange (C. tachiband). These latter eight species are all included in the
species C. reticulata in the new taxonomy. The three-species taxonomy will be used
here. The distinct types within C. reticulata, which had species status in the Swingle
taxonomy, are currently referred to as biotypes. All Citrus and related genera, includ
Poncirus, have a haploid chromosome number of n = 9, and diploidy (2n = 18) is the
normal somatic state (Lee, 1988b). For convenience, unless otherwise specified, the ter
'Citrus' will hereafter in this thesis imply Citrus, Poncirus and hybrids thereof.
1.2.2 Polyembryony.
The phenomenon of apomixis gives rise to embryos from somatic tissues and is
reasonably common amongst angiosperm species. Apomictic embryogenesis in the
nucellus tissues is found in the Citrus family, Rutaceae, and also in species of
Acanthaceae, Amaryllidaceae, Bombaceae, Cactaceae, Liliaceae, and Poaceae (Tisserat
et al, 1979). Nucellar apomictic embryogenesis resulting in polyembryony has been
subjected to detailed study in Citrus (Frost, 1926; Bacchi, 1943; Esan, 1973; Esen and
Soost, 1977; Kobayashi et al., 1978, 1979, 1981; Wilms et al., 1983) and related gener
(Wakana and Uemoto, 1987, 1988). This phenomenon results in multiple somatic
embryos in single seeds. In the Citrus species, this poly embryogenesis is common in t
C. reticulata biotypes, whereas C. medico and C. grandis are entirely monoembryonic
(producing zygotic embryos)(Torres, 1936; Frost and Soost, 1968). The phenomenon
is not universal within the C. reticulata group however. The trait is apparently contr
section 1.3
Introduction 1.7
by three interacting genes (Parlevliet and Cameron, 1959; Iwamasa et al., 1967; Deidda
and Chessa, 1982) but is influenced in its expression by environmental factors (Parlev
and Cameron, 1959), particularly those affecting pollination (Cameron and Soost, 1969;
Esen and Soost, 1977; Wakana and Uemoto, 1987). Although some cultivars are obligate
apomicts, many exhibit facultative poly embryogenesis. Thus, different seedling
populations produce a range of nucellar: zygotic seedling ratios (Frost and Soost, 1968
Some cultivars are entirely monoembryonic. While the so-called nucellar or
polyembryonic cultivars usually produce somatic nucellar progeny they always initiate
zygotic embryos which may survive to produce viable seedlings. The normal occurrence
however, is for the zygotic embryos to be suppressed by the nucellar ones. The number
of viable embryos produced per seed by members of these cultivars is variable and
strongly influenced by genetic and environmental factors (Cameron and Soost, 1969).
Poncirus trifoliata and its hybrids with Citrus are predominantly polyembryonic (Nishi
et al, 1974; Prasad and Ravishankar, 1983).
1.3 Apomixis and polyploidy
Apomixis in polyploid plants is a widespread phenomenon (Sullivan, 1976; Stebbins,
1980; Quarinef al, 1982; Sukhareva, 1984; Campbell et al, 1987; Lumaret, 1988).
Very often, apomictic polyploids derive from sexual diploids, although polyploidizatio
does not directly initiate apomictic reproduction (Stebbins, 1950). Apart from the
Rutaceae, there are apparently no documented cases of plants in which polyploidy is
initiated from somatic tissues during apomictic reproduction of diploids. In Citrus,
virtually all tetraploids originate from nucellar embryogenesis (Lee, 1988b), and arise
section 1.4
Introduction 1.8
from single nucellar cells in the ovule (Hutchison and Barrett, 1981). As nucellar
embryos are derived from somatic tissue, such tetraploids possess a perfectly duplicated
genome and are isogenic with their progenitors. A similar situation can arise through
selfing of unreduced gametes or zygotic doubling of selfed gametes (Lewis, 1980b), but
such tetraploids do not necessarily contain two identical genomes isogenic with their
parent (cf. Dunbier et al, 1975). Somatic chromosome doubling in meristematic tissues
is a very rare event in nature (DeWet, 1980; Lewis, 1980b), although it has been reporte
in Citrus (Frost and Krug, 1942). Such polyploidy is readily induced artificially throug
the use of mutagenic treatments, but the resultant plants are often chimeras with dispa
ploidy levels between adjacent histogenic layers (Sanford, 1983). This problem does not
occur in nucellar tetraploids which arise from a single cell (Sanford, 1983; Lee, 1988b)
I have adopted the term somatic polyploidy, as used by Sanford (1983), in reference to
Citrus tetraploids, to emphasise the duplicate nature of the genomes in question.
1.4 Characteristics of polyploids
The majority of polyploids are the so-called heterozygous types. Consequently, it is the
that have been the subject of the majority of research on polyploids. However, from one
species to the next, there is a diversity of genotypic relationships between the genomes
in such polyploids. These relationships are the result of the disparate genotypes of the
polyploids' progenitors, and are manifested in a wide range of physiological and gene
expression response differences between ploidy levels of the same species. Table 1.1
shows instances where such differences have been observed between tetraploids and their
diploid progenitors. Examples of many other such differences are presented in reviews
section 1.4
Introduction 1.9
Table 1.1
Cases of observed differences in physiology between
heterozygous tetraploids and their diploid progenitors.
plant species
studied
aspects of physiology exhibiting differences
between heterozygous tetraploids and diploid progenitors
Beta (sugar beet)
activity, mobility and numbers of allozymes of ACP, Spettoli et al.
EST, G D H , I N V and PER; water, sucrose and protein content
(1976)
Beta (sugar beet)
activity, mobility and numbers of allo2ymes of G D H ;
fresh weight and sucrose content
Spettoli & Cacco
(1977)
Clarkia
number of allozymes of GDH, MPI, PGI, PGM, SkDH
andTPI
Holsinger &
Gottlieb (1988)
references
Dactylis (orchard grass) activity and numbers of allozymes of M D H and P E R
Lumaret (1982)
Dactylis (orchard grass) numbers of allozymes of PGI
Lumaret (1986)
Galium
Gossypium (cotton)
Heuchera
activity of allozymes of PGI
Samuel et al.
(1990)
mobility of allozymes of ACO, MDH, PGM
Saha et al.
(1988)
activity, mobility and numbers of allozymes of PGI, Soltis & Soltis
P G M and TPI
(1989)
Hibiscus
activity of ADH, CAP and MDH; dry weight, total
protein and D N A content
Hoisington &
Hancock (1981)
Lolium (ryegrass)
activity, mobility and numbers of allozymes of PGI
Nielsen (1985)
Medicago (alfalfa)
water content and dry weight
Medicago (alfalfa)
buffer-soluble protein per unit DNA; RBC activity
Plantago
fertility; activity and number of allozymes of
A A T and S k D H
Pfeiffer et al.
(1980)
Meyers et al.
(1992)
Van Dijk & Van
Delden (1990)
Polygala
activity, mobility and numbers of allozymes of PGI
Solarium (potato)
numbers of allozymes of AAT, ADH, EST, MDH, PGI,
P G M , P E R and 6 P G D
Solarium (potato)
numbers of allozymes of PER; activity of S k D H allozymes Quiros &
McHale (1985)
Solarium (potato)
mobility of allozymes of P E R
Deimling et al.
(1988)
Stellaria
activity of allozymes of AAT, ME, SkDH and 6PGD
Cai & Chinnappa
(1989)
Lack & Kay
(1986)
Martinez-Zapater
& Oliver (1985)
section 1.4
Introduction
1.10
Table 1.1 contd.
Tolmiea activity, mobility and numbers of allozymes of PGI, PGM, Soltis &
Rieseberg (1986)
S k D H and TPI
Tragopogon mobility and numbers of allozymes of AAT, ACP, ADH, Roose & Gottlieb
C A P , EST, G D H , G 6 P D , M D H , M E , PER, PGI, P G M and S O D
(1976)
Tragopogon activity of allozymes of ADH Roose & Gottlieb
Vaccinium (blueberry)
activity of allozymes of I D H and 6 P G D ; numbers of
allozymes of M D H
(1980)
Vorsa et al.
(1988)
Vicia (vetch) activity and numbers of allozymes of AAT, AMY and EST Suso & Moreno
(1982)
Isozyme abbreviations
A A T - aspartate amino transferase ( = glutamate oxaloacetate transaminase)
A C O - aconitase
A C P - acid phosphatase
A D H - alcohol dehydrogenase
A M Y - amylase
C A P - cytosol aminopeptidase ( s leucine aminopeptidase)
E S T - esterase
G D H - glutamate dehydrogenase
G 6 P D - glucose-6-phosphate dehydrogenase
I D H - isocitrate dehydrogense
I N V - invertase
M D H - malate dehydrogenase
M E - malic enzyme
M P I - mannose-6-phosphate isomerase
P E R - peroxidase
PGI - phosphoglucose isomerase
P G M - phosphoglucomutase
R B C - ribulose 1,5-biphosphate carboxylase (= Rubisco)
S k D H - shikimate dehydrogenase
S O D - superoxide dismutase
TPI - triose phosphate isomerase
6 P G D - 6-phosphogluconate dehydrogenase
by Lewis (1980b), Gottlieb (1982), Levin (1983) and Oliver et al. (1983). Rare reports
of comparisons between homozygous tetraploids and heterozygous tetraploids and their
diploid progenitors reveal physiological differences between the types of polyploid,
between polyploid and diploid in Cochlearia (Gupta, 1981), Plantago (Van Dijk and Van
Delden, 1990), and in Rubus (Haskell, 1968).
section 1.4
Introduction 1.11
The usual observation of larger sized leaves, flowers and other organs in polyploid
plants, referred to as gigas characteristics is, at least partially, a result of increa
size in the polyploids (Randolph, 1941; Stebbins, 1950, 1971; Roy and Dutt, 1972;
Sanford, 1983). Cell size increases with DNA content (Randolph, 1941; Cavalier-Smith,
1978; Tal, 1980). Whereas there is most probably a direct causal, or nucleotypic,
relationship between cell size and the amount of DNA, there is also evidence that cell
size can be genetically determined (Cavalier-Smith, 1985; Nurse, 1985). For example,
Van Dijk and Van Delden (1990) found that colchicine-induced tetraploids of Plantago
media exhibited gigas characteristics but naturally occurring heterozygous tetraploids
the species did not. They argued that evolution in the natural population has selected
against large cell size. Gigas characteristics have been reported in Citrus tetraploids
(Soost and Cameron, 1969; Tachikawa, 1973; Barrett, 1974; Barrett and Hutchison,
1978; Lee, 1988b) and were used in the initial identification of tetraploids in my own
breeding programme (Lee et al., 1990), which were used in this study.
Whereas Cavalier-Smith (1978) suggested that growth rate is determined by DNA content
and cell size per se, there is ample evidence that genetic factors also come into play.
genetic effects of polyploidy are dependent upon mode of origin and are generally a
reflection of the level of heterozygosity. Due to poly-allelic genotypes, disomic
polyploids (allopolyploids) have the highest levels of heterozygosity. Such plants exhi
superior crop yield and growth performance (Stebbins, 1957, 1971; Lewis, 1980a; Zeven,
1980; Veilleux, 1985).
section 1.4
Introduction 1.12
Heterosis in heterozygous polysomic tetraploids, manifested as increased vigour has
observed in grapes (Vitis) (Olmo, 1952), potatoes (Solanum) (Mendiburu and Peloquin,
1977; Derailing et al, 1988), lucerne (Medicago) (Busbice and Wilsie, 1966; Groose e
al, 1989), wildrye (Psathyrostachys) (Berdahl and Barker, 1991), and various other
plants (Stebbins, 1957). Greater heterosis is generally observed with increasing
heterozygosity, i.e. more poly-allelic loci (Strickberger, 1985).
Because of their strictly mono-allelic and di-allelic nature, homozygous tetraploids
lucerne (Medicago sativa) are inbred (F=0.333) and exhibited reduced forage yield,
fertility and seed weight compared with their single cross and double cross progeny
(Groose et al, 1989). Numerous instances of growth retardation in homozygous
tetraploids compared with their diploid progenitors have been reported in cultivated
plants, including tobacco (Nicotiana) (Noguti et al, 1940), grapes (Vitis) (Olmo, 194
1952; Chadha and Mukherjee, 1975), tomatoes (Lycopersicori) (Nilsson, 1950; Albuzio
et al, 1978), melons (Cucumis) (Batra, 1952), apples (Malus) (Garner and Keep, 1954;
Beakbane, 1967), potatoes (Solanum) (Rowe, 1967), Citrus (Lee, 1988b; Lee et al,
1990), lucerne (Medicago) (Bingham, 1980; Groose et al, 1989), buckwheat
(Fagopyrum) (Murakami, 1952), crested wheatgrass (Agropyron) (Dewey, 1966, 1969),
barley (Hordeum) (Evans and Rahman, 1990) and various other grasses (Stebbins, 1950)
section 1.5.1
Introduction 1.13
1.5 Effects of genome duplication on physiology and
gene expression
Citrus nucellar tetraploids are readily identified and may be generated from a range of
cultivars (Lee, 1988 b ; Lee et al, 1990), and the resultant plants can be vegetatively
propagated. N o other group of organisms present a set of closely related genotypes in
which diploids and naturally-occurring somatic tetraploids are available for comparison.
The occurrence of Citrus tetraploids possessing duplicate genomes isogenic with their
diploid progenitors, provides a unique model for investigating the effects of genome
duplication on expression of specific genes, its effect on phenotype, and the relationships
of these effects between kindred genotypes. The only other ways to undertake such
studies are to use rare trisomic individuals (Smith and Conklin, 1975; Birchler, 1979,
1981, 1983; Birchler and Newton, 1981; Sidhu et al, 1984) or somatic polyploids
induced artificially, usually by colchicine application (Nakai, 1977; Setter et al, 1978).
Caution is required in interpreting results from trisomies because of the potential for
interactions between the duplicated gene and regulatory genes located on other,
nonduplicated chromosomes (Birchler, 1983). The possibility of mixoploid chimeras and
higher level euploids must be borne in mind w h e n using colchicine-induced polyploids.
Barrett (1974) produced tetraploids in Citrus by treating buds with colchicine and
observed that over 5 0 % of the plants were mixoploid chimeras.
1.5.1 Physiological effects.
Differences in physiology between diploids and their isogenic polyploid counterparts have
long been reported in the literature. Sullivan and Myers (1939) found higher dry matter
section 1.5.1
Introduction 1.14
and sugar content, but no difference in total nitrogen in colchicine-induced tetraploid
perennial ryegrass (Lolium) compared with its diploid progenitor. Noguti et al (1940)
made comparisons between various diploid tobacco varieties (both Nicotiana rustica and
N. tabacum) and their colchicine-induced tetraploids. All the tetraploids exhibited grea
dry matter, nicotine and organic acids content. Total ether extract and resin were
elevated in the tetraploids, but these plants contained less than half the total sugars
a third the reducing sugars of their diploid progenitors. Total nitrogen, calcium,
potassium, magnesium and soluble ash were higher in the tetraploids, whereas sulphur
and phosphorus contents were lower, compared with those in the diploids. Early
physiological studies of colchicine-induced tetraploid barley (Hordeum) (Chen and Tang,
1945) found imbibition rate and quantity were higher in tetraploid seeds, and oxygen
consumption and carbon dioxide production were lower in germinating tetraploid seeds
compared to their diploid counterparts. Dry matter, nitrogen (protein), ether extract
(lipoids), and ash content were higher in tetraploid seeds, and other nitrogen-free
compounds (carbohydrates) were lower. The cellular osmotic potential of young leaves
was elevated in the tetraploids, and transpiration rates were much lower, compared to th
diploids. Lower transpiration rates have also been observed in tetraploid tomatoes
(Lycopersicon) compared to their isogenic diploid counterparts (Tal and Gardi, 1976).
Higher nitrogen content in colchicine-induced tetraploids was also observed in cabbage
(Brassica) whereas auxin concentration was reduced (Avery and Pottorf, 1945).
Murakami (1952) investigated physiological differences between colchicine-induced
tetraploid buckwheat (Fagopyrum) and its diploid progenitor. Differences were found in
total nitrogen and total sugar between ploidy levels but these changed with development
stages during plant growth.
section 1.5.1
Introduction 1.15
For nine out of ten cultivars of Phlox drummondii, colchicine-induced tetraploids
exhibited lower net photosynthetic rates than their diploid counterparts (Bazzaz et al,
1982), and the degree of difference between ploidy levels was cultivar dependent.
Rathnam and Chollet (1980) observed that carbon dioxide fixation was higher in
colchicine-induced tetraploids of perennial ryegrass (Lolium) compared to the diploid
counterparts, and glycolate formation in the tetraploid was reduced.
Studies on specific biological compounds have also shown differences between diploids
and their isogenic tetraploid counterparts. Differences were found between tetraploids
and diploids for some non-terpenoid Citrus rind oil components (Cameron and Scora,
1968). Colchicine-induced tetraploids of Phlox drummondii produced several novel
glycoflavones not present in diploid counterparts (Levy, 1976), and conversely, some
glycoflavones present in the diploids were absent in the isogenic tetraploids. Similarly,
Haskell (1968) found both novel and absent flavonoids in colchicine-induced tetraploids
of raspberries (Rubus). Murray and Williams (1973) found that all colchicine-induced
tetraploids of Briza media produced the glycosylflavone orientin, rather than the similar
compound, vitexin, produced by the diploid progenitor. They also observed that esterase
activity in leaves of the tetraploid was four-fold that of the diploid, but there was no
difference in peroxidase activity. The observation that novel synthesis of orientin in
place of vitexin occurred in all the Briza tetraploids produced by Murray and Williams
(1973) suggests that doubling the gene dose altered the regulation of biosynthesis, rathe
than being the result of a novel gene.
Tal (1977) observed that leaf tissue of tetraploid tomatoes (Lycopersicon) had lower RNA
section 1.5.2
Introduction 1.16
and protein levels, per unit fresh weight and per unit DNA, than their isogenic diploid
counterparts.
1.5.2 Isozyme effects.
In its application as a method for phylogenetic investigation, isozyme analysis has been
used to determine the parental genotypes of disomic and heterozygous polyploids.
Numerous examples of this are cited in reviews by Carr and Johnson (1980), Gottlieb
(1982), and Crawford (1985), and specific cases are indicated by asterisk in the list at
beginning of the preceding section (pg. 1.9).
It seems logical to suggest that the presence of an identical duplicate genome should not
produce isozyme variants between somatic tetraploids and their diploid progenitors. The
absence of parental compounds in such tetraploids as cited earlier, may be the result of
altered gene dosage effects on biosynthesis. However, in some cases it is possible that
mutation may have occurred during polyploidization as a result of the mutagenic
properties of colchicine (Lapins, 1983), thus rendering some genes inoperative or
resulting in novel products. With strictly isogenic specimens, isozyme polymorphism
between ploidy levels could indicate (i) mutation, (ii) activation or deactivation of ge
as a result of the change in dose of some other regulatory gene(s), or (iii) production o
sufficient gene product in the tetraploid to elicit a visible reaction not detectable in
diploid zymogram, or vice versa. Conversely, gene dosage effects may be manifested
as isozyme band density differences (under carefully controlled conditions) between
isogenic euploids of differing ploidy. This project investigated such differences betwee
diploids and their nucellar tetraploid counterparts in Citrus and its hybrids.
section 1.5.2
Introduction 1.17
There are few examples in the literature of isozyme differences between diploids and
isogenic tetraploids. Button et al (1976) reported the absence of one weak peroxidase
band (of a total of 14 bands in the diploid) from each of three different nucellar te
'King' mandarins (Citrus). Yamashita (1977) found an additional minor peroxidase band
in nucellar tetraploid 'Natsudaidai' mandarin (Citrus). Different electrophoretic
mobilities in ribulose biphosphate carboxylase bands were reported by Garrett (1978),
between a diploid perennial ryegrass (Lolium) cultivar and its somatic tetraploid deri
by colchicine treatment of a diploid seedling. Nakai (1977) compared esterase and tota
protein electrophoreograms of colchicine-derived tetraploids with their diploid
counterparts, for numerous cultivars representing seventeen different species. Esteras
polymorphisms were detected in eight out of fifty-two accessions involving Brassica
pekinensis, Ipomoea triloba and Oryza saliva. This represented fifteen percent of the
plants investigated. Of twenty-four accessions studied, thirteen showed general protei
polymorphisms, representing fifty-four percent of the total. These occurred in Beta
vulgaris, Brassica pekinensis, Oryza sativa, O. glaberrina, Raphanus sativus, Triticum
monococcum and Vicia angustifolia. All plants in this work were raised from seed
derived from diploid and tetraploid parents, and each of these species is open pollina
The extent of polymorphism seems extraordinarily high and may be the result of
hybridisation rather than a ploidy effect per se. It is also possible that post-trans
modifications or different developmental stages of the diploid and tetraploid samples
account for some of the polymorphisms.
Nakai (1977) also evaluated electrophoretic band densities - esterase activity was sco
visually, and total protein was measured spectrophotometrically - in each of the fifty
section 1.5.2
Introduction 1.18
accessions. Where bands coincided (ie. Rf was similar between samples), various degre
of differences in band density between diploids and their tetraploid counterparts we
observed, indicating differential enzyme activity. In cabbage (Brassica), the tetrap
showed decreased esterase activity in most bands, however the usual observation acro
all other species was of increased esterase activity and protein in the tetraploids
to diploid counterparts.
In addition to the work of Nakai (1977), quantitative comparisons of isozymes betwee
isogenic tetraploid and diploid counterparts have been made with several other plant
species. Numerous studies of enzyme activity using densitometric (and biochemical)
methods under carefully controlled conditions are reviewed in Chapter 2. A high degr
of consistency in enzyme activity has been observed amongst plant tissue samples wit
the same gene dose. This has been the case for a variety of enzymes and plant specie
(Fobes, 1980; Birchler and Newton, 1981; Gottlieb and Higgins, 1984; Ferguson and
Grabe, 1986; Lumaret, 1986; Rosendahl et al, 1989). However, densitometric
differences have been reported between related species (Mitra and Bhatia, 1971).
DeMaggio and Lambrukos (1974) compared diploid and isogenic tetraploid ferns and
found various degrees of increased and decreased activity of eleven peroxidase subun
as a proportion of total peroxidase activity. They also found several novel peroxida
bands in the tetraploid. Total peroxidase activity per cell was slightly reduced in
tetraploids. Timko et al. (1980) found a similarly complex situation with esterase
subunits of castor bean (Ricinus). Twenty subunits showed variously increased and
decreased activity between isogenic haploids, diploids and tetraploids. Increases we
observed in total specific activity (ie. activity per unit total protein) of malate
section 1.5.2
Introduction 1.19
dehydrogenase, acid invertase, acid phosphatase, nitrate reductase and glutamate
dehydrogenase of tetraploid tomato (Lycopersicon) leaf tissue compared to their isog
diploids (Albuzio et al., 1978). Conversely, reduced activity was observed for ribul
biphosphate carboxylase/oxygenase, phosphoenol-pyruvate carboxylase, peroxidase and
glycolate oxidase. The degree of change differed for each enzyme. Levin et al (1979)
determined specific activity of alcohol dehydrogenase and activity per mg of sample,
Phlox drummondii seed from six diploid cultivars and their colchicine-induced tetrap
In each case alcohol dehydrogenase activity was higher in the tetraploid but the deg
of enhancement differed among cultivars.
Observations such as those above indicate a dramatic shift in the activity of enzyme
involved in metabolic processes, as a result of genome duplication. Although some of
these studies investigated specific activity of isozymes, most did not consider rela
gene expression per cell or per unit DNA. Because tetraploid cells are usually large
than diploid, the number of cells in a given sample size of tetraploid tissue is muc
smaller than in the corresponding diploid sample of the same size. Consequently the
amount of genomic DNA, and the amount of protein coded thereby, in a tetraploid tiss
sample will not be double that of a diploid tissue sample of the same size. To appre
the effect of gene dosage on gene expression, the quantity of DNA or number of cells
each sample must be taken into account.
The evidence supports the contention that chromosome doubling affects various enzyme
in different ways (Mac Key, 1987) leading to potentially unfavourable changes in the
regulation of metabolism (Albuzio et al, 1978; Levin, 1983). Mac Key (1987) points
section 1.6
Introduction 1.20
out that the activity of some enzymes depends upon dominance, and upon additivity in
others. Epistasis can also affect the activity of some enzymes (Strickberger, 1985).
Furthermore, activity of some enzymes is related to cell volume, and activity of othe
to areas of cell membranes. Accordingly, polyploidy could affect various enzyme
activities differently. As Stryer (1988) points out, although the major metabolic pa
have been elucidated, there is relatively little known about their regulation. Many
mechanisms of regulation occur. Important amongst these are the control of amounts of
enzymes and their activities (Stryer, 1988).
1.6 Project objective
There are few published reports of isozyme studies comparing Citrus diploids and
polyploids (Button et al, 1976; Spiegel-Roy et al, 1977), and none involving
quantification of enzyme activity or densitometry. Such a study would reveal much
information about the effects of gene dosage on expression of enzyme activity and cou
shed light on the cause of the observed vigour characteristics of Citrus nucellar
tetraploids. The study of differences between diploids and tetraploids in their expr
of a range of individual gene products may also give some indication of the degree of
complexity of the differences in metabolic activity between diploid and tetraploid
counterparts. The existence, or otherwise, of a consistent response in enzyme activit
across the range of enzymes, or consistency across a range of genotypes, would provid
information about the general effects of gene dose on gene expression.
The objective of this project was to investigate the effect of genome duplication in
section 1.6
Introduction 1.21
tetraploids, using Citrus and hybrids thereof. In particular, differences in gene
expression between diploids and their somatic tetraploid counterparts were studied, us
quantitative methods involving isozyme electrophoresis. Furthermore, elucidation of
relationships between quantitative gene expression and characteristics of reduced vigo
in Citrus tetraploids was sought.
To undertake such an investigation it was necessary to establish suitable laboratory
methods. The visualization of isozyme activity on electrophoretic media entails the
promotion of an enzymatic reaction in situ on the gel. If reliable quantification of th
isozyme activity was to be conducted, optimal sample handling and reaction conditions
had to be employed. It was necessary to make allowance for cell size differences
between diploids and tetraploids. This was achieved by measuring DNA concentration
of the samples and adjusting the quantitive isozyme data accordingly. Therefore,
appropriate methods of sample preparation, isozyme electrophoresis and quantification,
and DNA measurement were established and verified. The development of these
techniques is the subject of the following chapter. These methods were then applied in
pursuit of the above objective.
section 2.1
Methodology 2.1
Chapter 2
METHODOLOGY
2.1 Introduction
To address the objective delineated in Chapter 1, quantitative gene expression differ
between diploids and their somatic tetraploid counterparts was ascertained by measuri
isozyme activity using laser densitometry of electrophoresed isozyme bands. In order
account for differences in cell size between ploidy levels, the DNA content of each
sample was determined and isozyme activity was expressed in terms of the DNA
concentration. It was necessary to refine laboratory procedures and test their
appropriateness for the above applications. The particular cultivars and isozymes to
studied had to be selected and a suitable sample preparation method had to be chosen
validated. Also, growth studies had to be undertaken (see Chapter 4) to investigate t
relationship between gene expression and vigour.
To investigate differences in quantitative aspects of gene expression between Citrus
diploids and their somatic tetraploid counterparts, cellulose acetate gel electrophor
isozymes was employed. The darkness of the isozyme bands (i.e. their density) can be
measured with a densitometer. If there is a linear relationship between band density
isozyme band activity, then these techniques could be applied to assess the effect of
dosage on gene expression, using diploid and tetraploid Citrus cultivars. This is pos
section 2.1
Methodology 2.2
because, (i) isozymes are direct products of specific genes, and (ii) nucellar tetrapl
of Citrus are somatic and therefore possess a duplicated genome and are perfectly
isogenic with their diploid progenitors.
Unfortunately, quantification of isozyme activity is not straightforward. In consideri
quantitative aspects of gene expression all sampling units must be initially equivalen
All plants used in this study were grown under uniform glasshouse conditions. However,
fresh plant material may exhibit slight variability in moisture content between sample
and particularly between ploidy levels, as discussed in Chapter 1 (section 1.5.1). Thi
problem can be overcome by using dried plant material but the drying process must be
carried out in such a way that enzyme activity in the samples is not affected. This wa
achieved by lyophilization (freeze drying) of samples.
A problem concerning initial equivalence of sampling units arises when cell size is
considered. With the exception of rare instances involving disomic polyploids (Van Dij
and Van Delden, 1990), cells of tetraploids are usually much larger than cells of thei
diploid counterparts (refer Chapter 1, section 1.4)(see Fig. 2.8). Whereas the cell i
repository of the genome and the site of gene expression, sampling units must consist
equivalent numbers of cells for each sample, or some adjustment must be made in the
data collected to compensate for cell size differences. If not, comparisons of quantit
gene expression between ploidy levels cannot be validly made. This problem was
overcome by determining total DNA concentration of samples of uniform dry weight,
then expressing isozyme activity data in terms of DNA content.
section 2.1
Methodology 2.3
Another problem arises from isozyme polymorphism amongst the genotypes being
studied. Because isozyme electrophoresis reveals different allozymes from one genotype
to another, particular allozymes (individual isozyme bands) will be absent in some
cultivars. This situation makes it clearly impossible to compare such cultivars with
respect to that allozyme. Furthermore, even where a band of similar electrophoretic
mobility is detected in two or more cultivars, it cannot be assumed that they are the
product of identical allozymes (Coyne, 1981). Sequence variation between two alleles
may not result in different allozyme mobilities under a particular set of electrophoret
conditions, particularly where the different amino acids are of similar size and charge
(Richardson et al., 1986). In this study, diploid and tetraploid counterparts of the sa
progenitor genotype almost always revealed identical isozyme band mobilities indicating
that such plants are perfectly isogenic (a rare exception is discussed later). Therefor
it is possible to compare activity of particular allozymes between ploidy levels of
individual cultivars, but not between cultivars. Despite this constraint, it was possib
to compare cultivars by viewing the isozyme activity data holistically in a manner that
is described later.
Appropriate methodology for the electrophoresis, densitometry, lyophilisation and DNA
quantification procedures outlined above had to be developed and the validity of each
procedure for the particular application had to be confirmed. Consideration also had to
be given to the selection of appropriate cultivars and isozymes. These issues are
addressed in the following sections.
section 2.1.1
Methodology 2.4
2.1.1 Cultivar selection
Preliminary investigations to assess the various procedures to be employed were
conducted using leaf tissue samples from the lemon cultivar 'Eureka'. This cultivar was
selected because substantial quantities of suitable sample material were continuously
available. The cultivars used subsequently in this project were selected to cover a ran
of genetic types. Sixteen accessions were studied; diploid and tetraploid counterparts
eight different Citrus cultivars. The plant accessions used were:
accession ploidy notes
PA2X diploid accessions of the Parramatta cultivar of sweet orange;
PAH
tetraploid
used as both a rootstock and scion (also referred to as
P A in the text),
V02X2 diploid accessions of the Volkameriana lemon rootstock cultivar
V06
tetraploid
(also referred to as V O in the text),
SW2X2 diploid accessions of Swingle citrumelo rootstock cultivar; a
SW2
tetraploid
grapefruit x Poncirus trifoliata hybrid (referred to as
S W in the text)
TR02X diploid accessions of Troyer citrange rootstock cultivar; a hybrid
TR08
tetraploid
of orange and Poncirus trifoliata (referred to as T R )
MU2X diploid accessions of Murcott mandarin cultivar, (referred to as
MU1
tetraploid
MU)
CV2X1 diploid accessions of the mandarin rootstock cultivar Cleopatra
CV21
tetraploid
[Victoria strain], which is genetically distinct from other
mandarins (referred to as C V )
RL2X1 diploid accessions of Rough Lemon rootstock cultivar, which is
RL20
tetraploid
genetically distinct from other lemons (referred as R L )
BE2X diploid accessions of Benton citrange rootstock cultivar; a hybrid
BE14
tetraploid
of orange and Poncirus trifoliata (referred to as B E )
A full discussion on the origins of the accessions is presented in Appendix 4.
section 2.1.2
Methodology 2.5
2.1.2 Isozyme analysis procedures
Isozyme electrophoresis separates and reveals discrete molecular forms (allozymes) of
isozyme proteins as bands on the gel medium. A range of electrophoretic media are
available, with starch being most widely used in isozyme studies of Citrus and
poly aery lamide media also occasionally employed. Preliminary investigations (data n
shown) revealed that starch gels were not appropriate for use in isozyme analysis whe
relative enzyme dosage was to be evaluated by densitometry. The inconsistent optical
character of the starch medium and variation in gel thickness resulted in variability
optical density of the background. This introduced unacceptable 'noise' into the resu
of the densitometer scans of starch gels. Cellulose acetate and polyacrylamide media
provide a consistent background suitable for densitometry. More importantly, cellulos
acetate and polyacrylamide media permit precise control over the quantity of sample
loaded on the gel. Such precision is difficult to achieve with starch gels. Cellulose
acetate was the medium of choice for isozyme electrophoresis in this project. The eas
of preparation and fast running times of such gels is advantageous where large number
of tracks must be assessed.
Lo Schiavo et al (1980) resolved isozyme polymorphisms on cellulose acetate gels with
13 different types of plants for G6PD, 6PGD, MDH, ME, ADH, PGI, PGM and LDH
(lactate dehydrogenase)(refer Table 1.1 for key to isozyme names). The mechanics of
electrophoretic molecule separation on cellulose acetate gels differs from other medi
that no sieving occurs and migration rate is largely dependent upon charge density of
molecule species (Murphy et al, 1990). This does not usually pose a problem in
isozyme electrophoresis, as Richardson et al (1986) point out, because related allozy
section 2.1.3a)
Methodology 2.6
are generally very similar in size and shape, differing primarily in net charge.
Furthermore, separation characteristics are more significantly affected by the
electrophoretic conditions than the support medium (Richardson et al, 1986).
Consequently, the cellulose acetate medium was considered appropriate for this project.
2.1.3 Isozyme selection
a) Selection criteria
In selecting the specific isozymes to be incorporated in the project the following fact
were considered -
(i) only enzymes coded by nuclear DNA were used. This is the case for the vast
majority of enzymes, but supporting evidence had to be available. Only isozymes widely
reported in the genetics literature to demonstrate Mendelian segregation (indicating
nuclear origin) were used. Preferably, genetic information regarding Citrus should be
available.
(ii) satisfactory band density and resolution in each isozyme band had to be
achievable.
(iii) the staining reaction required to visualize the banding phenotype must not
involve linking enzymes. For some isozymes catalysis of the substrate cannot directly
lead to precipitation of a chromophore. Instead, an end-product of the primary reaction
section 2.1.3a)
Methodology 2.7
must cascade into a subsequent secondary reaction which produces the chromophore.
The catalysis of the secondary reaction requires the provision of another enzyme. The
is the possibility that, even if the linking enzymes were supplied in surfeit, the re
may be limited by factors other than the activity of the target enzyme. For example,
optimum pH of the primary reaction is often different from that of the secondary rea
(Vallejos, 1983). Approximately % of published isozyme protocols do not require linki
reactions. Suitable candidates are found in the Class 1 isozymes (oxidoreductases dehydrogenases), and the Class 3 isozymes (hydrolases).
(iv) the isozymes to be employed should resolve a limited number of bands and
overlapping of electromorphs should be minimized. Therefore, a maximum of 3
monomeric isozymes, 2 dimers, or 1 tetramer were preferred.
(v) the enzymes should be involved in a variety of major biochemical pathways,
thereby having the potential to effect significant impact on plant growth. It may be
argued that most enzymes perform important roles. However, if differential expression
is found between ploidy levels for enzymes involved in major pathways, differences i
plant growth are more easily implicated.
Examination of the literature indicated that caution in interpreting bands may be ne
because SkDH, in particular, and IDH and 6PGD to a lesser extent, are reported to be
prone to 'ghost' bands due to degradation products of primary allozymes (Kephart, 1990
section 2.1.3b)(i)
Methodology 2.8
b) Isozymes
Comparisons of banding patterns with those reported in the literature, showed that mos
isozymes were successfully separated under the cellulose acetate electrophoresis
conditions employed. With the exception of just one isozyme for one cultivar, the
tetraploid accessions always exhibited the same allozyme patterns as their diploid
counterparts. The banding patterns of the seven enzyme systems studied in detail are
presented below, schematically. Relative band widths, densities and locations are
accurately represented. A few enzymes, which satisfied the selection criteria, were
occasionally included in the methodological investigations but were not used subsequen
because satisfactory band resolution was difficult to achieve. The selected isozymes a
variously involved in metabolite synthesis and catabolism reactions, photosynthesis an
respiratory pathways. The functions and polymorphisms of the isozymes studied in detai
in this project are described below.
(i) Shikimate dehydrogenase - SkDH
E.C. 1.1.1.25
(= 3-Dehydroshikimate reductase)
SkDH catalyses the reversible reduction of 3-dehydroshikimate to shikimate in the
shikimate pathway which occurs in micro-organisms and plants. The pathway leads to
the production of chorismate from D-glucose. Chorismate is the precursor of the three
aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophane. In higher plants
the synthesis of many secondary metabolites relys on intermediates and end-products of
the shikimate pathway (Haslam, 1974). These secondary metabolites include most known
section 2.i.3b)(i)
Methodology 2.9
alkaloids and pigments. This pathway also provides the building blocks for the
biosynthesis of lignin and several vitamins. It is the principal one of only three ma
pathways of aromatic amino acid biosynthesis (Weiss and Edwards, 1980).
Cytosolic and chloroplastic forms of SkDH exist (Weeden and Gottlieb, 1980; Kephart,
1990). Weeden and Gottlieb (1980) demonstrated Mendelian segregation for the
chloroplast-specific SkDH allozymes in higher plants indicating coding by a nuclear g
They found two zones of activity in electrophoretic separations from higher plants, t
faster (anodal) being from the chloroplastic enzyme.
Moore and Castle (1988) identified one monomeric SkDH isozyme in Citrus. Table 2.1
summarises the Citrus literature pertaining to SkDH isozyme polymorphisms in the
cultivars in question. Similar tables are presented in the following sections on othe
isozymes. The orange cultivar Parramatta is grown only in Australia and is not
represented in the literature. However, there is general allozyme homology within
groups of closely related cultivars (Torres, 1983). Therefore, the information in the
below (and those in following sections) pertaining to oranges is applicable to Parram
Where numerous related cultivars within a group are known to possess common
allozymes, the number of such cultivars reported in the literature is indicated.
Superscripts indicate where different cultivars are reported to possess common allozy
Benton is not a widely grown cultivar and there are no reports of its isozyme
characteristics in the literature. The SkDH isozyme banding patterns observed under t
cellulose acetate electrophoresis conditions employed in this project are shown in Fi
2.1.
section 2.1.3b)(i)
Methodology 2.10
Table 2.1 Citrus S k D H isozyme references for selected cultivars
cultivar
orange
Volkameriana
Swingle
Troyer
Murcott
Cleopatra
Rough L e m o n
homozveous> heterozvgous
/
/a
/
/
/
/
/a
references
Ashari et al., 1989
Moore and Castle, 1988; Xiang and
Roose, 1988
Moore and Castle, 1988
Moore and Castle, 1988
Ashari et al., 1989
Moore and Castle, 1988
Moore and Castle, 1988
- indicates cultivars with reportedly identical allozymes
Fig 2.1 S k D H isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
Except for Murcott ( M U ) which was homozygous, the isozyme polymorphisms observed
in the experiments in this project, shown above, agree with the reports in the lit
with respect to their heterozygosity or homozygosity. However, Rough Lemon (RL) and
Volkameriana (VO) exhibited different genotypes. Parramatta (PA) and RL had
apparently similar but unique genotypes. Cleopatra (CV) and MU share the same
genotype, whereas VO, Swingle (SW), Troyer (TR) and Benton (BE) each possess unique
section 2.l.3b)(ii)
Methodology 2.11
SkDH genotypes. SW and TR exhibited four distinct bands suggesting two isozymes,
however the possibility of ghost bands must also be considered. The position of th
SkDH bands of the diploid of PA were consistently different from the tetraploid
(positions of the latter are indicated by '*' above). This conflicts with the expec
identical allozymes in the diploid and tetraploid accessions of a particular culti
to the somatic origin of the tetraploid (see section 1.5.2). The bands have been
nominally designated SkDH-a, SkDH-b, SkDH-c and SkDH-d in order from cathodal
(slow) to anodal (fast).
(ii) Isocitrate dehydrogenase (NADP*) - IDH
E.C. 1.1.1.42
IDH catalyses the conversion of isocitrate to a-ketoglutarate. The NAD-linked form
IDH is integral to the Kreb's cycle (Hall et al., 1981; Stryer, 1988) and is restri
the mitochondria (Smith, 1977; Hall et al, 1981). The NADP-linked form of IDH is
generally claimed to be cytosolic (Smith, 1977; Bohinski, 1983) although it has bee
isolated from mammalian mitochondria (Lai and Clark, 1979; Matlib et al., 1979). The
role of cytosolic NADP-linked IDH is not well understood (Hall et al, 1981) but th
a-ketoglutarate it generates is believed to be converted to glutamate for amino ac
synthesis (Bohinski, 1983). Isocitric acid from the Kreb's cycle moves out of
mitochondria to fuel this reaction; a process which may be associated with regulat
respiration.
The majority of IDH isozyme reports in higher plants, and all Citrus IDH reports, r
section 2.1.3b)(ii)
Methodology 2.12
to NADP-linked IDH. Using a gentle extraction technique followed by starch gel
electrophoresis, Torres et al (1982) could not detect the NAD-linked form in Citru
Mendelian segregation has been demonstrated for Citrus NADP-linked IDH (Torres et
al, 1982, 1985) indicating nuclear coding. A single dimeric isozyme has been repo
for a wide range of Citrus cultivars (Torres et al, 1985; Ashari et al., 1989). Ta
summarises the IDH literature for the cultivars in question.
Rough Lemon possess a different electromorph from other lemons (Soost and Torres,
1981; Torres etal, 1982; Torres, 1983; Ashari etal, 1988).
Table 2.2 Citrus I D H isozyme references for selected cultivars
cultivar homozygous heterozygous references
orange
/ (>24)* Soost and Torres, 1981; Torres et
al, 1982; Torres, 1983; Ashari
et al., 1988; Roose and Traugh,
1988
/(>15)* Xiang and Roose, 1988
Volkameriana
Swingle
/
Roose and Traugh, 1988; Xiang
and Roose, 1988
Troyer
/
Ashari et al., 1988; Roose and
Traugh, 1988
Murcott
/
Ashari et al, 1989
Cleopatra
/
Ashari et al., 1988; Roose and
Traugh, 1988
Rough L e m o n
/
Roose and Traugh, 1988; Xiang
and Roose, 1988
* - indicates the number of closely related cultivars possessing a c o m m o n allozyme composition
Fig 2.2 I D H isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
The IDH(NADP+) isozyme band patterns obtained in this project (Fig. 2.2) are generally
in agreement with the reports in the literature, except for Rough Lemon (RL) and
Swingle citrumelo (SW). Both of these cultivars revealed only a single major band. It
is possible that the cultivar strains used here differed from those in the literature,
cellulose acetate electrophoresis was incapable of separating the different allozymes.
faint anodal (fast) band, not reported in the literature, was consistently resolved. T
could have been a ghost band but its consistency, and the fact that the band position f
the two citranges, Troyer and Benton, differed from other cultivars, suggest that it is
real isozyme. The main bands were designated IDH-s (slow), IDH-m (medium), and
IDH-f (fast), and the fast faint band was nominally designated IDH-a. Although
homozygous, MU and CV band locations are referred to as M (medium) in the literature,
whereas this location otherwise relates to the middle band of heterozygous loci. This
convention was also used here. Bands present but not clearly resolved are indicated by
'N', and were not included in the quantitative studies reported later.
section 2.l.3b)(iii)
Methodology 2.14
(iii) Cytosol aminopeptidase - CAP
E.C. 3.4.11.1
(= Leucine aminopeptidase - LAP;
Cytosol aminopeptidase is the IUBNC preferred name (Webb, 1984))
CAP is a neutral aminopeptidase - one of a diverse enzyme group with varying affinit
for different amino acid substrates and different pH optima. CAP hydrolyzes dipeptid
liberating the N-terminal amino acids and so is active against a range of polypeptid
(Mikola and Mikola, 1986). It participates in regulating plant growth, development a
senescence through degradation of proteins to small peptides and amino acids for rel
ation to sites for biosynthesis of new compounds (Kaur-Sawlaney and Galston, 1986).
L-leucyl-jS-naphthylamide has been used as the substrate in reports of Citrus CAP iso
staining. Torres et al. (1982, 1985) demonstrated Mendelian segregation for Citrus C
and a single monomeric isozyme has been reported in numerous publications for a wide
range of Citrus cultivars. The CAP literature for the cultivars in question is summar
in Table 2.3.
In the work reported later, resolution of CAP was disappointing but band density was
good. The isozyme band patterns (Fig. 2.3) are in agreement with those reported in t
literature. The three cultivars with Poncirus trifoliata parentage (SW, TR and BE) h
allozymes distinct from those of the other cultivars. The narrow middle band in TR m
be artifactual, as may one or both of those in PA. The bands here are nominally
designated CAP-a ... CAP-c from cathodal (slow) to anodal (fast).
section 2.1.3b)(iv)
Methodology 2.15
Table 2.3 Citrus C A P isozyme references for selected cultivars
cultivar
references
orange
Volkameriana
Swingle
Troyeret al., 1988
Ashari
Murcott
Cleopatra
Rough L e m o n
homozvgous heterozvgous
/(>24)*
/(>24)* Torres etal, 1982; Torres, 1983;
Ashari et al. 1988, 1989
/
Xiang and Roose, 1988
/
/
/
•
Ashari et al., 1989
Ashari et al., 1988
Torres et al., 1982; Torres, 1983
* - indicates the number of closely related cultivars possessing a common allozyme composition
Fig 2.3 C A P isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
(iv) Glucose-6-phosphate dehydrogenase - G 6 P D
E.C. 1.1.1.49
The oxidative pentose phosphate pathway results in the production of N A D P H for
reductive biosynthesis reactions. It also produces ribose 5-phosphate which is ess
in the synthesis of many important biomolecules (Stryer, 1988) primarily nucleic a
and erythrose, a precursor to shikimic acid (Hall et al., 1981). G6PD catalyses the
section 2.l.3b)(iv)
Methodology 2.16
step in the pentose phosphate pathway by the dehydrogenation of glucose-6-phosphate to
6-phosphoglucono-lactone (Hall et al, 1981; Stryer, 1988).
Both cytosolic and chloroplastic isozymes of G6PD have been reported (Schnarrenberger
et al., 1973; Herbert et al., 1979; Levy, 1979). The chloroplastic enzyme is coded by
a nuclear gene (Weeden, 1981). In pea plants Anderson et al. (1974) found two G6PD
enzymes. These produce two electrophoretic bands - the faster (anodal) being the
cytosolic form, and the slower, chloroplastic. Conversely, with spinach leaf tissue,
Schnarrenberger et al (1973) showed that the slower (cathodal) isozyme was cytosolic
and the faster was chloroplastic. Both authors showed that the two isozymes were of
very similar molecular weights but differed in charge. The relative electrophoretic
mobilities of the cytosolic and chloroplastic isozymes may need to be evaluated for an
particular species (Herbert et al, 1979).
Fig 2.4 G 6 P D isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
There are no publications with regard to Citrus for this enzyme. In other plants two
dimeric isozymes are reported (Weeden and Wendel, 1989; Kephart, 1990). The banding
section 2.1.3b)(v)
Methodology 2.17
patterns obtained in this project suggest two isozymes (Fig. 2.4). The bands are
nominally designated G6PD-a ... G6PD-C from cathodal (slow) to anodal (fast).
(v) 6-Phosphogluconate dehydrogenase - 6PGD
E.C. 1.1.1.44
The NADP-linked decarboxylating form of 6PGD catalyses the third reaction of the
oxidative pentose phosphate pathway - the conversion of 6-phosphogluconate to ribu
5-phosphate (Stryer, 1988; Hall et al, 1981). Accordingly, its function is closely
associated with that of G6PD.
Two isozymes of 6PGD, one cytosolic, the other chloroplastic have been reported
(Schnarrenberger et al, 1973; Herbert et al, 1979). Schnarrenberger et al (1973)
showed that the two isozymes were of nearly identical molecular weight, but
electrophoretic separation suggested charge difference. The chloroplastic isozyme
migrates faster (anodal) in spinach (Schnarrenberger et al, 1973) and in Brassica
(Quiros, 1987). Weeden (1981) reports the chloroplastic form is coded by a nuclear
gene.
In higher plants the two isozymes are reportedly dimeric (Weeden and Wendel, 1989;
Kephart, 1990). This is possibly the case in Citrus (Hirai and Kozaki, 1981). Ashari
et al (1989) reported a single homozygous locus in sweet orange and a heterozygous
locus in Murcott mandarin. Complex isozyme band patterns (Fig. 2.5) were obtained i
the work presented later. All cultivars except the two mandarins (MU and CV) exhibi
section 2.l.3b)(vi)
Methodology 2.18
unique patterns, which were repeatable. However, interpretation of the band patterns
no bearing on the quantitative studies central to this project. The bands were nomin
designated 6PGD-a ... 6PGD-e from cathodal (slow) to anodal (fast).
Fig 2.5 6 P G D isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
(vi) Malate dehydrogenase (NAD) - MDH
E.C. 1.1.1.37
Four different MDH enzymes are recognized. Three of them (E.C. 1.1.1.38, E.C.
1.1.1.39 and E.C. 1.1.1.40) catalyse the decarboxylation of malate to pyruvate. The
three were all previously referred to as malic enzyme; the IUBNC preferred nomencla
is malate dehydrogenase (decarboxylating)(Webb, 1984). E.C. 1.1.1.40, which has been
widely referred to in isozyme analysis as malic enzyme, requires NADP as a coenzyme.
The other two utilize NAD.
The fourth isozyme, NAD-dependent malate deyhdrogenase E.C. 1.1.1.37, catalyses the
section 2.1.3b)(vi)
Methodology 2.19
reversible oxidation of malate to oxaloacetate. This reaction is involved in a nu
essential biochemical reactions occurring in the cytosol, mitochondria and peroxis
of leaves (Tolbert, 1981). Mitchondrial MDH(NAD) catalyses the final step in the
Kreb's cycle (Hall et al, 1981). Cytosolic MDH(NAD) takes part in the
malate/oxaloacetate shuttle whereby NADH and oxaloacetate are transported into
chloroplasts (Hall et al, 1981). Cytosolic MDH(NAD) also supports a malate shuttle
to the peroxisomes where peroxisomal MDH(NAD) supplies NAD(H) to the glycerate
pathway (Tolbert, 1981). The glyoxylate pathway utilizes MDH(NAD) in the
glyoxysomes of germinating seeds (Hall et al, 1981; Tolbert, 1981) but does not o
in leaf tissue.
Boulter and Laycock (1966) found Vicia chloroplast and mitochondrial MDH(NAD)
isozymes occupied the same band location on aery lamide gels. Many minor bands wer
revealed, most of which were shown to be complexes of mitochondrial MDH with othe
proteins. In Brassica species, Quiros (1987) showed that the fastest (anodal) isoz
of plastid origin.
MDH is dimeric and has been reported for a wide range of Citrus cultivars (Hirai
Kozaki, 1981; Ashari etal, 1989; Ben-Hayyim etal, 1982; Hirai etal, 1986; Xiang
and Roose, 1988). Three regions of MDH(NAD) activity have been identified (Torres
et al, 1982), the middle one of which shows two overlapping isozymes of similar
mobility and considerable variability (Torres et al, 1982; Soost and Torres, 1981).
These two Citrus MDH(NAD) loci have been widely reported (Ashari et al, 1988;
Moore and Castle, 1988; Roose and Traugh, 1988; Torres, 1983; Torres et al, 1985).
section 2.1.3b)(vi)
Methodology 2.20
The slower moving (cathodal) isozyme is designated MDH-1, and the faster MDH-2
(Torres et al, 1982). Torres et al. (1982) demonstrated Mendelian segregation for
MDH-1 and MDH-2 in Citrus, indicating nuclear coding. Table 2.4 summarises the
MDH-1 and MDH-2 literature for the cultivars in question.
The overlapping isozymes MDH-1 and MDH-2 (Fig 2.6) were also apparent in the work
reported later in this project. Two further zones were also detected, a slow (catho
zone generally consisting of a single band, but two close bands in the citranges (T
BE), and a very fast, faint pair of bands. The observed allozymes of MDH-1 and
MDH-2 are in agreement with the literature except for the trifoliates Troyer (TR)
Swingle (SW) which were heterozygous for MDH-2 as in the literature, but apparentl
homozygous for MDH-1. It may be that some bands have similar mobility on the
cellulose acetate gels and therefore are not discernible, giving the false appearan
homozygosity.
The two main isozymes are designated MDH-1 and MDH-2 in accordance with
convention in the literature, and their individual bands are designated s (slow),
(medium), and f (fast). MDH-If and MDH-2m occupy the same location but their
presence or absence are determined by inference from the specific allozyme patterns
each cultivar. The very slow zone bands (cathodal) are nominally designated MDH-a
MDH-b, and the fast zone bands (anodal), MDH-c and MDH-d.
section 2.1.3b)(vi)
Methodology 2.21
Table 2.4
Citrus M D H isozyme references for selected cultivars
MDH-1
cultivar
orange
Volkameriana
Swingle
Troyer
Murcott
Cleopatra
Rough Lemon
cultivar
orange
references
homozygous heterozygous
/ (>24)*
Hirai et al., 1986; Ashari et al,
1988, 1989; Roose and Traugh,
1988; Soost and Torres, 1981;
Torres, 1983; Torres etal, 1982
Moore and Castle, 1988; Xiang and
Roose, 1988
Moore and Castle, 1988; Roose and
Traugh, 1988
/
Ashari et al., 1988; Moore and
Castle, 1988; Roose and Traugh,
1988
Ashari et al., 1989
/
/
Hirai et al., 1986; Ashari et al.,
1988; Moore and Castle, 1988;
Roose and Traugh, 1988
Soost and Torres, 1981; Torres,
1983; Hirai et al, 1986; Ashari
et al., 1988; Moore and Castle,
1988
MDH-2
homozygous heterozygous
/ (>24)*
Volkameriana
Swingle
Troyer
/
Murcott
Cleopatra
/
Rough Lemon
/
/
references
Hirai et al, 1986; Ashari et al,
1988, 1989; Roose and Traugh,
1988; Soost and Torres, 1981;
Torres, 1983; Torres etal, 1982
Moore and Castle, 1988
Moore and Castle, 1988; Roose and
Traugh, 1988
Ashari et al, 1988; Moore and
Castle, 1988; Roose and Traugh,
1988
Ashari et al, 1989
Ashari et al, 1988; Moore and
Castle, 1988
Soost and Torres, 1981; Torres,
1983; Ashari etal, 1988
* - indicates the number of closely related cultivars possessing a c o m m o n allozyme composition
- indicates cultivars with reportedly identical allozymes
a
section 2.l.3b)(vii)
Methodology 2.22
Fig 2.6 M D H isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
(vii) Ribulose 1,5-biphosphate carboxylase - R B C
E.C. 4.1.1.39
(= RuBISCO)
The initial step in the Calvin cycle, whereby C02 is fixed during the dark reactions o
photosynthesis, is catalysed by RBC. C02 condenses with ribulose 1,5-biphosphate and
the resultant 6-carbon compound is immediately hydrolysed by RBC to yield two
molecules of 3-phosphoglycerate. When C02 concentration is relatively low RBC also
behaves as an oxygenase, yielding one molecule each of phosphoglycolate and
3-phosphoglycerate (Raven et al., 1986; Stryer, 1988). The former product is converte
to glycolic acid, the substrate for photorespiration. Hence, the same enzyme catalyse
major reactions in opposing metabolic pathways - the only known instance of such a
phenomenon (Miziorko and Lorimer, 1983). The regulation of RBC activity is a
section 2.l.3b)(vii)
Methodology 2.23
complex process influenced by capacity of RBC to exist in multiple forms, the inhibit
effects of sugar phosphates, modulation by the enzyme RuBISCO activase, the ratio of
ATP to ADP, light and temperature levels, and possibly other factors not yet elucidat
(Portis, 1992).
The RBC enzyme is comprised of eight large subunits and eight small subunits, the
former coded by chloroplastic DNA. The small subunit is coded by nuclear DNA
(Spreitzer, 1993). Heterogeneity has been demonstrated in the small subunit (Miziorko
and Lorimer, 1983; Daday and Whitecross, 1985; Daday et al, 1986), but the large
subunit is structurally highly conserved (Miziorko and Lorimer, 1983; Daday et al,
1986). Using isoelectric focusing, Garrett (1978) revealed two different isozymes of
RBC in ryegrass, one present in diploids, the other in tetraploids. Although there is
evidence that such variants may result from modification of a single gene product
(Miziorko and Lorimer, 1983), no differences were observed in the present study betwee
diploids and tetraploids of the same cultivar. Daday et al (1987) showed that, in
Medicago sativa, RBC levels are under the control of a nuclear gene and present evide
that it is the small subunit gene that is responsible.
RBC is the most abundant soluble protein in leaf tissues (Miziorko and Lorimer, 1983).
Its abundance is the key to its visualization in electrophoresis as the stain used to
it is a non-specific protein stain (Wendel and Weeden, 1989).
Handa et al. (1986) undertook detailed studies on RBC in Citrus. Using isoelectric
focusing of leaf tissue from 57 accessions of Citrus and related genera, they demonst
section 2.1.3b)(vii)
Methodology 2.24
the conserved nature of the large subunit by showing that all accessions, except the
distinct species Citrus medica (citron - 3 accessions), possessed the same allozyme.
Conversely, considerable heterogeneity was found amongst small subunit allozymes,
which displayed 5 different bands, singly or variously paired (nominally designated a
e by the authors, in order from cathodal (slow) to anodal (fast)). The 3 lemon scion
cultivars studied all possessed the same single band, c, but Rough Lemon displayed tw
bands, c and d. All 6 orange cultivars also possessed the c,d electromorph, as did the
scion mandarins similar to Murcott. Cleopatra mandarin displayed band d only. No
citranges or citrumelos were studied, but Poncirus trifoliata displayed a single and
band, a, which would presumably also be present in the hybrids of P. trifoliata.
Handa et al. (1986) found that the chloroplastic, large subunit of RBC migrated faste
(anodally) than the small subunit. This was also demonstrated in this project (see se
2.2.1a) later). The band patterns shown in Fig. 2.7 represent the small subunit only
use the band nomenclature of Handa et al. (1986). The band e occurred only in Citrus
medica (Handa et al. 1986), which was not involved in the work reported here.
Similarly, band b occurred only in a single, obscure species. Band a, present only in
Poncirus trifoliata, should possibly have occurred in the hybrids, Swingle, Troyer an
Benton. A poorly resolved band, slightly faster (anodal) than the others was observed
in these cultivars, but resolution was poor and density measurements were not recorde
from it. Conversely, however, band a of Handa et al. (1986) was slow (cathodal).
Assuming the two bands observed here coincide with the slower and faster bands, c and
d, respectively, of Handa et al. (1986), the observations are in agreement for RL, PA,
MU and CV. However, the VO band observed here differed from that of other lemons
section 2.1.4
Methodology 2.25
studied by Handa et al. (1986).
PA
VO
SW
TR
MU
CV
RL
BE
RBC
Fig 2.7 RBC isozyme band patterns of eight Citrus cultivars
electrophoresed on cellulose acetate gels
2.1.4 Effects of sample lyophilisation
Differences in moisture content between diploids and tetraploids have been reported in
the literature (Chapter 1, section 1:5.1). For comparisons of isozyme activity to be vali
initially equivalent sample weights must be uniform. Variability in moisture content
between samples would therefore present a problem. A preliminary study conducted to
determine whether this was the case in Citrus, revealed that the mean dry matter content
of five different diploid and tetraploid leaf samples were all significantly different
(P<0.01)(data not shown). Accordingly, there was clearly a need to use dried plant
tissue.
To ensure minimal impairment of isozyme activity during sample drying, the fast, heat-
free method of lyophilization offered the best option in this project. The process causes
water to sublime directly from the frozen sample without thawing. Therefore, it was
section 2.1.4a)
Methodology 2.26
necessary to ascertain the extent, if any, of loss of isozyme activity due to the
lyophilization procedure employed.
a) Effects of lyophilisation on proteins
Drying of plant tissue by lyophilization has been shown to result in loss of various l
and medium molecular weight compounds (Van Sumere et al, 1983). However, little
research has been conducted on the effects of lyophilization on enzymes in whole tissu
Fujita et al. (1987) observed 50-70% loss of activity of certain enzymes when whole
Drosophila flies were rapidly frozen in very cold acetone (<—90°C) prior to
lyophilization. However, Tarczynski and Outlaw (1987) found that by handling
lyophilized plant tissue under low humidity, they avoided protein degradation by
endogenous proteases.
The majority of studies on the effects of lyophilization on proteins have been conduct
with more or less purified proteins or mixtures of proteins. In native proteins, water
molecules are bound to amino acid residues by hydrogen bonds. By removing water
molecules, lyophilization alters hydrogen bonding characteristics of proteins resultin
structural changes and denaturation (Hanafusa, 1985; Matsuda, 1985). Such changes are
reversible if the protein is returned to a similar native environment (Matsuda, 1985).
Studies of the effects of lyophilization on the structure of proteins, including enzym
have shown varying results. Dissociation of the catalase tetramer into monomer subunit
during lyophilization has been a consistent observation (Tanford and Lovrien, 1962;
Deisseroth and Dounce, 1967, 1969; Hanafusa, 1969; Sichak and Dounce, 1987).
section 2.1.4b)
Methodology 2.27
Lyophilization caused no degradation or alteration in isoelectric points of the protei
rattlesnake venom (Egen and Russell, 1984), although the authors concede that undetecte
changes could have occurred in tertiary structure. Thirion et al. (1983) showed that
lyophilization resulted in only minor changes in the secondary and tertiary structure
haemoglobin, and no detectable alteration to the quaternary structure. Hoshi and
Yamauchi (1983) showed that lyophilization of US globulin resulted in partial
dissociation of the hexamer, a decrease in sulfhydryl content and an increase in disul
bonds. Some degree of degradation following lyophilization has also been observed in
lysozyme (Yu and Jo, 1973), and myosin (Yasui and Hashimoto, 1966; Hanafusa, 1969).
Conversely, alcohol dehydrogenase self-associated upon lyophilization to form large
polymers (Ross et al, 1979).
b) Effects of lyophilisation on isozyme activity
Little work has been undertaken to investigate the effects of lyophilization on isozym
activities. Sichak and Dounce (1987) observed conformational alteration of both the
monomer product of catalase lyophilization, and the undissociated tetramer. Moreover,
they found that, after rehydration in buffer, the monomer retained most of its catalyti
and peroxidatic activity, and the tetramer exhibited enhanced activity. Hellman et al.
(1983) observed that lyophilization of L-asparaginase caused dissociation of the enzym
tetramer into monomer subunits which completely reassociated and regained full activit
upon rehydration in near neutral buffers of low ionic strength. However, Marlborough
et al (1975) did not achieve full recovery of L-asparaginase activity under such
conditions.
section 2.1.5
Methodology 2.28
Carbonic anhydrase was shown to undergo denaturation upon lyophilization but regained
full activity when redissolved in aqueous buffers (de Jesus et al, 1979). Yasui and
Hashimoto (1966) found that ATPase activity of myosin resuspended after lyophilization
ranged from 12% to 86% of initial activity, depending upon the ionic strength of the
initial buffer containing the protein and was improved by the inclusion of sucrose in t
solution. Similarly, Hanafusa (1969) observed reduced ATPase activity of myosin
(between 30% to 92% of initial activity), dependent upon the contents of the reconstitution buffer.
In this study, several different experiments were undertaken to determine whether
lyophilization would alter isozyme activity in Citrus. Experiments were conducted
entailing a number of isozymes, and a representative example is presented in the Method
section following (section 2.2.2). It was shown that lyophilisation caused no adverse
effect on isozyme activity.
2.1.5 Densitometry
Isozyme activity was measured in this project by laser densitometry of electrophoresed
bands. This procedure was subjected to rigorous assessment (see section 2.2.3). The
bands are the result of chromophores (dyes) precipitated on the gel as a product of
isozyme-catalysed reactions (Vallejos, 1983; Richardson et al, 1986). If a surfeit of
substrates and other necessary reagents is available, the concentration of chromophore
a band will be a function of enzyme activity (Rosalki and Foo, 1984; Ros Barcelo, 1987)
section 2.1.5
Methodology 2.29
Catalysis, and the resultant chromophore precipitation, procedes only while an enzyme
is active - the greater the activity, the more chromophore is precipited. As discussed
the beginning of this chapter, for densitometry to provide a valid method of studying
quantitative aspects of gene expression, a linear relationship must be shown to exist
between measured band density and isozyme activity. Assessment of the density of
electrophoretic bands has been widely applied to quantify enzymes and other proteins.
At the simplest level, this can be achieved by assigning visual rating systems to band
density (Smith and Conklin, 1975; Chyi and Weeden, 1984; Sidhu et al, 1984).
Detailed discussions on the densitometric characteristics of general protein stains app
to electrophoretic gels are presented by Cornell (1989), and Neuhoff et al. (1990). No
such information is available regarding stains produced by in situ enzymatic reactions
employed to visualize isozymes. Nonetheless, as shown in Table 2.5, densitometric
instruments have been used to quantify numerous isozymes and other proteins in gel
electrophoresis studies. All such studies assume of a direct relationship between band
density and protein concentration or enzyme activity. Because they degrade quickly,
activity of a given isozyme is synonymous with its concentration - the more enzyme
present the more substrate that can be catalysed before degradation occurs, and hence t
more chromophore precipitated. A direct relationship between band density and isozyme
activity forms the foundation of the main technique employed in this project.
Accordingly, I have paid particular attention below to reviewing publications in which
similar techniques were used. In the Methods section following (section 2.2.3) I report
on experiments performed to validate the procedure employed in this project.
Methodology 2.30
section 2.1.5
Table 2.5
Examples of studies entailing measurement of isozyme activity or protein
concentrations using densitometric quantification of electrophoretic bands
enzyme/protein reference
acid phosphatase
'Fobes (1980)
alcohol dehydrogenase *Mitra and Bhatia (1971)
alkaline phosphatase Burlina and Galzigna (1976); Rosalki and Foo (1984, 1989); Secchiero
et al. (1989); Steinberg and Rogers (1987)
aspartate aminotransferase Rosendahl et al. (1989)
esterase *Fobes (1980); *Mitra and Bhatia (1971); Rosendahl et al. (1989);
*Timko et al. (1980)
glutamate dehydrogenase *Mitra and Bhatia (1971)
isocitrate dehydrogenase *Mitra and Bhatia (1971)
lactate dehydrogenase Triveni and Rao (1986); Kim and Yum (1985); Yum and Kim (1989)
malate dehydrogenase 'Danzmann and Bogart (1982); *Mitra and Bhatia (1971)
peptidase Rosendahl et al. (1989)
peroxidase "DeMaggio and Lambrukos (1974); *Fobes (1980); Ros Barcelo (1987)
6-phosphogluconate Gasperi et al. (1983)
dehydrogenase
phosphoglucose isomerase 'Gottlieb and Higgins (1984); Linde et al. (1990); 'Lumaret (1986)
ribulose biphosphate carboxylase Tarczynski and Outlaw (1987, 1989)
superoxide dismutase Sevilla et al. (1984, 1988)
other proteins Autran and Galterio (1989ab); Bauer et al. (1985); Csiba and
Szecsenyi-Nagy (1989); Dannenberg and Kessler (1986, 1988); Graml
et al. (1989); Hillier (1976); Honeycutt et al. (1989); Ramos et al.
(1985); Sato et al. (1986); Sullivan and Johnson (1989); Tichy
(1985)
general protein Autran and Galterio (1989ab); "Birchler and Newton (1981); Bauer et
al. (1985); Csibaand Szecsenyi-Nagy (1989); DeMaggio and Lambrukos
(1974); Ferguson and Grabe (1986); Honeycutt et al. (1989); Kusama
et al. (1984); Mansur-Vergara et al. (1984); Neuhoff et al. (1990);
Ohmori et al. (1985); Romagnolo et al. (1990); *Timko et al. (1980);
Zeineh & Kyriakidis (1986)
* - asterisks indicate gene dosage studies with polyploids or polysomics.
section 2.1.5a)
Methodology 2.31
a) Comparisons of densitometry with other quantitative methods
The densitometric method has been proven reliable through studies, with a variety of
enzymes and proteins, entailing quantification by chemical assays and subsequent
comparison with results from densitometric quantifications (Table 2.6). Similarly, Mit
and Bhatia (1971) measured isozyme band densities for alcohol dehydrogenase, malate
dehydrogenase and glutamate dehydrogenase from Triticum species of four different
ploidy levels. Activity of the three enzymes for each of the four species was also
determined by spectrophotometric methods. With both methods, increasing activity with
higher ploidy level was observed for all three enzymes.
Table 2.6
Correlations between electrophoretic band densities and concentration of enzymes and
proteins quantified by various means.
enzvme/protein
alkaline phosphatase
method of
quantification
sequential heat-inactivatioon
correlation with
electrophoretic
band densitv
reference
r=0.98
Rosalki & Foo (1984)
peroxidase
benzidine assay
r>0.99
Ros Barcelo (1987)
carbonic anhydrase
pure enzyme sample cone.
r>0.99
lipoprotein
cholesterol assay
r=0.99
Tarczynski & Outlaw
(1987)
Gambert et al. (1988)
monoclonal I g M
radial immunodiffusion
laser nephelometry
sedimentation analysis
radial immunodiffusion
laser nephelometry
radial immunodiffusion
laser nephelometry
r=0.854
r=0.841
r=0.954
r=0.786
r=0.718
r=0.924
r=0.419
Tichy (1985)
refractometry
biuret method
B C G method - autoanalyser
calculated
r>0.99
r>0.99
r>0.99
r=0.99
Bauer et al. (1985)
M
n
monoclonal IgG
n
monoclonal IgA
n
total proteins
n
serum albumin
globulins
ii
ii
n
n
it
"
it
it
n
section 2.1.5b)
Methodology 2.32
b) Different approaches to densitometry
Various approaches have been applied to the quantification of proteins by densitometry
of electrophoretic bands. The most conservative approach taken to densitometric
quantification of bands on electrophoretic gels is the comparison of bands within a si
track, ie. from the same sample. This method overcomes the need for stringent control
over sample size. The method has been employed with general protein stains where,
within each track there are numerous bands available for calculation of density ratios
It has been used to characterize cultivars of wheat (Autran and Galterio, 1989a), peren
ryegrass (Ferguson and Grabe, 1986), soybeans (Honeycutt et al, 1989) and rice
(Kusama, 1984). However, the most common use of densitometry of electrophoresed
proteins entails direct comparison between samples in adjacent tracks within a single
This approach has been employed to differentiate between genotypes or species (eg. Mit
and Bhatia, 1971; Rosendahl et al, 1989; Linde et al, 1990), and between different
tissues (eg. Triveni and Rao, 1986; Rosalki and Foo, 1989; Secchiero et al, 1989).
Experiments reported in the Methods following (section 2.2.3) showed that comparisons
between tracks within a gel, but not between gels, was a valid procedure under the
conditions employed in this project.
(i) Internal standards. If a linear relationship can be demonstrated between band dens
and the concentration of a purified reference protein, a precise aliquot of that prote
be included on each gel as an internal standard to facilitate absolute densitometric
quantification of unknown samples. This approach has been used in the determination
of concentrations of various animal proteins (Hillier, 1976; Bauer et al, 1985; Ramos
et al, 1985; Sullivan and Johnson, 1989). Sato et al. (1986) used a variant of this
section 2.1.5c)
Methodology 2.33
approach by using lysozyme as an internal standard to quantify soybean globulins.
(ii) Subfraction apportionment. Another technique involves estimation of the total
concentration of a protein in question using biochemical assays, then applying
densitometry to the electrophoresed subfractions. The relative concentrations of each
subfraction are determined by comparing band densities, and each is quantified by
reference to the concentration of the total protein. This method has been used to measu
various proteins and isozymes in human tissues (Rosalki and Foo, 1984, 1989; Tichy,
1985; Gambert et al, 1988), and peroxidase isozymes in plants (Ros Barcelo, 1987).
(iii) Comparative densitometry. Another approach, comparative densitometry of
electrophoresed isozyme bands, has proven to be a useful technique for investigating
dosage effects of duplicated identical gene loci in polyploids and polysomics, as descr
in Chapter 1. Isozymes studied by this method are listed in Table 2.5 (indicated by
asterisks). This procedure entails comparing band densities of adjacent isozyme tracks
of samples of different ploidy levels. If the samples are quantitatively equivalent (se
below) the band densities of the different ploidy levels can be compared directly, ie.
absolute quantification of the isozyme activity of each sample is unnecessary. This
approach was applied in later investigations in this project.
c) Linearity of band density response
For densitometry to be valid, it was necessary to determine the range of sample
concentrations within which isozyme band density showed a linear response to isozyme
activity (see section 2.2.3 in Methods). The relationship between enzyme activity and
section 2.1.5c)
Methodology 2.34
the density of its electrophoresed bands should be assessed for each enzyme individuall
Differing response slopes for the relationship between band densities and protein
concentrations were observed for various proteins (Hillier, 1976; Ramos et al, 1985).
Linearity of response slopes and low variability amongst repeated assays of each enzyme
will indicate reliability of the method. Dilution series experiments have shown linear
responses for several proteins including the enzymes alcohol dehydrogenase (Mitra and
Bhatia, 1971), carbonic anhydrase (Tarczynski and Outlaw, 1987), peroxidases (Ros
Barcelo, 1987), alkaline phosphatase (Rosalki and Foo, 1989) and neutral endopeptidase
(Sullivan and Johnson, 1989).
(i) Peak height vs. curve area. Determinations of band density from densitometric scans
can be derived in two ways, (a) the peak height of the curve, and (b) the area under the
curve. The majority of studies utilize curve area. This approach poses a problem which
is usually overlooked. Rarely are adjacent bands on gels clearly separated and the tail
of the resultant densitometer curves are fused. Sophisticated computer software, such a
LKB Gelscan XL, which can integrate the curve data to determine the putative individual
curve areas, are laborious to use. Often however, merged band curves are simply
separated by a vertical line coinciding with the trough between the peaks, hence
truncating one tail of each curve. Areas determined from such truncated curves will lea
to underestimation of the protein responsible for the bands. Recent examples of this
occur in Gambert et al. (1988) and Secchiero et al. (1989).
Although curve areas have been shown to increase linearly with protein concentrations,
peak height may be a more reliable estimator of band density than area because it
section 2.1.5c)
Methodology 2.35
overcomes the problem discussed above. Linear relationships have been demonstrated
between densitometric peak height and concentrations of various animal proteins (Hilli
1976; Ramos et al., 1985; Csiba and Szecsenyi-Nagy, 1989). Peak height data was also
shown to be well correlated with esterase, aspartate aminotransferase and peptidase
activity in mycorrhizal fungi (Rosendahl et al, 1989). The majority of cases cited
employed polyacrylamide as the electrophoretic gel medium. However, densitometric
studies have been successfully conducted using cellulose acetate gels with isozymes
including alkaline phosphatase (Burlina and Galzigna 1976; Rosalki and Foo, 1984, 1989
Secchiero et al, 1989), lactate dehydrogenase (Kim and Yum, 1985; Yum and Kim,
1989), and 6PGD (Gasperi et al, 1983), and with various other proteins (Bauer et al,
1985; Tichy, 1985; Graml etal, 1989).
In order to assess the validity of using isozyme band densities to ascertain relative
isozyme activity under the conditions employed in this project, several investigations
undertaken (see Methods section 2.2.3). Initially, a series of experiments was performe
using ranges of sample weights and a variety of electrophoretic conditions. The
experiments aided in the selection of appropriate buffers, running durations and elect
currents for best band resolution of various isozymes. These experiments also indicate
linear relationships between sample size (and by implication, isozyme concentration) a
band densities. Several experiments were then conducted in which sample supernatants
were diluted to achieve a range of concentrations. Highly significant positive correla
between concentration and band densities were observed for all isozyme bands
investigated. Subsequently, a similar approach was used to obtain calibration data for
of the isozymes to be used in later investigations. Using these data, appropriate sampl
section 2.1.6
Methodology 2.36
concentrations were ascertained for application in the following parts of the project.
These calibration data ensured confidence in the results of the ensuing work.
2.1.6 DNA quantification
At the beginning of this chapter the inappropriateness of directly comparing sampling
units from different ploidy levels was introduced. The cell is the site of gene expres
Therefore, because cells of tetraploid plants are larger than those of diploids (Fig.
sampling units must consist of equivalent numbers of cells for each sample, or equival
amounts of genomic DNA amongst samples of different ploidy level, or some adjustment
must be made in the data collected to compensate for cell size differences. This is
necessary for valid comparisons of different ploidy levels using quantitative isozyme
procedures. The problem was overcome by determining total DNA concentration of
samples of uniform dry weight, then expressing isozyme activity data in terms of DNA
content.
Initial investigations showed that purification of DNA from crude plant material was
accompanied by variable loss (>20%), and hence was not an appropriate approach to
quantification. A method was required which would permit DNA estimation from crude
sample extracts. Two methods of DNA quantification were employed in this project
(Methods section 2.2.4).
section 2.1.6
Methodology 2.37
diploid t
10/rni
i tetraploid
Fig. 2.8 Size comparison of diploid and tetraploid cells
(from root tip cells of T R 0 2 X and T R 0 8 )
section 2.1.6
Methodology 2.38
In preliminary studies of differential isozyme activity between diploid and tetraploid
Citrus plants, DNA concentrations of samples were measured using a densitometric
procedure, similar in many respects to that used in isozyme quantification (Pulleybank
et al, 1977; Horz et al, 1981; Ribeiro et al, 1989). Experiments aimed at adapting this
method to Citrus leaf material are presented in the Methods section 2.2.4a).
Because of concerns that the photographic steps in this procedure could potentially
introduce inaccuracy into the results, a fluorometric method of DNA quantification (Lee
and Garnett, 1993) was employed for the major study subsequently undertaken. This
method of Brunk et al. (1979), which is based on the Kapuscinski and Skoczylas (1977)
technique, had not previously been employed with crude extracts from plant material.
Detailed experimentation was conducted to validate this application of the method, and
the results are presented in the attached paper, Appendix 5 (Lee and Garnett, 1993).
section 2.2.1
Methodology 2.39
2.2 Methods
2.2.1 Isozyme analysis procedures
Throughout this project the Helena Super-Z electrophoresis system was employed using
Titan III cellulose acetate gels (usually 76 x 60 mm). The proprietary gels are suppli
on a rigid, transparent mylar backing, and are of highly consistent quality. The gels
prepared prior to use by immersion in a suitable gel buffer solution. The gel and tray
buffers most frequently used are presented in Table 2.7, but various other buffers whi
were used during the project are described in the text.
To achieve good isozyme band resolution, samples must be prepared in a suitable
extraction buffer and must be free of undissolved plant debris. Various buffer formula
were investigated and the four most successful (see Appendix 1) were used at various
stages in this project. The general procedure entailed weighing a precise amount (± 0.
mg) of dried, ground leaf tissue into a 1.5 ml Eppendorf tube. This was suspended in
an appropriate volume of extraction buffer and agitated briefly. Tubes containing the
suspensions were stored in crushed ice during the process. The suspensions were
centrifuged for approximately three minutes at ~ 13,000 X g RCF to pellet debris. Ten
microlitres of supernatant was drawn from each sample with a micropipette and loaded
into wells of the Helena Super-Z sample well plate. The sample loading system consists
of a series of laser-etched grooved tips each of which pick up a 0.25 ^1 aliquot of sa
from the sample wells for loading onto the gel. The manufacturer claims that the tips
accurate to ± 5%.
section 2.2.1
Methodology 2.40
Table 2.7
Buffer systems and duration of electrophoresis of the main isozymes studied.
isozvme
IDH
eel buffer
0.1MTEM7*
G6PD
n
n
II
M
6PGD
SkDH
CAP
MDH
RBC
0.1MTC7
trav buffer
0.2M TC7*
0.2M TC7
duration
120 min.
90 min.
120 min.
120 min.
M
M
75 min.
II
H
90 min.
II
n
75 min.
- T E M 7 = Tris-EDTA-maleate-MgCl2 p H 7.4 buffer 1
- T C 7 = Tris-citrate p H 7.0 buffer
J
Richardson et al.
(1986)
Electrophoresis was always conducted inside a closed tray apparatus held at 4°C in a
laboratory refrigerator. The duration of electrophoresis depended upon the
the isozyme to be examined and the voltage applied. For the isozymes most f
studied, Table 2.7 presents the durations of electrophoresis at 100 volts (
gel). Sample concentrations and further details of sample handling and occa
alterations to the above procedures are described in the text.
Isozyme bands were revealed on the cellulose acetate gels by applying an agar overlay
containing appropriate reagents or by immersing gels in reagent solutions a
in Appendix 2. With the exception of gels stained for cytosol aminopeptidas
ribulose biphosphate carboxylase which were maintained at room temperature,
proceeded in a dark laboratory oven at 37°C until maximum band density was
After staining, all gels were fixed by immersion in 10% acetic acid, air dr
in the dark.
section 2.2.1a)
Methodology 2.41
a) Identification of the cytosolic RBC isozyme
The large subunit and small subunits of RBC were differentiated on cellulose acetate
electrophoresing samples of both whole leaf tissue and samples of chloroplasts, from
cultivars MU, PA and VO (refer section 2.1.1). Chloroplasts were extracted from fresh
leaf tissue according to the method of Dowling et al (1990)(Protocol 2, Part A).
Samples of cut leaf tissue were mixed with isolation buffer consisting of 0.35M sorb
50mM Tris-HCl pH 8.0, 5mM EDTA, 1.0% BSA and 0.1% 2-mercaptoethanol, and
homogenized with a polytron. The resultant suspension was filtered through Miracloth®
then centrifuged at 1 000 x g for 15 minutes at 4°C. The supernatant was pipetted of
for use as the whole tissue sample. The pellet was resuspended in isolation buffer,
loaded onto a 52%-30% sucrose step gradient, then centrifuged at 80 000 X g for 60
minutes at 4°C. The resultant chloroplast phase was drawn off and sonicated briefly
rupture the chloroplasts. The whole tissue and chloroplast samples were loaded onto a
cellulose acetate gel in the order:
MU whole, MU chloroplast, PA whole, PA chloroplast, VO whole, VO chloroplast
then electrophoresed under the conditions described above and stained for RBC using
amido black (Appendix 2).
The resultant gel (Fig. 2.9) showed a zone of intense staining at the extreme anodal
(fast) in all samples. The whole tissue samples also showed cathodal (slower) bands,
these were absent in the chloroplast samples. These observations confirm the finding
Handa et al. (1986) that Citrus chloroplastic RBC migrates faster (anodally) than th
cytosolic RBC isozymes. The chloroplastic band was not considered in subsequent
studies carried out in this project.
section 2.2.2
Methodology 2.42
chloroplast chloroplast chloroplast
whole
| whole | whole |
MU
*
MU
•
PA
PA
*
*
VO
VO
*
*
chloroplastic R B C
cytosolic R B C
cytosolic R B C
ongm
Fig. 2.9 Comparison of ribulose 1,5-biphosphate carboxylase bands
from whole tissue and chloroplast samples of three Citrus cultivars.
2.2.2 Investigation of the effect of lyophilization on isozyme band density
The effect of sample lyophilisation on isozyme band density was investigated in several
experiments involving SkDH, IDH, MDH and malic enzyme, to ascertain whether the
procedure would adversely affect isozyme activity. The following is a typical exampl
section 2.2.2a)
Methodology 2.43
a) Investigation of the effect of lyophilization on MDH band density
Ten leaf tissue samples (excluding midribs) of 10 ± 0.1 mg fresh weight each were tak
from a single Eureka lemon leaf. The leaf was fresh, from a recent but dormant flush.
Five of the samples were ground fresh and five were lyophilized prior to grinding. Th
samples to be freeze dried were placed into Eppendorf tubes and plunged into liquid
nitrogen. The tubes were transferred directly from liquid N to bell-jars and applied
a Dynavac FD1 freeze dryer which had been pre-evacuated. Pressure was reduced to 20
torr within one minute, to 10 torr by 2 - 3 minutes and the samples never thawed.
Drying was conducted at initial pressure <0.25 torr reducing to <0.20 torr.
Lyophilization proceeded for approximately 24 hours. All of the samples had a dry
weight of 3.5 ±0.1 mg.
Samples were prepared for electrophoresis by grinding in porcelain wells with crushed
glass in 100 /d of extraction buffer (CSIRO formula - Appendix 1). The homogenates
were transferred by Pasteur pipette to 1.5ml Eppendorf tubes and stored in ice to awa
centrifugation as described above. Electrophoresis was conducted as described previou
(section 2.2.1), and the samples and apparatus were kept chilled throughout.
The gel and tray buffer was 0.1M Tris-citrate pH 7.0 and electrophoresis was carried
at 200V for 30 minutes at 4°C ambient. Three gels were run each loaded with all ten
samples (fresh treatments in tracks 1-5, lyophilized in tracks 6 - 10). The gels were
stained for MDH (Appendix 2) as described earlier and bands were scanned with an LKB
Ultroscan laser densitometer (refer section 2.2.3) to measure density in absorbance
section 2.2.3
Methodology 2.44
Absorbance units (AU) data from the darkest (fastest) band was subjected to two-way
analysis of variance (ANOVA), with sample preparation (fresh vs lyophilized) as one
factor and gels as the other. The five tracks of each treatment served as replicates.
Fresh samples weighed 10 mg, and dried samples weighed 3.5 mg, therefore the fresh
samples contained 6.5 mg of endogenous water (6.5 [A). Because 100 fil of extraction
buffer was used, the fresh samples contained 106.5 /xl of moisture compared to 100 fii
for the dried samples. Accordingly, band density (AU) data was adjusted upwards by
a factor of 1.065 for the fresh samples. Both adjusted AU and actual AU values were
analysed separately as described above.
2.2.3 Assessment of densitometry procedures
The relative activities of gene products synthesised by diploid Citrus cultivars and t
somatic tetraploid counterparts, were compared as a means of studying gene dosage
effects. This was achieved by subjecting electrophoretic gels stained for isozyme acti
to densitometric scanning. An LKB Ultroscan XL Enhanced Laser Densitometer with
helium-neon laser light source of 633 nm fixed wavelength was used in this project. The
device offers a range of programable parameters for scanning and curve integration. The
track of each electrophoresed sample was typically scanned three times with a gap of 1.
mm between passes (X-width = 2.4 mm), and scanning points of each pass (Y-step) were
usually set at 20 /mi intervals. To maximize precision only that section of each track
containing the isozyme bands was included in the scan. The instrument plots a curve
representing the section of track scanned, with peak heights proportional to the band
section 2.2.3a)
Methodology 2.45
densities. It measures band density in absorbance units (AU) and calculates the area
under the curve (as AU X mm).
The following experiments are typical of the many investigations undertaken to confirm
linearity of isozyme band density responses with enzyme activity, as discussed earlier
(section 2.1.5c)). The first and second demonstrated response linearity. The second
experiment also assessed the reliability of the applicators used to load sample aliqu
onto the gels. Finally, a calibration experiment is presented which established the
appropriate sample concentrations to be used for each isozyme in all subsequent work.
a) An experiment to assess the effects of a series of concentrations
of sample extract on band densities of I D H , S k D H and M D H .
Fresh Eureka lemon leaf tissue was ground in liquid nitrogen and suspended in Peakall
formula extraction buffer (Appendix 1) at a concentration of 1245.00 mg fresh wt./ml
then centrifuged for 3 minutes. The supernatant was diluted with extraction buffer to
yield a dilution series of the following concentrations:
343.45, 383.08, 433.04, 498.00, 585.88, 711.43, 905.45, 1245.00 mg fresh wt./ml.
Three gels were run in the manner described earlier in section 2.2.1. Samples were
loaded in ascending order and electrophoresed at 200V for 30 minutes. Gels were staine
for IDH, MDH and SkDH (Appendix 2) and the best resolved gel of each isozyme was
scanned with the laser densitometer to ascertain peak height and curve area of each
isozyme band. The relationship between sample concentration and band density was
analysed by linear regression.
section 2.2.3b)&c)
Methodology 2.46
b) An experiment to test sample applicator reliability
This experiment used 12 samples (the applicators having 12 individual tips) of the
following concentration series: 161.04, 174.46, 190.32, 209.35, 232.61, 261.69,
299.07, 348.92, 418.70, 523.38, 697.83, 1 046.75 fresh wt. mg/ml.
The samples were loaded in order on four cellulose acetate gels. On two gels a new
sample applicator not used in the preceding experiments was employed (applicator 1), a
on the other two the previously used applicator was employed (applicator 2). One gel
from each applicator was electrophoresed in the TC/TEM buffer system for IDH, the
other two were subjected to the reciprocal buffer system for MDH. All gels were
electrophoresed at 200V for 30 minutes, stained for IDH and MDH and the resultant
bands scanned with the laser densitometer as described above. The use of a series of
increasing concentrations permitted further investigation of the relationship between
enzyme dose and band density, using linear regression.
c) Isozyme activity calibration
The experiments such as those detailed above all showed a linear response of isozyme
band peak heights with increasing isozyme concentration for IDH, MDH and SkDH. To
confirm that similar relationships exist with the other isozymes to be studied in
subsequent parts of the project, calibrations were performed using sample dilution seri
The resultant response profiles were used to ascertain the optimum sample concentratio
for each isozyme to ensure that band density values were directly proportional to isoz
activity.
section 2.2.3b)&c)
Methodology 2.47
Lyophilized leaf tissue of tetraploid Rough Lemon was prepared in Soltis + extraction
buffer (Appendix 1) as described previously, at a concentration of 125 mg dry matter/m
Further buffer was added to aliquots of the supernatant to produce the sample dilution
series shown below. The dried sample concentrations were roughly equivalent to the
lower end of the fresh sample concentration series used in the preceding experiments,
although the lowest concentrations below are more dilute than those previously used.
sample cone.
/xg DM/fil
12.5
25.0
37.5
50.0
62.5
75.0
87.5
100.0
112.5
125.0
0.25 /xl load applicator
/xg DM/double load
6.25
12.50
18.75
25.00
31.25
37.50
43.75
50.00
56.25
62.50
Gels were loaded with these samples in sequential order, electrophoresed and stained for
the seven isozymes (Appendix 2) central to this project (see section 2.1.3b)). Initial
with the exception of gels to be stained for IDH, the samples were double-loaded (ie.
applications with a 0.25 /xl applicator tip) to deliver the sample loads shown above.
was known to produce faint bands and so was triple-loaded. The experiment was
repeated twice, with sample order randomized. In the initial run, SkDH and RBC
maximum peak heights did not exceed 1.5 AU and there was no declension in the
response slope (refer section 2.3.2c) and Fig. 2.14). Accordingly, samples were
increased to quadruple-loaded for the subsequent runs for these isozymes, to increase
band density. In the initial MDH run the darkest band (MDH-2f) was quite dark and
section 2.2.4a)
Methodology 2.48
produced only a shallow response slope. This suggested that the reaction may have been
limited in this band, possibly due to excessive potential enzymatic activity. Furthermor
the other MDH bands exhibited response declensions near the 50 /xg load. Accordingly,
the subsequent runs for MDH were single-loaded. The relationship between sample
concentrations and band densities were graphed. Identification of linear response region
was used to ascertain appropriate sample concentrations for use in subsequent
investigations.
2.2.4 DNA quantification methods
Two methods of DNA quantification were used (refer section 2.1.6 above). Experiments
were conducted to optimise both methods for use with crude extracts of Citrus leaf
material.
a) DNA quantification by densitometry of electrophoretic bands
Several initial experiments were conducted to determine the most appropriate sample
handling methods to prepare leaf tissue for crude extract DNA electrophoresis. Briefly,
lyophilization of tissue had no detectable adverse effect on DNA yield compared to fres
tissue, although it was observed that strict control of the temperature and pressure
environments before and during lyophilization was important. This finding is in
agreement with that of Le Cam et al (1989). A variety of tissue macerating enzymes
was investigated, but none improved DNA yield. Sonication treatments all resulted in
loss of DNA band resolution, presumably by fragmenting the molecules. The best results
section 2.2.4a)
Methodology 2.49
were obtained when lyophilised tissue, was suspended in TE8 buffer (lOmM Tris-HCl
pH 8.0, lmM EDTA) with 100 /xg/ml proteinase K (to inhibit endogenous nucleases) and
incubated at 37°C for 1 hour. The sample was then centrifuged briefly (3000 X g) and
the supernatant collected as crude extract for electrophoresis. Sample concentrations
were selected as appropriate for each experiment and generally ranged between 20 and
50 mg dry matter/ml.
When ethidium bromide associates with nucleic acids it exhibits enhanced fluorescence
when exposed to ultraviolet (UV) radiation. The technique of DNA quantification by
densitometry of electrophoretic bands relies upon the measurement of this fluorescenc
Crude extract was electrophoresed in an agarose gel containing ethidium bromide. The
resultant gel was illuminated with UV radiation and photographed. The DNA bands on
the photographic negative were then scanned with a densitometer to measure their
density. Internal standards of known DNA concentration were included in each gel. The
DNA concentration of unknown samples was calculated by interpolation with the
calibration curve of the internal standards. There was a concern that the method coul
be prone to variability because of the large number of steps involved and the opportu
for inconsistencies between runs at each step. However, the technique has been shown
to be reliable (Prunell et al, 1977; Prunell, 1980; Moore and Sutherland).
Investigations were conducted to assess the relative electrophoretic mobilities of DN
RNA. Both DNA and RNA enhance ethidium bromide fluorescence. Crude extracts of
Citrus tissue were electrophoresed (see methods below) and the gel was photographed
before and after incubation in a solution of RNase A, an enzyme which digests RNA.
section 2.2.4a)(i)
Methodology 2.50
Only the DNA bands remained after incubation and comparison of the two images
showed that all RNA fragments migrated anodally much faster than DNA (cfi Fig. 2.19)
The electrophoretic conditions employed are such that minimal disruption of DNA
molecules occurs and slow migration results in a single concentrated DNA band.
(i) Assessment of the relationship between DNA concentration
and electrophoretic band density
The DNA molecular weight Marker II (Boehringer Mannheim Cat. No. 236 250) consists
of X phage DNA fragments cleaved with Hindlll restriction endonuclease. This marker
contains 8 fragments, in equimolar concentrations, of sizes 23 130, 9 416, 6 557,
4 361, 2 322, 2 027, 564, and 125 base pairs. It was found that whereas these
fragments could be separated by electrophoresis in a VA% agarose gel, a 2% gel would
concentrate all the >4 Kbp fragments of Marker II into a single band. Earlier
experiments had shown that this gel concentration also produced a sharply resolved
band from crude extracts of Citrus leaf tissue.
The wells in a 2% agarose gel containing 0.167 /xg/ml ethidium bromide were loaded
with samples of DNA Marker II prepared in TE8 buffer at a concentration of 10 ng
DNA//xl (>4Kbp). Sample volumes ranged between 0.5 and 10 /xl such that the 11
sample loads were, in order, 5, 10, 20, 30, , 100 ng >4 Kbp DNA. Each sample
was mixed with 5/xl of bromophenol blue in 50% glycerol, to increase sample density
act as a tracking dye. Three separate electrophoretic runs (a, b and c) were conduct
at 80 volts for Vh hours. Run c did not include the 5 ng and 100 ng tracks. Gel and
tray buffer consisted of 89mM Tris, 89mM boric acid and 2mM EDTA, pH 8.8 (TBE
section 2.2.4a)(ii)
Methodology 2.51
buffer - Ausubel et al, 1987).
Gels were illuminated using transmitted UV radiation and photographed with Polaroid
type 665 positive/negative film, through an orange filter attached to the camera lens.
negative image thus obtained was scanned with the laser densitometer as used in the
isozyme studies. The relationship between DNA concentration and band density was
analysed by linear regression after cubic transformation of the data.
(ii) Quantification of DNA in Citrus crude extracts
by densitometry of electrophoretic bands.
In order to investigate the applicability of the above methods to quantification of DNA
in crude extracts, samples of diploid and tetraploid Swingle citrumelo (accessions SW2
and SW2, respectively) lyophilized leaf tissue were prepared by incubating 10 mg of dry
matter in 1 ml of TE8 buffer containing 9 mg sodium dodecyl sulphate (SDS) and 50 /xg
of proteinase K. A 2% agarose gel containing 0.167 /xg/ml ethidium bromide was loaded
with 5 /xl of the extract, equivalent to 50 /xg of dry matter, per well. Three aliquots
each sample were loaded in the first 6 wells, in alternating order, diploid, tetraploid
diploid, ...., tetraploid. The last 5 wells were loaded with a dilution series of Marke
II such that the >4 Kbp DNA loads were, in order, 10, 20, 30, 40 and 50 ng per well.
The gel was electrophoresed as above, photographed and the negative subjected to
densitometry.
The experiment above (designated, run a) was repeated with tissue from different
accessions of diploid and tetraploid Swingle citrumelo (SW2X1 and SW1, respectively).
On this occasion (run b), each sample load was equivalent to 25 /xg dry matter; half of
section 2.2.4b)
Methodology 2.52
that in run a. The Marker II >4 Kbp DNA loads were 10, 15, 20, 25 and 30 ng. All
tracks were in the same order as in run a. The Marker II data was transformed and
analysed by linear regression. The DNA concentration of the crude extracts was
calculated using the Marker II concentrations as an internal standard.
b) Quantification of DNA in Citrus crude extracts by fluorometry
Although the densitometric method of DNA quantification yielded satisfactory results,
more direct and efficient technique was sought for use in the final investigation in t
project. Experiments conducted to confirm the validity of the method have been
published in the Journal of Biochemical and Biophysical methods (Lee and Garnett, 1993
an offprint of which is attached (Appendix 5).
The method is based on that of Brunk et al. (1979). It involves measuring the increase
in fluorescence caused by successive additions of precise aliquots of DNA-containing
solutions to a 4',6-diamidino-2-phenylindole (DAPI) solution (Fig. 2.10). A series of
10/xl aliquots of the unknown crude extract solution were added first, followed by a s
of 10/xl aliquots of purified DNA of known concentration. The fluorescence intensity o
the solution increases linearly with DNA concentration. The quotient of the unknown an
known response slopes is multiplied by the known concentration to determine the
unknown DNA concentration. Minor quenching of fluorescence due to cellular
components or buffer is automatically corrected by the use of the internal standard (B
et al, 1979). The technique relies on the linear relationship between fluorescence and
DNA concentration. The response rate differs with DAPI concentration, but for any give
DAPI solution between 20 and 200 ng/ml the response is linear as DNA concentration
section 2.2.4b)
Methodology 2.53
70
• crude extract (unknown)
O pure standard (23.35 ng DNA/ml)
20
40
60
80
100
crude sample <—|—> pure standard
S A M P L E / S T A N D A R D A L I Q U O T (\i\)
Fig. 2.10 D N A estimation method. Fluorescence intensity (I) of 100 ng/ml
D A P I increases linearly with succeeding 10 /xl aliquots of solutions containing
D N A . Crude extract of lemon (lower slope) and pure citrus D N A (upper)
increases in the range 5 - 1000 ng/ml (Kapuscinski and Skoczylas, 1977). This linearity
is maintained while DNA concentration does not exceed 10 times the DAPI concentration
(Brunk et al, 1979). Refer to Appendix 5 for full details of the method and the
experiments.
section 2.3.1a)
Methodology 2.54
2.3 Results and Discussion
2.3.1 Investigation of the effect of lyophilization on isozyme band density
a) Investigation of the effect of lyophilization on MDH band density
Lyophilisation caused no reduction in isozyme band density, which indicated its
suitablility for sample preparation for quantitative densitometry on cellulose acetate g
The density (AU) of the darkest MDH band from lyophilised leaf tissue (refer Methods
section 2.2.2a)) was 0.489±o.oi5(s.e.), and was not significantly different ((P=0.521)
from the adjusted band density for fresh tissue, which was 0.502±0.015 AU. The
unadjusted data produced similar results.
Experiments were also conducted with SkDH, IDH and malic enzyme (ME), and again,
lyophilization did not cause any reduction in isozyme band density. Therefore,
lyophilization of Citrus leaf tissue following grinding in liquid nitrogen was determine
to be an appropriate preparative procedure. Contrarily, Loomis (1974) reported
browning of tissues during lyophilization. However, such browning can result from
thawing of samples during drying. Deisseroth and Dounce (1969) demonstrated that
different conditions during lyophilization significantly affected the degree of
conformational change and subunit dissociation in catalase. Ultra-low temperatures were
strictly maintained at all stages throughout the procedure used here and dried samples
were stored at -80°C. Lyophilisation was subsequently adopted for routine preparation
of bulk leaf tissue samples.
section 2.3.2a)
Methodology 2.55
The mean AU value of one of the gels (0.541 +0.018) was significantly greater (P=0.02)
than that of the other two (0.483+0.018 and 0.462+o.oi8)(l.s.d.005=0.054). This
observation indicated that comparisons should not be made between separate gels.
Tarczynski et al. (1989) observed similar lack of comparability between gels. The
problem can be addressed by including a common sample on each gel (Dannenberg and
Kessler 1986; Rosendahl et al, 1989). However, the more conservative approach of not
directly comparing density values between gels was adopted in this project. Subsequent
experiments herein were designed such that all treatments to be compared were applied
to single gels, and separate gels were only used as replicates or as treatments that w
not being used for critical comparisons (in particular, the various different isozyme
2.3.2 Assessment of densitometry procedures
a) An experiment to assess the effects of a series of concentrations
of sample extract on band densities of I D H , S k D H and M D H .
This experiment supported the notion that, within the limits of the range of isozyme
concentration in the samples, there is a direct relationship between isozyme concentr
and band density, measured as peak height. All isozyme bands exhibited a significant
positive correlation between band density and sample concentration (Table 2.8) and R2
values showed that at least 80% of the variation in band densities could be explained
the sample concentrations. Typical densitometric scans of the isozyme bands are shown
in Fig. 2.11 (refer Methods section 2.2.3a)). Allozyme nomenclature accords with that
of Volkameriana (VO) lemon as defined in section 2.1.3b). Densitometric data from both
section 2.3.2a)
Methodology 2.56
MurllMI
t-i.lti 1.10 .. ],ll t.U.
I t-iln • 1.1! • •••• • S.1S I
IDH
i.io
s.w
i.to.
AU
1,41
1.50
ill
111
f-p»utlo» Oil
h-
Miorbuil
III
110
-•
M l v l • lit
«-HI»I M l .. J.0» ».0.
111
Y-«to» • 121
111
Hi
I-«l»p "1/1
111
I • 10 iltrmii I
I «-ii« • 2.lt »-••• • 5.0! I
MDH
AU
:.ut:
112
110
r-FMiltn I M I
lit
I
III
lit
•
120
I-ttifl • 110
122
»-itoo • 110
1»
I2t
l-Kto '1/2
121
130
I • 20 ticroM l
SkDH
S-litli l.» .. 2.52 I.U.
I «-im • 1.15 A-ni > 2.20 I
AU
'-pnitlM <••!
*
l-llirl • 110
f-Itop • 12!
M i l ) < 1/2
I • 20 ileum I
Fig. 2.11 Typical densitometric scans of IDH, M D H and S k D H
isozymes of Eureka lemon
section 2.3.2a)
Methodology 2.57
IDH-m and IDH-a
•
•
2.5
IDH-m density (AU)
IDH-a density (AU)
2.6
2.7
2.8
2.9
3
3.1
conc.(log mg/ml)
MDH-a and MDH-2f
0.8
•
•
MDH-a density (AU)
MDH-21 density (AU)
—1
<
0.6
•
•
H—'
_£_
CD
•
//
s^m
CD
V
0.4
•
Cfl
CD
LL
•
0.2
•
2.5
2.6
2.7
2.8
2.9
3
3.1
3
3.1
conc.(log mg/ml)
SkDH-a and SkDH-c
0.4
•
•
ID
SkDH-a density (AU)
SkDH-c density (AU)
0.3 -
<
0.2
a
CD
Q.
0.1
2.5
2.6
2.7
2.8
2.9
conc.(log mg/ml)
Fig. 2.12 Relationship between sample concentration and isozyme band densities
of I D H , M D H and S k D H from Eureka lemon leaf tissue samples
section 2.3.2a)
Methodology 2.58
Table 2.8
Linear regression statistics of the relationship between isozyme band densities
(measured as peak height - A U ) and sample concentrations of electrophoresed
Eureka lemon leaf tissue samples stained for I D H , M D H and S k D H
mean peak P-value of coefficient F-value of the
the slope
of variation* regression model
height - A U n
R2
IDH-m
IDH-a
0.6125
0.1300
8
8
0.8730
0.9139
0.0007
0.0002
MDH-a
MDH-2f
0.1425
0.6013
8
8
0.8658
0.8801
0.0008
0.0006
14.498
9.212
38.72
44.03
SkDH-a
SkDH-c
0.2075
0.2175
8
8
0.7911
0.8626
0.0031
0.0009
16.073
15.237
22.73
37.66
14.284
14.742
41.26
63.70
* - coefficients of variation for each set of eight values
peaks of each isozyme (only the two large M D H peaks were used) were analysed by
linear regression of peak height (AU) against sample concentration. The responses are
plotted in Fig. 2.12 (abscissa has been log transformed). The regression statistics ar
presented in Table 2.8. Similar relationships were revealed between curve area and
concentration (data not shown). The direct relationship between sample concentration a
band density indicated that densitometry would be a suitable method for quantifying
electrophoresed isozyme activity.
Fig. 2.12 reveals a tendency for erratic responses at lower concentrations. It is show
below that this effect was caused by anomalous loading characteristics of the sample
applicator used, and corrective measures were subsequently taken.
section 2.3.2b)
Methodology 2.59
b) An experiment to test sample applicator reliability
The tips of the Helena Super-Z loading system sample applicators are extremely delicate
and prone to damage if mishandled. Close examination of the response curves of peak
height against sample concentration in the above experiments showed a perturbation at
the lower end. This effect occurred in all experiments conducted. As each isozyme
constituted a separate gel the effect was apparently repeatable. In each case, samples
were loaded in ascending order of concentration, using applicator 2. In this experiment
(refer Methods section 2.2.3b)) the effect was seen in the responses when applicator 2
was used, but not applicator 1. This observation plus the striking similarity in the
perturbation pattern in each previous instance where applicator 2 was used, indicated t
the loading tips on applicator 2 were not each delivering the same sized aliquot of
sample. Applicator 1 seemed to be functioning satisfactorily.
Isozyme band patterns were the same as those in the preceding experiment. At the lowest
sample concentrations, only the main band of each isozyme (IDH-m and MDH-a) was
sufficiently dense to yield reliable data. In the plots of peak height against sample
concentration (Fig. 2.13), applicator 2 showed the same erratic pattern at the lower end
as seen in the previous experiment. Applicator 1 exhibited no such pattern. The effect
of the erratic sample loading of applicator 2 was to reduce the goodness of fit of the
linear regression model for this applicator compared with applicator 1 (Table 2.9).
Nevertheless, there was a strong correlation between peak height and concentration of t
samples for all treatments. Curve area data showed similar correlation relationships to
those of peak height.
Methodology 2.60
section 2.3.2b)
applicator 1
applicator 2
IDH-m
IDH-m
-0.2
2.2
2.4
2.6
2.8
"•
IDH-m density (log AU)-applicator 2
2.2
2.4
2.2
2.8
cone.(log mg/ml)
conc.(log mg/ml)
applicator 1
applicator 2
MDH-2f
MDH-2f
-1.2
2.6
2.4
2.6
2.8
cone.(log mg/ml)
3
2.2
2.4
2.6
2.8
3
cone.(log mg/ml)
Fig. 2.13 Relationship between sample concentration and band densities of I D H
and M D H isozymes from Eureka lemon leaf tissue samples delivered by two
different applicators (log transformations of cone, and density (AU) performed - actual
concentrations were clustered around lower end because of anticipated greater response variability
at low sample concentrations)
section 2.3.2b)
Methodology 2.61
Table 2.9
Linear regression statistics for the relationship between isozyme band densities
(measured as peak height - A U ) and sample concentrations of electrophoresed
Eureka lemon leaf tissue samples stained for I D H and M D H , using two different
sample applicators
mean peak
coefficient F-value of the
2
height - A U
n
R of variation* regression model
IDH-m applic. 1 0.2342 12 0.9753 11.317 394.78
I D H - m applic. 2
0.1808
12
0.9591
12.132
234.52
MDH-a applic. 1 0.2000 12 0.9584 11.638 230.30
0.2042
12
0.9383
M D H - a applic. 2
15.126
152.19
* - coefficients of variation for each set of twelve values
The inaccuracy in sample load was not considered severe, however several gels were run
with identical samples in each well to determine which tips were inaccurate. The
applicator tips are removeable, so a special applicator was constructed for subsequen
in this project using only those tips shown to deliver similar volumes. This applicato
was regularly checked for accuracy. Furthermore, for experiments in later parts of the
project, sample order was randomized between replicates.
This experiment confirmed the linear relationship between concentration of isozyme
sample and band density as measured by the LKB Ultroscan laser densitometer within the
range of concentrations used. It could not be assumed, however, that the same
relationship would be exhibited by all isozymes. It was necessary to confirm the
existence of a direct linear response between band density and isozyme concentration
each isozyme to be used for subsequent investigations in this project. That calibratio
work is reported in the following section.
section 2.3.2c)
Methodology 2.62
c) Isozyme activity calibration
As could be expected from the previous observations, peak height (absorbance) increased
linearly with sample load, reflecting increasing enzyme activity. However, after an ini
positive gradient, some bands exhibited declension in response slope, resulting in a
plateau. This plateau indicates a limitation to the staining reaction. The cause was
probably associated with the exhaustion of the substrate or some other reagent in the
staining formula under the conditions employed. Certainly it was not caused by any limi
in the capacity of the gel matrix to accumulate the chromophore (formazan, in most
cases), because ancilliary studies had shown band densities sufficiently dark to exceed
maximum capacity (AU > 4.0) of the densitometer (eg. for malic enzyme). The sample
load at which the declension point occurred was deemed to be the maximum sample
concentration which should be used in isozyme quantification experiments. Ideally,
sample loads should correspond to a site on the initial gradient below the declension
point, yet sufficiently high to ensure adequate band density to achieve reliable
densitograms.
The band density responses for the calibration experiment (section 2.2.3c)) are shown a
solid squares in Fig. 2.14. The experiment was repeated twice, with sample order
randomized - the data are shown as circles in Fig. 2.14 (for clarity, only two of the t
cases are shown). Allozyme nomenclature accords with that of Rough Lemon (RL)
(section 2.1.3b)).
For subsequent isozyme electrophoresis, samples of 25 mg dry matter were suspended
in 250 fxl of Soltis + extraction buffer (Appendix 1), and incubated at room temperatur
section 2.3.2c)
Methodology 2.63
SkDH-c
SkDH-b
0.3
•
o
0.25
SkOH-b - double-loaded
SkOH-b - quadruple-loaded
• SkCH-c - double-loaded
o SkOH-e - quadruple-loaded
0.25 0.2
<
S
0.2
<
^T 0.15
sz
•J? 0.15
xz
00
CD
•
o
5 0.1
•
Z£
•
CO
CD
01
u>l
Q.
Q. 0.05
0.05
50
0
100
150
dry matter (jig)
0
50
100
150
dry matter (ug)
CAP-b
• CAP-P - double-loaded
o CAP-b - double-loaded
1.5
cu
SZ
ca
cu
0.5
Q.
20
40
60
dry matter (jxg)
EDH-m
0.5
IDH-a
0.2
• IOH-m - triple-loaded
] o IOH-m - triple-loaded
o
• • IDH-a - triple-loaded
• o IDH-a - tnple-loaded
o
o
0
0.4
0
0.15
0
0
<
~
0.3
sz
D)
<
0
•
.gj 01
cu
sz
'(U
f 0.2
o
•
0
o
•
0
•
•
•
•
•
J*
ca
cu
ca
cu
Q_
^o.os
0.1
20
40
60
dry matter (jig)
80
100
•
•
20
40
60
80
dry matter (uej)
Fig. 2.14(a) Relationships between sample concentration (expressed
as sample dry matter equivalent) and band densities
for allozymes of S k D H , C A P and I D H
100
Methodology 2.64
section 2.3.2c)
G6PD-b
02 j
G6PD-C
• -
0.4
• G6PD-b - double-loaded
O G6PO-b - double-loaded
• G6PD-C - double-loaded
o G6PD-C - double-loaded
•
•
•
•
— 0.15 Z)
Z>
<U
SZ
o
0
•
•
0
0
°-0.05
•
o
o
o
O
0.2
•
•
o
0
0
•
a. 0.1 -
•
o
•
CO
CO
•
o
o
sz
•
•
•
•
i
<
•
P
ak height
<
0.3
•
.
o
°
o
0
o
0
1
.
20
r
1
40
60
n
,
,
_1_
1
20
dry matter (u.g)
.
1
40
60
dry matter (ng)
RBC-d
0.3
• RBC-d - double-loaded
o RBC-d - quadruple-loaded
0.25
< °-2
| 0.15
sz
:*:
co Q i
O.
•
o
•
o
0.05
50
150
100
dry matter (jig)
6PGD-d
6PGD-C
u.t
6PGD-C - double-loaded
6PGD-C
•
• O
•
•
0
•
o
v.c •
•
o
O
•
6PGD-d - double-loaded
6PGD-d - double-loaded
^0.15
Z>
<
•
0
•
•
•
•
0
0
0
O
0
•
0
O
CD
height (AU)
- double-loaded
o
•
o
n> 01
0
CO
cu
°- 0.1
o
O
0
20
40
dry matter (ng)
60
O B
•
•
°- 0.05
•
()
•
CD
SZ
CO
CD
•
O
•
a
0
)
20
40
dry matter (ng)
Fig. 2.14(b) Relationships between sample concentration (expressed
as sample dry matter equivalent) and band densities
for allozymes of G 6 P D , R B C and 6 P G D
60
Methodology 2.65
MDH-Is
section 2.3.2c)
MDH-a
a MDH-a - double-loaded
0 MDH-a - single-loaded
O 0
t
0.15
o
o o
s-. 0.2 r
o
a
a
a
a
a
0
Q.
a
o
a
a
ca
clo.05
0
a
0.05
0
o
0 o a
a
SCD °-1
O 0
0
<
a
0
03
CD
SZ
O
height
t
a
•5,0.15 -
0
S
0
Z)
<
MDH-ls- double-loaded
MDH-1s • single-loaded
*
0
o
0.25
n
.
1
L.
20
,
1
40
60
20
dry matter (ng)
40
60
dry matter (ng)
MDH-lm
a MDH-lm - double-loaded
o MDH-lm-single-loaded
0.2
Z)
< 0.15
sz
CD
sz
0.1
CO
CD
Q.
0.05
20
- a MDH-ll - double-loaded
o MDH-1 (- single-loaded
0.3
a
Z)
<
sz
rzn
CD
sz
a
•
0
0
o
0.1
a
a
CO
CD
CL
a
CO
CD
a
n
o o o
-
a
0.2
a
a
a
a
a
a
a
a
a
0.1
Q.
- 0
0.05
o
a
a
o
-
CD
sz
zz
_ a MDH-21 - double-loaded
o MDH-21 - single-loaded
o o o o 0
a
o o
< 0.15
sz
MDH-2f
o
o
3
60
dry matter (ng)
MDH-If
0.2
40
o
I
20
.
I
.
I
40
dry matter (ng)
-
60
n
I
20
.
I
.
I
40
dry matter (ng)
Fig. 2.14(c) Relationships between sample concentration (expressed
as sample dry matter equivalent) and band densities
for allozymes of M D H
60
section 2.3.3a)(i)
Methodology 2.66
for 10 minutes. After centrifuging, the clear fraction was pipetted off and immediately
chilled in ice prior to gel loading and electrophoresis under refrigeration as described
earlier in this chapter (section 2.2.1). A single 0.25 /xl applicator load of this extra
equivalent to 25 /xg of dry matter. In accordance with the principles defined above and
in view of the declension points for the various isozymes in Fig. 2.14, the following
sample loads were subsequently employed:
isozvme
SkDH
IDH
CAP
G6PD
6PGD
MDH
RBC
load /xg D M equivalents
75/xg
triple
double
50/xg
25/xg
single
50/xg
double
single
25/xg
25/xg
single
75 /xg
triple
The lack of coincidence in band densities between the gel runs for I D H and G 6 P D , which
entailed similar sample concentrations in each run, is an example of the occassional
divergence in response between gels which was reported earlier.
2.3.3 DNA Quantification
a) DNA quantification by densitometry of electrophoretic bands
(i) Assessment of the relationship between DNA concentration
and electrophoretic band density
Transformed DNA band density data showed a positive linear response to increasing
DNA Marker II sample concentration.
section 2.3.3a)(i)
Methodology 2.67
The image of the gel from run a (Fig. 2.15) and the densitometer trace from its 100 ng
track (Fig. 2.16) show the dense >4 Kbp band and the faster (anodal) 2 322 and 2 027
bp bands (refer Methods section 2.2.4a)(i)). The faint 564 bp band was difficult to
discern and the 125 bp band had migrated off the gel (the individual DNA fragments had
been differentiated in earlier studies - data not shown). Plots of the response of pea
height and curve area to DNA load (Fig. 2.17) show curvilinearity for both parameters.
Second order polynomial regression revealed very high R2 values in each case (see
below). Variability between runs was very low for peak height data but greater for cur
area. The response curves could be linearised by cubic transformation of peak height
curve area data. The transformed data (Fig. 2.18) were analysed by linear regression
the R2 values were very high:
peak height R2 curve area R2
untransformed transformed untransformed transformed
data
data
data
data
run a 0.9935 0.9937 0.9974 0.9819
run 6 0.9906 0.9969 0.9973 0.9718
rune 0.9963 0.9922 0.9977 0.9830
The response rate (regression slope) for transformed peak height was consistent betwe
runs, but less so for curve area. Furthermore, the peak height regressions, unlike tho
of curve area, intercepted both x and y axes close to 0 (Fig. 2.18). Numerous similar
studies (data not shown) confirmed that the cubic transformation of peak height data
consistently gave a good linear correlation with DNA concentration. Experiments
involved both Marker II DNA and Citrus crude extract samples.
section 2.3.3a)(i)
Methodology 2.68
564 bp band
2027 & 2322 bp bands
> 4 Kbp bands
Fig. 2.15 D N A bands from a series of concentrations of D N A Marker II
(5, 10, 20 .... 100 ng D N A > 4 Kbp) electrophoresed in a 2 % agarose gel
f-positiM (ul
I-
-•
t-*twt « U
t-stop » 78
t-tttp "1/2
1 « » ricrom )
Fig. 2.16 Densitometric scan of bands from D N A Marker II
electrophoresed in 2% agarose gel (gel a, 100 ng track)
section 2.3.3a)(i)
Methodology 2.69
peak height response
Marker II >4 Kbp D N A
•-• runaAU
"A-ArunOAU
•—• runcAU
0
20
40
60
80
100
DNAng
curve area response
Marker II >4 Kbp D N A
•-• run a area
A - A run b area
•—• run c area
x
<
Cfl
CD
>
o
0
20
40
60
80
100
DNAng
Fig. 2.17 Relationship between D N A concentration and band density,
measured as densitometric peak height and curve area
(Marker II > 4 Kbp D N A electrophoresed in 2 % agarose)
section 2.3.3a)(i)
Methodology 2.70
transformed (A3) peak height response
Marker II >4Kbp DNA
CO
<
<
CO
<
•t—'
SZ
CD
.c
ca
cu
Q_
20
40
60
80
100
DNAng
transformed (A3) curve area response
Marker II >4 Kbp DNA
A
1.2 • run a area 3
A
A runftareaA3
• runcarea A 3
CO
<
•
/
s/y
e
£
x
Z>
CO
<
0.8
-
0.6
-
0.4
-
/k
• ,
/A
<
CD
//
A
/ '•
v_
03
CD
>
0.2
o
0 ,, •,/?
20
40
i
1
60
80
100
DNAng
Fig. 2.18 Relationship between D N A concentration and transformed (*3) band
density, measured as densitometric peak height3 and curve area3
(Marker II > 4 Kbp DNA electrophoresed in 2% agarose)
section 2.3.3a)(ii)
Methodology 2.71
(ii) Quantification of DNA in Citrus crude extracts
by densitometry of electrophoretic bands.
Because there was a consistent correlation between a series of known Marker II DNA
concentrations and the transformed band densities, it was able be used as an interna
standard to quantify DNA in an unknown crude extract. The image of the gel (Fig. 2.1
of run a (refer Methods section 2.2.4a)(ii)) revealed denser DNA bands in the tetrap
samples (2nd, 4th and 6th tracks) than the diploid. Run b was similar. The peak heig
responses to Marker II DNA load were curvilinear (2nd order polynomial R2 = 0.9943
and 0.9984 for runs a and b, respectively). The responses became linear when height
data were cubed (Fig 2.20), with R2 = 0.9962 and R2 = 0.9946 for runs a and b,
respectively. The regression slopes differed between the two runs, possibly due to
differences in the photographic stage. The regression models (a + bx) for the cubed
were, -0.017765 + 0.010259 x x and -0.065104 + 0.016860 X x for run a and
b, respectively. The DNA load in a crude sample band can be calculated by applying t
appropriate regression model values from the markers on the same gel, using the
formula,
(Vy) - a
x=
b
where x is the DNA load, y is the peak height of the crude sample band, a is the Mar
II regression intercept and b is the slope. This gives a value for the DNA load in
nanograms (the units used for the Marker) which can be divided by the dry matter
equivalent of the sample aliquot (in micrograms) to determine the DNA concentration
the dry matter (as ng//xg = /xg/mg). The DNA concentrations, thus calculated, in this
experiment are shown in Table 2.10.
section 2.3.3a)(ii)
Methodology 2.72
{
R N A bands
D N A band
m
A
m
m
m
/ft
2X4X2X4X2X 4X
Fig. 2.19 Image of 2 % agarose gel showing bands of electrophoresed D N A and
R N A from diploid and tetraploid Citrus tissue, and D N A bands from Marker II
transformed (A3) peak height response
Marker II >4Kbp D N A
• runaAUA3
- • run&AUA3
-
0.5
CO
<
<
CO
<
0.4
•
/m
m /
0.3
sz
CO
0.2
CD
.*:
SZ
03
CD 0.1 Q.
n
/
' .
.
t
10
• /
.
i
,
20
30
DNAng
,
,
.
i
40
.
.
,
.
i
, ,
50
Fig. 2.20 Relationship between D N A concentration and transformed (A3) band
density, measured as densitometric peak height3
(Marker II > 4 Kbp D N A electrophoresed in 2 % agarose)
section 2.3.3a)(ii)
Methodology 2.73
Table 2.10
Concentrations of D N A in diploid and tetraploid Citrus tissue samples
calculated using densitometric data from electrophoretic bands of D N A
run a replicates mean coefficient of variation
xxg D N A / m g D M
1
2
3
diploid
1.7147 1.6319 1.6880
1.6782
tetraploid 1.7656 1.7573 1.7738
1.7656
of 3 replicates
2.518
0.275
run b replicates mean coefficient of variation
_!
2
3
/xg D N A / m g D M
diploid
1.6490 1.7601 1.7066
1.7052
tetraploid 1.7248 1.8577 1.7772
1.7866
of 3 replicates
3.258
3.746
A s indicated by the denser bands (Fig. 2.19) the tetraploid samples in both runs had
higher DNA content per unit dry matter. Given that the tetraploids had twice the quan
of DNA per cell, the above data imply that mean cell size of the tetraploids was sligh
less than twice that of diploids. The low coefficients of variation indicate that wit
gel image, variability was low. However, as evident from the regression slope
differences (Fig. 2.20), some variation between images may occur. For quantification
of DNA however, the incorporation of Marker II as an internal standard should overcome
the effect of variation between gel images. Whereas the two runs represented samples
from different accessions of the Swingle citrumelo hybrid, there was remarkable
similarity in DNA concentration between the two diploid accessions, and between the t
tetraploids. This experiment demonstrated the suitability of the method for DNA
quantification.
The experiments on fluorometric quantification of D N A are presented in Appendix 5.
section 2.4
Methodology 2.74
2.4 Conclusion
Whereas the techniques employed in this project were all established procedures, the
particular application of each for the purposes of this project was novel. Although
densitometry of electrophoretic bands has been used elsewhere to measure enzyme
activity, its application with cellulose acetate gels is rare. Densitometry of
electrophoretic bands on cellulose acetate gels to compare isogenic plants of different
ploidy levels has not been reported in the literature, and the use of these techniques to
examine a range of related genotypes may never have been attempted. Determination of
DNA concentrations using densitometry of electrophoretic band images is usually applied
to small DNA fragments or highly purified DNA. I found no references in which the
method was applied to quantification of DNA in crude plant extracts. Whereas DAPI
fluorescence has been shown to be a highly sensitive method of determining DNA
concentration in purified preparations and in protozoans, this technique also had never
before been applied to crude extracts of plant material.
Because of the novel application of these techniques for the purposes of this project, it
was necessary to rigorously assess each procedure to confirm its validity. However, the
initial tissue preparation process also had the potential to influence the outcomes of the
quantitative methods. To overcome the effects of endogenous moisture in samples,
drying was required. Therefore, it was also necessary to investigate the effects of the
lyophilization drying process. In particular, effects on isozyme activity due to
lyophilization, could not be tolerated.
section 2.4
Methodology 2.75
This chapter has presented a selection of the many experiments conducted to validate the
various procedures to be applied in the subsequent parts of the project. The following
chapter reports on the investigation of the effects of genome duplication on quantitative
expression of the seven isozymes examined above, in the diploid and tetraploid accessions
of the eight Citrus cultivars described. That investigation utilized the methods describe
above.
section 3.1
Ploidy and Gene Expression 3.1
Chapter 3
THE RELATIONSHIP BETWEEN PLOIDY AND
GENE EXPRESSION IN CITRUS
3.1 Introduction
Responses of quantitative gene expression to genome duplication will be affected by
gene-specific mechanisms. Trisomic aneuploid plants which possess a duplicate copy of
a single chromosome are similar in cell size to their diploid counterparts (Birchler, 1979).
Such plants provide some interesting insights into the effects of duplication of genes
without an associated change in gross cell morphology that occurs in polyploids. Fobes
(1980) studied allozyme concentrations of tomato (Lycopersicon esculentum) trisomies
using densitometry of electrophoretic bands. In plants with an extra gene for the
allozymes A C P - 1 (acid phosphatase), PER-2 and PER-4 (peroxidase) a proportional
increase in concentration of these allozymes was observed in the trisomies relative to
diploids. However, no increase was found in EST-1 (esterase) concentration in the
trisomic with an extra gene for this allozyme (Fobes, 1980). In studies of maize plants
with various doses of the 1L chromosome region, Birchler (1979) observed that activities
of G 6 P D , 6 P G D , I D H and E S T were negatively correlated with 1L dosage. H e
concluded that a regulatory gene affecting these isozymes was located on the 1L
chromosome. The levels of M D H and P G M (phosphoglucomutase) were unaffected by
1L dosage because neither the genes for these isozymes, nor their regulatory genes, are
located on that chromosome. However, it is k n o w n that the gene coding for A D H
section 3.1
Ploidy and Gene Expression 3.2
(alcohol dehydrogenase) is located on 1L, but the concentration of this enzyme did not
increase with 1L dosage. It was subsequently shown that this gene dosage compensation
effect was the result of a regulatory gene also located on 1L, closely linked to the AD
locus (Birchler, 1981). In trisomies with an extra ADH gene but not the regulatory
gene(s), ADH activity was proportional to ADH gene dose. Conversely, Birchler and
Newton (1981) showed that when the entire genome is duplicated, expression of
numerous proteins is generally proportional to gene dosage, although a number exhibited
enhanced or depressed concentrations.
Commensurate with a near doubling of cell size, other differences in cell anatomy occur
with increased ploidy. Cell surface area in tetraploids is approximately 1.58 times th
of diploids (Levin, 1983). Therefore, relative to diploids, there is a reduction in cel
surface to volume ratio in tetraploids, which has "manifold repercussions" in enzyme
activities and physiology generally (Levin, 1983). Weiss et al. (1975) demonstrated tha
differences in cell geometry between ploidy levels resulted in fundamental changes to
activities of certain enzymes. It was found that, whereas the activity of some enzymes
was dependent upon cell volume, activity of others related to cell surface area, and as
result, cell size influences physiology through its effect on the balance of enzyme
concentrations.
Price et al (1973) demonstrated a direct correlation between DNA content, cell volume
and nuclear volume in higher plants. This relationship has been found throughout a rang
of plant species (Cavalier-Smith, 1985). The number of pores per unit area of nuclear
membrane is fairly constant throughout the plant kingdom (Maul, 1977). Therefore, the
section 3.1
Ploidy and Gene Expression 3.3
ratio of nuclear membrane area to cell volume will be relatively less in tetraploids
compared with diploids, as will the ratio of the number of nuclear pores to DNA content.
This will affect the relative rate of RNA transport to the cytoplasm (Cavalier-Smith,
1978), which, it is argued, will have ramifications in gene activity and physiology (Levi
1983), and in growth rate (Cavalier-Smith, 1978).
It seems that, in cases of genome duplication, responses in quantitative gene expression
may be affected by both genetic factors and cellular anatomy. The objective of the work
reported in this chapter was to investigate the effect of genome duplication on quantita
gene expression by employing a range of nucellar Citrus tetraploids. The evidence
reviewed in Chapter 1 (section 1.5.2) and above indicated that, for a given species,
genome duplication affected the degree of expression of particular enzymes differently.
The scarce information available (refer Nakai, 1977; Levin et al., 1979) shows that
where genome duplication is considered for a number of intraspecific genotypes, the
effect on gene expression is generally, but not universally, consistent for a particular
enzyme. Therefore, it was postulated that doubling of gene dose in Citrus should
produce a consistent response in quantitative gene expression (as measured by isozyme
band density) across all cultivars for a given gene, but that the response may differ
between genes, due to regulatory effects. The work presented in this chapter aimed to
test this hypothesis.
The large scale experiment reported in this chapter provided evidence of an interplay
between cell anatomy and genetic factors. A genotypic influence on quantitative gene
expression was also demonstrated.
section 3.2a)
Ploidy and Gene Expression 3.4
3.2 Methods
Several small scale studies were conducted in an attempt to gain an overview of
quantitative gene expression differences between diploid and tetraploid counterpart
work revealed that some enzymes were differently expressed between the counterparts
in some cultivars, but no consistent response patterns were apparent. A representat
example of this work is summarised below. In order to further investigate this sit
a large scale experiment was subsequently conducted, which revealed a complex
relationship between ploidy, cultivars and enzymes, with respect to quantitative ge
expression.
a) Small scale study
Crude extracts for isozyme analysis were prepared from small samples (2-3 leaves)
diploid and tetraploid pairs of the Volkameriana, Troyer and Swingle accessions, V0
& V06, TR02X & TR08, SW2X1 & SW1, and SW2X2 & SW2 (refer Chapter 2,
section 2.1.1). The samples were lyophilised as describe earlier (see section 2.2.2
prepared for electrophoresis at a concentration of 25 mg dry matter in 50 /xl of S
extraction buffer (Appendix 1). Aliquots of 0.25 /xl of each of the eight samples
loaded in random order on cellulose acetate gels and electrophoresed (see section 2
Two gels were run concurrently for each of the isozymes IDH, G6PD and MDH
(Appendix 2). The exercise was then repeated with new samples, the treatment order
rerandomised, and the additional isozymes SkDH and 6PGD included.
Densitometric peak heights (AU) of the most prominent isozyme bands were recorded a
section 3.2b)
Ploidy a n d
Gene
Expression 3.5
previously described (section 2.2.3). The DNA concentration of each sample was
determined by the densitometric method (sections 2.2.4a)(ii) and 2.3.3a)(ii)).
The simplest approach to presenting comparative data between diploid and tetraploid
counterparts is to express the relationships as ratios. Diploid-.tetraploid ratios (2X:4X
of isozyme band peak heights for each cultivar pair were calculated. Similarly, the
coinciding 2X:4X ratios of DNA concentrations were calculated. The isozyme peak
height ratio was divided by the DNA ratio to yield the ratio of 2X:4X isozyme band
densities per unit DNA. If genome duplication has no effect on quantitative gene
expression, all ratios of isozyme activities per unit DNA between diploid and tetraploid
counterparts would be 1.0.
b) Large scale study
(i) Sample collection and preparation. This experiment employed 16 accessions,
comprising diploid and tetraploid counterparts of eight cultivars (refer section 2.1.1), v
PA2X&PA14, V02X2&V06, SW2X2 & SW2, TR02X & TR08,
MU2X & MU1, CV2X1 & CV21, RL2X1 & RL20, and BE2X & BE14.
Fully expanded leaves from current or most recent flushes were selected such that they
were as close as possible to physiologically uniform. For the tetraploid accessions, the
University glasshouse collection from which the leaves were taken represented the major
portion of the entire population (the only other plants of these accessions being the
original parent plants and a few cuttings of each held at Bundaberg Research Station).
All source plants had been vegetatively propagated. With the exception of the accessions
section 3.2b)
Ploidy and Gene Expression 3.6
of Volkameriana, which grew vigorously providing copious foliage, all suitable leaves
were collected from every plant of each tetraploid accession. Accordingly, the bulk
samples used in this work constituted, for the tetraploids at least, a large proportion
the total population of suitable leaves from these accessions. Having selected all suit
leaves from the tetraploids of each cultivar, a similar number of leaves was collected
from the diploids (of which there were always more available).
The bulk leaf tissue samples were ground in liquid nitrogen then lyophilized (refer sec
2.2.2a)) and the < 150 /xm sieve fraction was collected. This material was stored at
-80°C.
(ii) Isozyme and DNA analysis. Samples were taken from these bulk tissue collections
and subjected to total DNA estimation by the DAPI fluorometry method (refer Appendix
5 and section 2.2.4b) DNA determinations were repeated up to nine times for each
cultivar, and the DNA concentration data was subjected to one-way analysis of variance
to ascertain whether sample concentrations differed. Separate samples were prepared at
a concentration of 25 mg dry matter per 250 /xl of Soltis + extraction buffer (Appendix
1) for isozyme electrophoresis followed by laser densitometry as described earlier.
(iii) Experiment design. Each gel was loaded with five separate aliquots of the diploid
and five of the tetraploid of a single cultivar, in random order. At any one time seven
gels were electrophoresed, one for each of the isozyme systems studied - so the same
random order of diploid and tetraploid applied for the seven isozymes for the one culti
on that occasion. The procedure was repeated for each of the eight cultivars studied,
section 3.3a)
Ploidy a n d G e n e
Expression 3.7
rerandomised on each occasion. The experiment was replicated three times (Series 1-3).
The exercise thus consisted of:
w
x
X
5 separate tracks per gel (A, B, C, D, E) ^
i i A i i /^v J AV\
r
constitutes 1 gel of 10 tracks
6
2 ploidy levels (2X and 4X)
J
8 cultivars (PA, V O , S W , TR, M U , C V , R L , B E )
X
7 isozymes (6PGD, G 6 P D , IDH, M D H , S k D H , C A P , R B C )
and this entire series was subsequently repeated twice, to give
x 3 replicates (Series 1, Series 2, Series 3).
(iv) Data analysis. The data for diploid:tetraploid isozyme band density ratios per unit
D N A were analysed by one-way A N O V A .
A detailed justification for using this
procedure is presented later (section 3.3b)(iii)).
3.3 Results and Discussion
a) Small scale study
The majority of isozyme bands showed no elevation or depression of gene expression
resulting from genome duplication. However, expression of several allozymes was
clearly affected, but no consistent patterns were apparent.
(i) DNA concentrations. The DNA concentrations in both sets of samples (Table 3.1)
support the earlier finding (section 2.3.3a)(ii)) that Citrus tetraploids generally contain
slightly more D N A per unit dry matter than their isogenic diploid counterparts.
section 3.3a)
Ploidy and Gene Expression 3.8
Table 3.1
D N A concentration in samples used for the small scale study.
dry matter
sample
first
second
v% D N A / m g
diploid
V02X2
TR02X
SW2X1
SW2X2
2.348
1.318
1.484
1.437
2.385
1.508
1.706
1.665
Mg DNA/mg dry matter
sample
first second
tetraploid
2.393 2.381
V06
TR08
1.564 1.643
SW1
1.518 1.658
SW2
1.573 1.679
(ii) Isozyme band density per unit DNA.
mean
2X:4X
ratio
0.991
0.881
1.004
0.954
Although density comparisons cannot be
reliably made between different gels (refer section 2.3.1a)), band density ratios for
diploid and tetraploid sample pairs on one gel should be comparable with the equivalent
ratios of the same cultivar sample on another gel. Because these ratios define the rel
degree of isozyme activity per unit DNA, they represent the relative magnitude of gene
expression between comparable diploid/tetraploid counterparts. Ratio values less than
indicate isozyme activity was enhanced in the tetraploid and conversely for values gre
than 1.0. The notion, ratio of diploid .-tetraploid isozyme band density per unit DNA,
central to this project, and for convenience I will coin the symbol p to occasionally
to this concept in the following discussions.
The mean diploid:tetraploid isozyme band density ratios per unit DNA (p) and their
coefficients of variation for the four pairs of accessions are presented in Table 3.2.
'normal range' (two standard deviations either side of the grand mean) was 0.471-1.414,
and 3.3% of values fell outside this range. The range of values between 0.9 and 1.1
represents approximately 20% of the 'normal range' and it was considered that, if p fel
section 3.3a)
Ploidy and Gene Expression 3.9
Table 3.2
Mean diploid:tetraploid isozyme band density ratios per unit D N A
and their coefficients of variation for G6PD, IDH, M D H , SkDH and 6 P G D
from V O , T R and S W accessions.
TR02X:TR08
mean CV.
0.558 21.61
SW2XLSW1
mean CV.
G6PD-C
V02:K2:V06
mean C.V.*
1.272 33.45
1.254
55.65
SW2X2:SW2
mean CV.
0.983 19.05
IDH-m
0.982
23.52
0.771
28.77
0.892
14.79
1.053
8.24
MDH-a
MDH-ls
MDH-lm
MDH-2s
MDH-2f
0.896
0.988
0.881
nil
0.952
20.95
1.199
0.780
nil
0.791
0.838
10.60
0.652
0.884
nil
0.974
0.954
10.55
0.970
0.954
nil
0.985
0.974
10.49
SkDH-a
SkDH-c
SkDH-d
0.939
0.878
nil
6.77
1.076
nil
1.094
3.81
6PGD-b
6PGD-d
0.904
nil
17.06
6.72
8.07
0.48
4.15
22.77
34.95
23.63
0.911
nil
0.817
10.87
0.917
0.979
9.03
1.21
2.89
1.127
nil
9.84
10.19
10.57
5.24
2.45
24.56
20.59
8.74
1.071
nil
0.919
31.77
0.951
nil
20.61
13.70
- isozyme band nomenclature is in accordance with that described in Chapter 2
- 'nil' signifies absence of a band in that location.
- * coefficient of variation
between 0.9 and 1.1 there was no marked effect of ploidy on isozyme band density.
The data indicated that a complex situation exists where isozyme activity per unit DNA
was variously enhanced, reduced or unaffected in tetraploids relative to their diploi
counterparts. In a minority of cases isozyme activity was enhanced in the diploid
(p > 1.1). More frequently, isozyme activity was enhanced in the tetraploid (p < 0.9),
in the majority of cases there was no marked difference (p «1). No consistent effect of
enhanced or reduced activity for any particular isozyme band was apparent across all
genotypes. Nor were there any consistent effects within any cultivar.
section 3.3a)
Ploidy and Gene Expression 3.10
Consistency of response was expected between the two Swingle accessions (which should
be genotypically identical); however this was not the case. Furthermore, coefficients o
variation was unacceptably high (>20%) in several cases.
The plant material used was derived from very small initial samples. It is possible tha
the occasional inconsistencies and high coefficients of variation were caused by
differences in the physiological states of the samples. All further work was conducted
on bulked samples comprised of larger numbers of leaves (see section 3.2b)(i)). As will
be demonstrated in following sections, when this approach was adopted, variability
between replicates was generally low. Furthermore, depending upon developmental
stage, different polymorphisms between diploids and their isogenic tetraploids have
occasionally been found (DeMaggio and Lambrukos, 1974; Yamashita, 1977). There is
a significant body of evidence that expression of isozyme variants changes with
developmental stage or environment (Scandalios, 1974; Freeling, 1983), and there is
evidence of this phenomenon in Citrus (Warner and Upadhya, 1968). Accordingly,
samples were subsequently collected from uniform tissue types for all plants studied.
Strict control was exercised over the leaves sampled, such that only fully expanded le
from current or most recent growth flushes were selected. Source plants were all grown
in a glasshouse under uniform management conditions.
An important observation from the study above was that genome duplication has some
effect upon quantitative gene expression. This was demonstrated by the observation that
diploid:tetraploid ratios of isozyme activity per unit DNA (p) were considerably great
or less than 1 in certain cases. Because the data sets were small and lacked replicatio
section 3.3b)(i)
Ploidy and Gene Expression 3.11
statistical analysis was not feasible. These observations warranted further investigati
to see if some systematic effect could be identified.
b) Large scale study
This experiment showed that different groups of related cultivars exhibited distinct
isozyme activity responses to genome duplication. The evidence suggested that this
response may derive from a cellular anatomy factor which is genetically determined.
(i) DNA concentrations. The mean DNA concentrations of the 16 accessions covered
a broad range (Table 3.3). However, with the exception of the cultivars VO and TR,
the mean ratios of diploid:tetraploid counterparts were close to 1.0. This implied that
for the majority of cultivars, tetraploid cell size was close to double that of the dip
(the exceptions, VO and TR are discussed below).
Occasionally, anomalous values were observed in some DNA estimations. Sample size
for DNA estimations was only 10 mg (refer Appendix 5) and the anomalies possibly
originated during sample weighing (the balance employed was subsequently found to
occasionally give inaccurate readouts). Accordingly, additional determinations were
conducted for certain accessions, and therefore, numbers of determinations (n) vary.
The data sets for each accession were subjected to outlier analysis using the r ratio
method of Dixon (1950, 1951), applying the critical values at a = 0.10 in accordance
with Sokal and Rohlf (1981). Data sets where an outlier has been removed are indicated
(*) in Table 3.3, and the very low coefficients of variation amongst the remaining value
Ploidy and Gene Expression 3.12
section 3.3b)(i)
Table 3.3
M e a n D N A concentrations and related data of sixteen accessions
of diploid and tetraploid Citrus.
accession
D N A concentration
iixg/mg dry matter
ploidv
n
PA2X
PA14
2X
4X
7
7
4.2025
V02X2
V06
2X
4X
5*
10
4.2257
SW2X2
6
6
4.7808
SW2
2X
4X
TR02X2
TR08
2X
4X
6
6*
2.3737
MU2X
MU1
2X
4X
CV2X1
CV21
2X:4X ratio
of D N A cone.
CV.
3.7837
15.65
8.16
}
1.1107
5.4022
2.91
9.87
0.7822
4.3470
12.30
15.06
1.6661
13.58
9.86
4*
4*
11.3913
11.8030
0.60
2.36
2X
4X
5
5
15.1859
14.5167
2.41
1.77
}
}
}
}
}
RL2X1
RL20
2X
4X
5
5
4.6568
5.1158
11.40 1
10.84 J
BE2X
BE14
2X
4X
4*
5
4.3701 1.76
4.0766
7.30 '
1.0998
1.4247
0.9651
1.0461
0.9103
1072
°
- n indicates number of determinations conducted
- * indicates an outlier was removed
- C V . - Coefficient of Variation amongst determinations
in each case attest to their uniformity.
Analysis of variance of the D N A data showed that the tetraploid V 0 6 had significantly
higher DNA concentration (P<0.01) than its diploid counterpart V02X2. Conversely,
the tetraploid TR08 had significantly lower DNA concentration (P<0.01) than the
diploid TR02X. There were no significant differences in DNA concentration between
section 3.3b)(ii)
Ploidy and Gene Expression 3.13
any of the other diploid/tetraploid pairs of cultivars. This finding was important in
of the subsequent discovery that the cultivars VO and TR represent the opposite extrem
of p values (see section 3.3b)(iii)).
(ii) Isozyme bands. The number of individual bands varied between cultivars and
between isozymes, and the total number of data points (individual band AU values) was
4,051 (excluding nine missing values), derived from 168 separate gels (8x7x3; refer
section 3.2b) (iii)). The mean density (AU) of each set of five separate diploid bands
calculated and divided by the mean of the five tetraploid bands on the same gel to
determine a 2X:4X ratio of band densities for each gel. This ratio was then divided by
its respective DNA ratio (Table 3.3) to derive the 2X:4X band density ratio per unit
DNA 06), as described above.
Appendix 3 (41 pages) presents the isozyme data for every band, the means and
coefficients of variation for diploid and tetraploid samples from each gel, and the
individual p values. These data are summarised on the following pages. The mean
isozyme band densities per /xg DNA of the three Series, for each of the 16 accessions a
shown in Table 3.4. Table 3.5 presents the 2X:4X ratios of band density per unit DNA
for diploid and tetraploid counterparts (p) showing the values from each of the three
Series (these are the data shown in bold print in Appendix 3) and their means are shown
in Table 3.6. In only three of the 154 mean data sets (< 2% of cases) was the coefficie
of variation (CV) greater than 20% (Table 3.6). Eighty percent of the CVs were below
10%, indicating the reliability of the individual data. Similarly, CVs of the five sam
bands in each gel (Appendix 3) were also low.
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section 3.3b)(ii)
Ploidy and Gene Expression 3.16
Table 3.6
M e a n s of 2 X : 4 X ratios per unit D N A from electrophoresis Series 1 - 3, and their coefficients of variation
mean
CV
6PGD-a
6PGD-b
6PGD-C
6PGD-d
6PGD-e
1.442
1.480
1.479
1.185
1.324
4.875
5.570
5.369
19.188
G6PD-a
G6PD-b
G6PD-C
1.390 30.758
rDH-s
-
nil
1.187 19.365
1.448 5.295
N
0.987
MDH-a
MDH-b
MDH-ls
MDH-lm
MDH
-2s
MDH-lf/-2m
-2f
MDH
MDH-c
MDH-d
CV
2.020 9.187
1.914 10.056
nil
1.678 14.880
N
1.713 18.732
nil
N
IDH-m
[DH-f
IDH-a
mean
-
nil
1.203 9.745
nil
nil
nil
1.061 11.907
0.993 3.844
1.330 15.418
1.740 28.990
2.034
1.855
1.849
1.588
10.082
12.856
5.277
3.341
nil
CV
0.862
0.870
0.878
1.001
1.004
6.979
4.079
13.296
4.233
8.451
nil
0.761 4.894
0.821 6.876
nil
nil
0.950 3.196
N
N
nil
1.730 18.500
nil
CV
mean
CV
nil
nil
1.112 2.176
1.315 5.554
nil
nil
0.661 3.961
0.677 5.222
1.073 1.465
1.095 11.952
1.077 18.267
1.073 9.537
0.619 4.197
0.655 8.319
0.623 7.261
0.982 1.298
0.843 5.350
0.705
0.623
0.621
0.568
nil
0.902
0.904
0.874
1.004
0.823
mean
nil
-
nil
nil
CV
0.671 0.564
1.909
2.912
2.036
8.733
1.169 12.785
0.878 4.639
N
mean
0.567
0.805
0.570
0.562
0.651
0.639
0.632
0.653
1.512 11.130
1.714 12.297
1.632 10.840
1.464 1.412 5.548
1.996 1.468 -
nil
nil
3.808
2.874
2.258
15.871
.
1.066 3.086
1.059 3.299
1.088 -
nil
0.715 10.125
nil
nil
nil
1.070 6.366
1.000 2.121
nil
N
nil
nil
nil
1.153 7.672
1.192 6.210
nil
nil
nil
nil
0.981 2.480
0.791 1.963
0.770 0.759 5.996
0.977 3.016
CAP-a
CAP-b
CAP-c
1.030 .
0.973 8.051
1.035 -
1.691 9.136
1.602 2.737
RBC-c
RBC-d
1.908 .
1.099 0.643
1.640 7.375
1.668 8.127
nil
nil
0.980 0.425
0.742 4.955
nil
nil
nil
0.648 0.983
N - indicates band not clearly resolved or too faint to reliably measure
1.090 6.365
1.556 22.466
0.719 3.549
0.593 3.351
nil
nil
1.321 6.742
nil
nil
nil
0.903 0.799 2.692
nil
N
0.657 3.963
0.565 17.521
0.658 5.948
0.583 -
1.942 9.173
1.552 5.797
5.419
1.219
2.128
8.876
6.665
nil
nil
nil
nil
-
1.295
1.277
1.289
1.205
1.352
1.452 2.036
nil
4.261
4.130
3.680
CV
1.328 4.669
0.962 10.879
-
nil
mean
1.007 3.099
0.959 0.074
16.559
7.315
nil
1.360 12.832
0.955 10.946
0.774 7.203
0.914 14.756
0.880 0.906 15.338
SkDH-a
SkDH-b
SkDH-c
SkDH-d
nil
mean
nil
1.810 7.365
N
nil
nil
1.795 14.756
1.211 5.037
1.144 5.371
N
nil
1.165 5.043
0.955 11.249
mean
0.842 9.694
0.863 4.691
nil
nil
nil
nil
0.820 2.570
0.861 1.053
0.561 0.668 7.206
0.894 7.754
0.810 9.106
N
nil
0.918 2.282
0.858 1.402
nil
nil
nil
nil
nil
1.298 3.211
1.185 6.681
CV
nil
0.765 1.527
0.795 2.270
nil
nil
0.882 4.874
1.017 3.148
1.602 3.279
nil
nil
1.078 7.877
nil
0.879 7.702
1.284 7.962
nil
0.892 4.068
1.729 7.897
1.340 2.191
nil - indicates band absent
These data show that the observed effects are repeatable, but provide no obvious
explanation for the apparently erratic differences in the values of p between cultivars
any given band, and between isozyme bands for any given cultivar. However, the means
of the three Series (Table 3.6) allow the ratio data to be scrutenized to distinguish
systematic effects. Although it is probable, there is no certainty that any individual
isozyme band occupying the same location (Rf value) for two or more different cultivar
is the result of the same allozyme. Furthermore, few isozyme bands exhibited activity
section 3.3b)(ii)
Ploidy and Gene Expression 3.17
across all eight cultivars. Because it may therefore be imprudent to directly compare
individual values for bands of similar mobility across all the cultivars, the data must be
viewed holistically. With this in mind, some interesting patterns begin to emerge from
the mean ratio values in Table 3.6.
Where a particular band is represented in several cultivars, there was often a wide range
of ratios observed amongst the cultivars. This indicates that there was no consistent
effect on isozyme expression, regardless of cultivar, resulting from genome duplication
- which contradicts the hypothesis proposed at the beginning of this chapter. However,
each cultivar exhibits similar ratios for most of its isozymes - for example, the Troyer
citrange (TR) ratio data are consistently less than 0.9, which indicates that, for every
band, isozyme activity per unit DNA was greater in the tetraploid than the diploid.
Conversely, for Volkameriana lemon (VO), all ratio values were considerably greater
than 1.1, i.e. isozyme activity per unit DNA was consistently lower in the tetraploid
relative to the diploid. By contrast, the ratio values for Murcott mandarin (MU) appear
to be distributed around 1.0. This situation becomes clearer when the frequency
distributions of the ratios for each cultivar are plotted (Fig 3.1).
The frequencies of the ratios in Table 3.5, rounded to one decimal place, are tabulated
for each cultivar in Table 3.7. Far from being randomly distributed, as appeared to be
the case in the data of the small scale study, the frequency distributions (Fig. 3.1) of t
ratio data each show some central tendency.
Ploidy and Gene Expression 3.18
section 3.3b)(ii)
Table 3.7
Frequencies of 2X:4X ratios of isozyme band density per unit D N A
for isozyme data in Table 3.5.
ratio
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
total nl
of bands §
PA
VO
SW
TR
MU
CV
RL
BE
-
-
3
-
-
-
-
-
-
-
-
-
-
4
14
21
13
4
1
1
30
22
6
2
-
-
5
7
17
19
1
*
-
-
-
-
2
11
5
5
3
9
4
5
1
-
-
1
2
2
9
9
4
10
5
2
5
2
49
51
-
2
1
1
-
-
-
-
-
-
-
-
2
1
3
9
9
3
5
3
1
-
-
-
-
-
-
1
-
-
-
-
-
1
4
1
7
11
18
6
3
2
1
1
-
1
3
14
13
3
-
2
1
-
1
1
-
-
-
2
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
58
63
37
36
57
55
§ - total number of bands per cultivar for which band density ratio data was recorded (cf. Table 3.5)
* - ratios which did not occur (ie. frequency = 0) are indicated by a dash
section 3.3b)(ii)
Ploidy and G e n e Expression 3.19
30
—
PAIreq.
PA
25
\
& 20
c
cu
3
cr 15
ffi
i-
—
TR
25
cr
30
i
TR»«>
&
c
CO
20
3
\
cr 15
cu
•^
< 10
10
; i
0.
5
5
0
()
/ M
n
1
0.5
1.5
2
0
2.5
0.5
i
. ... • . Ws . i\. . .
1.5
ratio
on
ou
30
—
25
&
BE
BEtac,
20 .
15
CU
()
30
1
0.5
1.5
2
0
25
3
cu
o? 15
/'
0
0.5
VO
fc 20 '.
c
01
3
cr
15 "
03
i. I
0
2.5
25 ;
1
/
5
2
in
A
10
1.5
oU '.— voire*
/\
/\\
I
1
1
ratio
SW
»—
0.5
ratio
•
5
" 20
c
10
5 "
SWtre^
25
RL
_i
cr
\1
0
Rllieq.
cu
1 1
/
10
5
—
& 20
c
cu
3
cr 15 '
A
/!
/
3
UJ
CD
2.5
25
•
c
0)
cr
2
ratio
1
1.5
l
10
5 "
Q
2
2.5
0
<1.0
ratio
0.5
1
A\
1.5
2
2.5
ratio
>1.0
«1.0
JU
ou
25
—
—
MUta).
03
c:
i
n
3
.
R
\
>
u
; I
\
\ . ^A
,J
0.5
ratio
lb
IU
5
5 [n
CV
20
5* 20
c
t 15
i 10
25
MU
•
CVfceq.
1.5
2.5
J-i
• A
\
0.5
1.5
2.5
ratio
Fig. 3.1 Frequency distributions of the 2 X : 4 X ratios of isozyme band densities per
unit D N A from eight Citrus cultivars (3 groups have been informally assigned by relating the
approximate centre of the distribution to the ratio value of 1)
section 3.3b)(iii)
Ploidy and Gene Expression 3.20
(iii) Analysis of the data. Because different allozymes may exhibit similar Rf values, an
because rarely was any particular isozyme band present in all cultivars, it was impracti
to compare the ratio values of different cultivars for specific bands (ie. means across t
rows in Table 3.6). Furthermore, because variances of the three Series differ from one
band to another in Table 3.5 (cf. CVs in Table 3.6), it was also invalid to use a factori
analysis of variance (cultivars X ploidy) with the Series as replicates. However, it was
earlier affirmed that the low coefficients of variation verify the reproducibility of the
data. These factors presented a problem with regard to how the data should be analysed.
From inspection of the frequency distributions of the 2X:4X isozyme band density ratios
per unit DNA in Fig. 3.1, it appeared that both the means and the variances may differ
amongst cultivars. It will be shown later that this was indeed true. Because the means
of the eight distributions differ (Table 3.8), a Kolmogorov-Smirnov test (Harrison and
Tamaschke, 1984) could not be applied to compare cultivar distributions because this test
is known to be vulnerable to differences between means. However, skewness and
kurtosis of the data sets (Table 3.8) show that the distributions are non-normal and diff
from each other in shape. Although these irregularities lead to problems in the analysis
of the data, the distributions clearly fall into three distinct groups as shown in Fig 3.
(i) those with centres of distribution less than 1.0, (ii) those with centres approxima
1.0, and (iii) those with centres greater than 1.0. Similar relationships were observed i
the earlier studies with respect to individual isozyme bands, however, in this case they
relate to a consistent response amongst all the various isozymes in each individual
cultivar. Statistical analysis of the distribution means was required to confirm whether
the cultivars could legitimately be assigned to the above groups.
section 3.3b)(iii)
Ploidy and Gene Expression 3.21
Table 3.8
Statistics of the frequency distributions of 2X:4X ratios of
isozyme band densities per unit D N A for eight Citrus cultivars.
cultivar
PAVOSWTRMUCVRL BE
n 49 51 58 63 37 36 57 55
mean 1.3143 1.7196 0.9103
Std. dev. 0.2979 0.2433 0.1210
Skewness 0.6960* 0.1563N 0.6408*
kurtosis-0.0949*-0.6268* 0.8388*
0.6587
0.0855
0.7421*
0.5328*
1.1054 1.1000
0.2368 0.1912
2.4284* 0.0000N
5.5172*-0.2930*
* - skewness or kurtosis significant (P<0.05)
1
N - skewness or kurtosis not significant (P<0.05) I
1.2806
0.2356
0.8472*
1.7540*
0.8873
0.2582
2.2124*
5.1313*
- ref. Appendix Table A 6 , in
Snedecor and Cochran (1967)
Analysis of variance procedures ( A N O V A ) assume that the data are normally distributed
and treatment groups have equal variances (Cochran, 1947; Cochran and Cox, 1957;
Eisenhart, 1947). However, ANOVA is quite robust against non-normality, particularl
where sample sizes are large (Cochran, 1947; Li, 1964; Snedecor and Cochran, 1967;
Harrison and Tamaschke, 1984). ANOVA is also relatively robust to differences in
variances between treatments (Cochran, 1947; Cochran and Cox, 1957; Harrison and
Tamaschke, 1984). Bartlett's test (Steel and Torrie, 1960) was used to compare cult
variances. This procedure is very sensitive and, predictably, indicated that the v
differed significantly (P < 0.05). Inspection of the frequency distributions (Fig.
standard deviations (Table 3.8) suggested that the SW and TR cultivars may be the
of this difference, due to apparently lower variances. When these two data sets were
removed Bartlett's test indicated that the variances of the remaining distribution
significantly different:- Bartlett's test x25 = 3.669 cf. x25,o.os = HI
section 3.3b)(iii)
Ploidy and Gene Expression 3.22
If the inequality of variances was ignored, a one-way ANOVA model could be used to
compare the cultivar means. The variances of TR and SW are smaller than those of all
other cultivars, therefore the pooled variance will be greater than these two indivi
variances. Consequently, if the pairwise LSD values, which are calculated from the
pooled variance, indicate significant differences between TR or SW and any other
cultivar, then these differences would be genuine. Furthermore, if differences indic
between groups of cultivars are highly significant (ie. P < 0.01 rather than P < 0.05
would imply real differences despite the violation of the assumption of equal varian
Owing to the robustness of the ANOVA, this procedure is acceptable when used to obtai
comparisons between means of pairs of data sets. This allows the cultivars to be grou
according to differences between their means. The one-way ANOVA, with the three
Series as replicates, provides for the calculation of individual LSD's between any tw
cultivars. Table 3.9 shows the ANOVA table for the frequency data, and the means of
the frequency distributions of the eight cultivars in ranked order with the LSD0
01
va
for adjacent pairs.
The ANOVA indicates highly significant differences (P<0.01) between cultivars such
that the mean frequencies of 2X:4X ratios of pooled isozyme band densities per unit
DNA differ as follows - TR <^ BE, SW <^ CV, MU < RL, PA < VO. This ranking
of the cultivars revealed by the ANOVA, produces a pattern of significant differences
which is in accordance with the groupings informally assigned in Fig. 3.1. The
important distinction exists between the group of cultivars TR, BE and SW (for which
the mean frequencies of 2X:4X band density ratios per unit DNA are < 1.0), which are
significantly different (P<0.01) from the cultivars CV and MU (mean p frequencies
section 3.3b)(iii)
Ploidy and Gene Expression 3.23
Table 3.9
A N O V A table, means and exact pairwise LSD's for frequency data of
DNA-corrected 2X:4X isozyme band density ratios of eight Citrus cultivars.
source d.f. M.S. F
treatments
error
7
398
5.80431
0.04652
cultivar mean 2X:4X ratio
TR
0.65873 af \
BE
0.88727 b h "
SW
0.91034 b J
1.10000 c h "
CV
MU
1.10541 c J
RL
1.28596 d h "
PA
1.31429 d ii
VO
1.71961 e 1 "
124.77 **
LSDrj.oi
0.10302
0.10507
0.11845
0.13069
0.11786
0.10876
0.11167
f - values followed by different letters differ significantly (P<0.01)
= 1.0), which are in turn significantly different (P < 0.01) from the cultivars RL, P A and
VO (mean p frequencies >1.0). Thus, the cultivars can be assigned to three broa
groups.
It is interesting to examine the two cultivars at the extremities of the groupings, viz. T R
and VO. These are the only cultivars where the DNA concentrations of diploid/t
pairs differed significantly (P < 0.01). The 2X:4X ratios of DNA concentration
cultivars were 1.4247 and 0.7822, respectively (Table 3.3). The DNA ratios for
other six cultivars are all close to 1.0. The mean p values for TR and VO will
be strongly influenced by their DNA ratios, unlike the other cultivars. However
the data sets for TR and VO are calculated without the use of the DNA correctio
factors, the mean uncorrected 2X:4X ratios of isozyme activity are 0.934 for T
1.342 for VO. When compared with the mean DNA-corrected 2X:4X ratios of isozyme
section 3.3b)(iv)
Ploidy and Gene Expression 3.24
activity of the other cultivars (Table 3.9) it is clear that TR and VO still fall with
broader cultivar groupings as assigned above. This is not intended to imply that the
DNA ratio values for TR and VO may be incorrect, simply that they have not influenced
the assignment of these cultivars to their respective broad groups.
^Interpretation. The important outcome of the analysis is the clear distinction betwee
the three groups of cultivars. The fact that the mean values of CV and MU, designated
= 1.0, are actually slightly greater than 1.0, is of no intrinsic importance to the
conclusions arising from the analysis (see below). Nor are the outcomes that TR <^ BE
and SW, and that VO > RL and PA, of consequence to the conclusions below. The
important difference is that the two cultivar groups (TR, BE, SW cf. RL, PA, VO) are
significantly different from each other and from MU and CV.
The highly significant (P<0.01) differences between the three groups of cultivars, wit
mean ratios < 1.0, =1.0 and > 1.0, respectively, become fundamentally important when
the taxonomic relationships between the cultivars is examined. The cultivars TR, BE an
SW, with mean ratio <1.0, are distinctly different from the other cultivars, being
Poncirus trifoliata hybrids (two citranges, i.e. orange x P. trifoliata, and a citrume
i.e. grapefruit X P. trifoliata, respectively). These cultivars exhibit trifoliate lea
morphology whereas the others are unifoliate. The cultivars MU and CV, with mean
ratios =1.0, are both mandarins (Citrus reticulata). The group of cultivars with mean
ratios > 1.0, comprises two lemons and an orange. Under the Swingle taxonomy these
three cultivars constitute two distinct species (see Chapter 1, section 1.2.1), wherea
the new three-species taxonomy they are considered to be biotypes of hybrid origin, C.
section 3.3b)(iv)
Ploidy and Gene Expression 3.25
reticulata X C. grandis in the case of orange, and in lemon, a trihybrid of C. medica X
C. grandis X Microcitrus sp. (Barrett and Rhodes, 1976). Therefore, the three groups
of cultivars, as defined by mean ratios, are each taxonomically discrete. The three
groups of cultivars are also morphologically distinct and the isozyme polymorphisms
described in Chapter 2 show differentiation between the groups (Fig. 3.2). In light of
these findings, it is not surprising that the results of the small scale study were baf
given that the work included only VO, TRO and SW.
The mean isozyme band densities per fig DNA for all isozyme bands of all 16 accessions
were presented in Table 3.4. Pursuant to the observations made above, it is interesting
to look closely at the relationships between individual isozyme band densities per unit
DNA for each diploid/tetraploid pair of accessions. These relationships are depicted in
Fig. 3.3 which uses the data from Table 3.4. The distinction between the three groups
of cultivars described above is reflected in the slopes of the lines - upwards in the
trifoliates (i.e. greater band densities in the tetraploids), almost horizontal in the
mandarins, and downwards in the orange/lemon group (lower band densities in the
tetraploids). The general level of isozyme activity per unit DNA is considerably lower
in the two mandarins compared with the other cultivars, emphasising the demarkation
between the groups. In particular, the diagrams illustrate the remarkable consistency in
response of relative isozyme activity between ploidy levels across the range of isozymes
- rarely do response slopes run counter to the trend for the cultivar group in question.
In each case where a response slope does run counter to the cultivar trend it is RBC
isozymes that are involved. Considering the importance of this enzyme in plant
physiology further investigation of this anomaly may be warranted.
section 3.3b)(iv)
Ploidy and Gene Expression 3.26
mean ratio frequency
relative to 1.0
TR
<0.1
BE
>1.0
= 1.0
SW
CV
MU
RL
PA
VO
6PGD
G6PD
m
S
IDH
-2f
-2m & -If
-2s
-lm
-Is
MDH
SkDH
CAP
a?
d
RBC
Fie. .3.2 . Isozvme.band patterns (cf. Chapter 2) of the three cultivar groups
i § aKijifiiiiisilix.
( V N Q S-rl/nv) V N Q llun JBC! Alisusp p u e q S L U A Z O B
( V N Q 6-rf/nV) V N Q H
sd /viisuap &ufeo 5'JJ'ZOS
section 3.4
Ploidy and Gene Expression 3.28
The coincidence of the three cultivar groups with three distinct taxonomic groups
indicates a clear genetic differentiation with respect to the effect of genome duplicatio
on quantitative gene expression. Considering the diversity of physiological functions of
the various isozymes, and the fact that activity of each isozyme may be regulated by a
number of factors, it seems unlikely that epistasis, the control of dosage of regulatory
genes or concentrations of regulatory compounds, would be responsible for the observed
relationships. Apparently, some physiological or cellular anatomical feature which can
have a more or less general effect on the quantitative expression of genes is involved.
The observations above indicate that this feature is genetically influenced.
It would be interesting to investigate the nature of this feature which can exert a gener
effect on quantitative gene expression. It seems reasonable to implicate differences in
cellular morphological characteristics of the two ploidy levels, as described in the
Introduction (section 3.1).
3.4 Conclusion
Quantitative gene expression has been examined above in terms of DNA concentration.
Although it was explained earlier that DNA concentrations were used to adjust isozyme
activity data to compensate for cell size differences, this must not be misconstrued to
suggest that relative cell sizes between ploidy levels are exactly the same for all Citru
genotypes. Cell size is, to some extent, genetically determined (Nurse, 1985). It may
be that the differences observed above between Citrus groups with respect to the effect
of genome duplication on general quantitative gene expression, are associated with
section 3.4
Ploidy and Gene Expression 3.29
differences between the groups in regard to relative cell sizes between ploidy levels,
which in turn may be genetically influenced. The most extreme cultivars with regard to
relative DNA concentration between ploidy levels, viz. VO and TRO, exhibited highly
significant differences in relative isozyme activity between diploid and tetraploid, and
relationship between concentrations of one cultivar were the inverse of the other. These
two cultivars are the most phylogenetically disparate of the eight (Scora, 1988) and it is
intriguing to surmise that they may represent the extremities of a continuum of the eight
cultivars, with different relative cell sizes between ploidy levels of each cultivar. To
investigate that would require detailed study of actual cell sizes, and is beyond the scop
of this thesis.
The observations from this work disproved the hypothesis proposed at the beginning of
the chapter that, for a given gene, doubling of gene dose should produce a consistent
response in quantitative gene expression across all cultivars. The above interpretation o
the data provides an explanation for the observed responses. As was predicted by the
hypothesis, different responses were demonstrated for different isozymes. The
manifestations of polyploidy are not simply an effect of genome duplication, but are
influenced by the degree of heterozygosity of the constituent genomes (refer Chapter 1,
sections 1.1 and 1.4). Furthermore, by investigating somatic polyploids from closely
related genotypes, this study showed that the different manifestations of polyploidy are
dictated by only slight differences in the genotype, not merely by gross differences in th
degree of heterozygosity.
The effect of tetraploidy on growth of Citrus plants, relative to that of diploids, could
section 3.4
Ploidy and Gene Expression 3.30
possibly be influenced by the observed differences in quantitative gene expression. If thi
was the case it might be expected that a similar grouping of the cultivars as seen above
could also be found with respect to growth characteristics of the plants. This is the
subject of the following chapter.
section 4.1
Ploidy and Growth 4.1
Chapter 4
THE RELATIONSHIP BETWEEN PLOIDY
AND G R O W T H IN CITRUS
4.1 Introduction
The primary aim of my ongoing tetraploid Citrus breeding activities is to produce a range
of tetraploid rootstock varieties to complement the commonly used diploid rootstock
cultivars.
There is considerable evidence that tetraploidy in Citrus impairs vigour
(Barrett and Hutchison, 1978; Lee, 1988b) and tetraploid rootstocks have the potential to
confer dwarfed growth habit on normal diploid scion cultivars. It is believed that
dwarfed Citrus trees m a y offer important agronomic advantages over normal trees. T o
this end a variety of approaches has been investigated over the years, but no dwarfing
strategy has yet been commercially adopted (Lee et al., 1990). The goal of the present
project was to augment m y tetraploid rootstock breeding program by gaining an insight
into the genetic characterisitcs of somatic tetraploidy in Citrus and its hybrids. Also, it
was hoped that a molecular screening protocol could be developed which would enable
differentiation between potentially useful dwarfing tetraploid rootstocks and those that
would result in normal tree growth. This would facilitate considerable savings in cost
and time in the field trials which must be conducted to assess the rootstocks.
Tree growth rate data are prone to variability (Pearce, 1976), and agronomic research
and plant breeding with tree crops is notoriously longeval. Therefore, obtaining reliable
section 4.1
Ploidy and Growth 4.2
growth rate data typically takes years. Variation between plants in the occurrence and
duration of growth flushes, and irregular dormant periods make short-term studies
difficult to conduct. I have found that Citrus plants propagated from cuttings and throug
tissue culture are variable in their growth characteristics. Conversely, plants propagate
by budding onto rootstocks generally exhibit more uniform growth. However, scion
growth can be influenced by rootstock. My work is the only program being conducted
in Australia with Citrus polyploids. Many of the tetraploids have not yet reached fruitin
age, so seedling plants were not available for growth studies or for use as rootstocks to
conduct comparative investigations with the accessions studied in the preceding chapter.
The only Citrus experiment ever published which measured growth from replicated
treatments of tetraploid and diploid rootstocks investigated 17 scion cultivars propagat
on Poncirus trifoliata (Mukherjee and Cameron, 1958). Extreme variability was
observed and no clear conclusion could be made with regard to the effect of tetraploid
rootstocks on scion growth. As will be shown later, highly reproducible dwarfing was
achieved in my own studies when tetraploidy in the rootstocks was assured through
rigorous confirmation by microscopy. It is possible that such screening was not carefull
done in the Mukherjee and Cameron (1958) experiment and that some presumed
tetraploids were in fact zygotic diploid hybrids. All other published accounts of growth
differences between diploid and tetraploid Citrus comprise empirical and non-replicated
studies (for review see Lee, 1988b; Lee et al, 1990).
Differences between cultivar groups at the genetic level were revealed in the preceding
chapter. It was hypothesised that a relationship would exist between growth and those
section 4.2a)
Ploidy and Growth 4.3
observed gene expression effects of genome duplication. This would be demonstrated if
similar groupings of the cultivars as were found previously for isozyme activity were al
revealed with respect to growth characteristics. Accordingly, studies on the growth
characteristics of tetraploids were conducted, and are the subject of this chapter.
Nursery plants for the isozyme work reported in the previous chapter were considerably
disfigured by regular pruning for sample collection, and were not considered suitable for
use in growth studies. Short-term growth rate experiments were attempted with the 16
diploid and tetraploid accessions examined in the preceding chapter using both in vitro
and in vivo vegetatively propagated material. These were unsuccessful due to extreme
variability. A field trial to investigate the growth effects of Troyer tetraploid rootst
was established at the commencement of this project. Early results of this experiment
are presented below. A field study of the growth characteristics of the 16 diploid and
tetraploid accessions used in the preceding chapter is also presented.
4.2 Methods
a) In vitro growth studies
Numerous tissue culturing protocols for Citrus and its hybrids have been published
(Button and Kochba, 1977; Kochba and Spiegel-Roy, 1977; Spiegel-Roy and Vardi,
1984; Vardi, 1988). The high degree of control possible over the in vitro environment
offered a potentially useful approach for studying growth. Callus culturing has been
achieved with a range of genotypes (Murashige and Tucker, 1969; Vardi, 1987).
Accordingly, several initial investigations, an example of which is presented below, wer
section 4.2b)
Ploidy and Growth 4.4
conducted to ascertain the usefulness of callus cultures for growth rate studies.
This study aimed to produce callus cultures of both diploid and tetraploid accessions of
the cultivars Volkameriana, Troyer and Poncirus trifoliata. Explants consisted of a
freshly excised axillary bud complete with a portion of stem sliced longtitudinally. Ca
formation has been shown to be enhanced by the absence of sucrose (Giladi et al., 1977)
in the culture medium and by wounding of explant source (Altman and Goren, 1977).
The medium of Chaturvedi and Mitra (1974) with 0.7% agar was used and a separate
treatment incorporating this medium with nil sucrose was included. Another treatment
entailed explants from sites on the donor plants which had been wounded (by removal of
the leaf lamina) four days prior to collecting the explants. All explants were surface
sterilised and placed on autoclaved medium in individual culture phials then stored in
darkness at constant 25°C. The experiment was replicated three times.
The diameters of the resultant calluses were measured after 38 days and the data were
subjected to factorial analysis of variance (cultivars x ploidy x treatment X reps).
b) In vivo nursery growth studies
Difficulties were also encountered with in vivo glasshouse studies. Three separate
replicated experiments were attempted using cuttings of the 16 accessions employed
above. In each case extreme variability was encountered in adventitious root production
and shoot growth.
The propensity of Citrus cuttings to produce adventitious roots varies extraordinarily
section 4.2c)
Ploidy and Growth 4.5
between cultivars (Salomon and Mendel, 1965; Sabbah et al, 1992), and was also shown
to vary with the age and size of the explant used (Salomon and Mendel, 1965).
Furthermore, Sagee et al (1992) showed that differences in levels of endogenous growth
regulatory compounds (IAA and ARP) between individual Citrus stem cuttings was
associated with differences in propensity to produce adventitious roots. The effect of
such extraneous factors would make a nonesense of any comparisons between diploid and
tetraploid counterparts of different cultivars, if the plants were propagated from cut
Even when I propagated plants from apparently uniform material, which were then
grown-on for several months in the nursery to equilibrate, the degree of variation in
growth rates between individual plants from the same accession was found to be so high
that meaningful results could not be obtained (data not shown).
c) An experiment to investigate the effect of tetraploid rootstocks
on the growth of various diploid Citrus scion cultivars.
The following experiment is a long-term study to investigate the dwarfing effect of
tetraploid Troyer rootstocks on a range of Citrus scion cultivars. Plants were propaga
at the commencement of this project and planted in the Bundaberg Research orchard eight
months later.
Because tetraploid seed for rootstock propagation was only available from the cultivar
Troyer, the range of cultivars used in the previous chapter could not be studied in thi
work. Trees of six scion cultivars (Valencia and Navel oranges, Imperial and Murcott
mandarins, Marsh grapefruit, and Villafranca lemon) were each propagated on diploid
and tetraploid nucellar Troyer citrange rootstock seedlings. The tetraploid seedlings
section 4.2d)
Ploidy and Growth 4.6
selected from a large nursery rootstock seedling population using morphological
characteristics as described by Lee et al. (1990), and tetraploidy of every plant was
confirmed by microscopy using a modification of the method in Lee (1988a). The diploid
rootstock seedlings were from a normal nursery population. The 12 treatment
combinations (6 scions x 2 rootstocks (diploid and tetraploid)) were replicated four
times. The treatment order within each replicate was randomly assigned, and each
replicate constituted a separate row of trees planted on a level site of consistent, d
euchrozem soil type protected on all sides by windbreak plants. The trees were planted
at normal commercial spacing (5.3 metres within rows and 6.7 between) and managed
with standard commercial practices. Nearly two years after planting, and at
approximately six-monthly intervals thereafter, measurements were taken of canopy
height from the ground to the highest extremity, canopy width at the widest extremity,
and trunk girth ten centimetres above the bud union, from every tree. Data from this
randomized complete blocks design was analysed by two-way ANOVA.
d) A field study of growth differences between diploid and tetraploid
counterparts of eight Citrus cultivars
The only practical means of studying growth of the tetraploid accessions used in the
preceding chapter was to go back to the original tree of each in the arboretum at
Bundaberg Research Station and measure growth in the field. Even this approach was
fraught with difficulty because, as Davenport (1990) points out, individual stems of C
spend most of the time in a dormant condition interspersed by 2-4 intermittent periods
of growth of a month or so, each year. This is further compounded by asynchronous
flushing or resting of adjacent shoots on the same plant. Nevertheless, a study of the
section 4.2d)
Ploidy and Growth 4.7
growth patterns of the 16 accessions was conducted using the arboretum trees.
For the reasons presented earlier, the only reliable source of plants for a growth stud
incorporating the eight tetraploid accessions employed in the previous chapters was the
original seedling trees in the Bundaberg Research Station arboretum. Only one tree of
each accession could be used (i. e. the original tree on its own roots) because all oth
trees propagated from the originals are on diploid rootstocks. All the original trees
of the same age having been field planted three years before the following study was
conducted and had become well established.
The original tetraploid trees of the accessions used in the experiments reported in
preceding chapters are growing in a single uniform block on a euchrozem soil surrounded
by windbreak plantings. The accessions are of the cultivars Troyer (TR) and Benton
(BE) citranges, Swingle (SW) citrumelo, Cleopatra (CV) and Murcott (MU) mandarins,
Rough Lemon (RL), Paramatta (PA) orange, and Volkameriana (VO) lemon, as described
earlier. For comparison, one diploid tree of each cultivar was selected from an adjoin
block of trees of similar age to the tetraploids. Growing conditions, soil type and
management practices were identical for all trees.
Although citrus trees produce growth flushes sporadically two to four times throughout
the year, the Spring flush following the Winter dormancy period is reasonably
synchronous across all varieties. The Spring flush was therefore studied to ensure that
growth being measured was occuring under the same seasonal conditions for all 16
accessions.
section 4.2d)
Ploidy and Growth 4.8
Only non-flowering shoots were studied, which presented a few problems, particularly
in the diploids, because nascent floral shoots cannot be distinguished from vegetative
shoots at the time of bud burst. Floral shoots are typically less vigorous than vegetat
shoots. When a selected shoot turned out to be floral, that data was no longer included
in the study.
Ten buds were labelled on each tree at the time of budbreak and observed at regular
intervals of approximately three days. In Citrus, the termination of shoot growth is ea
distinguished because the terminal bud suddenly whithers and abscisses. At this stage t
shoot has almost attained its full length for the current flush, but the leaves have no
expanded. The growth period duration of each flush was recorded and the shoots were
left growing until leaves were fully expanded and hardened off. At maturity, each shoot
was cut from the tree, at its point of origin. Shoot lengths and number of internodes
were recorded, then the shoots were oven dried at 60°C and total dry weight (leaves +
stems) was recorded.
The number of shoots available from each accession differed considerably (due to
discounting of flowering shoots, and abscission of some vegetative shoots). Accordingly
the data was analysed by one-way ANOVA and pairwise comparisons made between the
diploid and tetraploid counterparts. Significant differences between counterparts of ea
cultivar were thus determined and the effect of ploidy on growth of each of the cultiva
was examined.
section 4.3a)
Ploidy and Growth 4.9
4.3 Results and Discussion
a) In vitro growth study
The use of tissue cultures was found to be an inappropriate approach to conducting
growth studies.
There were no significant differences in callus sizes between diploids and tetraploids
any of the cultivars or treatments. Many of the cultures, particularly those of
Volkameriana (both diploid and tetraploid) failed to grow. Within individual treatments
there was a very high degree of variability in callus sizes. The ANOVA indicated that
Volkameriana produced significantly smaller (P<0.01) calluses than the other cultivars,
except in the case of the wounded treatment. This would have been the result of the
failure of most of the cultures of this cultivar to grow. There was a significant
interaction (P < 0.01) between cultivar and treatment. In P. trifoliata, with the absenc
of wounding, treatments with the sucrose medium produced significantly (P < 0.01) larger
calluses than the nil-sucrose. For Troyer, the nil sucrose treatment resulted in
significantly (P<0.01) smaller calluses than the other three treatments, all of which
contained sucrose in their media. This finding was contrary to that of Giladi et al.
(1977)(see section 4.2a)).
A high degree of variability in growth characteristics between individual Citrus callus
cultures and between genotypes in vitro is commonly observed (Prof. J.W. Grosser,
University of Florida - pers. comm.). This proved to be the case in a number of studies
I conducted (data not shown), and may arise due to differential selection for growth und
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section 4.3b)
Ploidy and Growth 4.11
b) An experiment to investigate the effect of tetraploid rootstocks
on the growth of various diploid Citrus scion cultivars.
Trees with tetraploid rootstocks exhibited significantly smaller (P<0.01) mean canopy
heights, widths, and trunk girths compared to trees with diploid rootstocks. Mean canopy
height of trees on tetraploid rootstocks was 22.6 centimetres shorter than that of tree
diploid stocks. Mean canopy width was 23.3 centimetres less and mean trunk girth was
2.59 centimetres smaller in trees on tetraploid stocks compared with those on diploid.
There was no significant interaction between rootstocks and scions for any of the
parameters.
Table 4.2 presents mean data for canopy height and width, and trunk girth for each of
the 12 treatments at the most recent date prior to the time of writing, 44 months after
planting.
The highly significant differences in the growth rates are surprising in a young field
experiment but serve to demonstrate several points. Even at this early age, the tetraplo
rootstocks have had a retarding effect on scion growth in terms of canopy height and
trunk girth. Trunk girth is a particularly useful gauge of tree growth (Pearce, 1953).
The actual size differences between trees on diploid rootstocks and those on tetraploids
are relatively small. This is because the trees are young and have not assumed their
mature growth habit. Greater size differences are anticipated as the trees get older,
because observations of other (non-experimental) trees indicate that those with tetrapl
rootstocks stop growing earlier than those on diploid stocks. The differences between
rootstock treatments became apparent at an early stage (Fig. 4.1).
section 4.3b)
Ploidy and Growth 4.12
Table 4.2
M e a n canopy heights and widths, and trunk girths of six Citrus cultivars
each with diploid and tetraploid Troyer citrange rootstocks, at 4 4 months after planting.
Valencia Navel Imperial Murcott Marsh Villafranca
orange orange
mandarin mandarin grapefruit
lemon
canopy height ( m )
diploid rootstock
2.338§ 2.200
2.813
2.475
2.488
3.180
tetraploid rootstock 2.050
2.025
2.363
2.363
2.163
3.175
(rootstock by scion interaction not significant; mean rootstocks L S D 0 0 1 = 0.1413)
canopy width (m)
diploid rootstock
2.575
2.613
2.313
tetraploid rootstock 2.175
2.550
2.200
(rootstock by scion interaction not significant;
2.150
3.025
3.984
1.925
2.813
3.600
001
mean rootstocks L S D
= 0.1950)
trunk girth (cm)
diploid rootstock
22.63
22.38
23.75
22.00
31.50
34.82
tetraploid rootstock
21.50
20.00
20.63
19.88
26.90
32.63
(rootstock by scion interaction not significant; mean rootstocks L S D 0 0 1 = 1.394)
Mean
2.582a*
2.356b
2.776a
2.544b
26.18a
23.59b
§ - values for each rootstock/scion treatment are means of four replicates
* - pairs of values followed by different letters differ significantly (P<0.01)
It m a y be that general growth retardation in the tetraploid rootstock impedes scion
growth. Alternatively, synthesis of a growth regulatory compound produced in the
roots, but effective in the canopy, may possibly be modified by genome duplication.
Whatever the exact cause, retardation of growth of the scions on tetraploid rootstocks,
compared with identical scions on diploid rootstocks of the same genotype as the
tetraploid, indicates a physiological difference stemming from genome duplication.
The early results from this experiment demonstrate that tetraploidy in Troyer rootstocks
reduces vigour of diploid scions, but it provides no information about the growth effects
per se of tetraploidy, nor about possible differential responses to tetraploidy of the
various cultivars studied in the previous chapter. It was therefore necessary to compare
the growth characteristics of diploid and tetraploid trees of the eight cultivars in que
section 4.3b)
Ploidy and Growth 4.13
HEIGHT
20
25
30
35
40
45
50
45
50
45
50
age (months)
WIDTH
20
25
30
35
40
age (months)
GIRTH
25
30
35
40
age (months)
Fig. 4.1 Effect over time, of diploid vs. tetraploid rootstocks
upon mean canopy height and width and mean trunk girth
section 4.3c)(i)
Ploidy and Growth 4.14
c) A field study of growth differences between diploid and tetraploid
counterparts of eight Citrus cultivars
Complex growth differences were observed between diploid and tetraploid counterparts
of the eight cultivars. A pattern of concurrence was apparent between some of the
findings of isozyme activity in Chapter 3 and those here. The cultivars TR and VO
exhibited the greatest differences in isozyme activity between diploid and tetraploid.
diploids of these cultivars showed greater growth than their tetraploid counterparts.
Conversely, the cultivars MU and CV, in which there was little difference in isozyme
activity between ploidy levels, exhibited greater growth in their tetraploids.
The differences in growth characteristics between the diploid and the tetraploid of each
cultivar are shown in Fig. 4.2. The order of cultivars is the same as that assigned by t
differences in band density ratios determined in Chapter 3 (see pg. 3.23). In each case,
values of the y axis are the difference between the diploid of each cultivar and its
tetraploid counterpart, such that where the diploid exceeds the tetraploid for the
parameter in question, the ordinate has a positive value, and conversely, is negative
where the parameter is greater for the tetraploid than the diploid. Where there was a
significant difference (P<0.05) between ploidy levels of particular cultivars, LSD bars
are presented.
(i) Flush duration. The mean duration of the flush growth was significantly (P<0.01)
longer in the trifoliate diploids TR and SW (by more than 16 and 8 days, respectively)
than in their tetraploid counterparts (Fig 4.2(a)). Similarly, the diploid of the trifo
cultivar BE grew for more than five days longer than the tetraploid (P< 0.075). The
section 4.3c)(ii)-(iv)
Ploidy and Growth 4.15
unifoliates, except VO, exhibited the converse situation to the trifoliates, with lon
flush durations in the tetraploids than in the diploids (Fig. 4.2(a)). MU and RL tetr
mean growth durations exceeded their diploid counterparts by 12 days (P<0.01) and 6
days (P < 0.05), respectively. CV and PA tetraploids exceeded their diploid counterpa
by more than five days mean growth (P<0.1 and P<0.075, respectively).
(ii) Flush length. The mean total length of TR and VO diploid flushes was significant
longer (P<0.01 and P<0.05, respectively) than that of their tetraploid partners (Fig.
4.2(b)). Conversely, mean flush length of the tetraploid CV was greater than that of
diploid (P<0.01). The PA tetraploid also, showed a trend (P< 0.075) towards greater
length than its diploid counterpart (Fig. 4.2(b)). The other cultivars exhibited no
significant differences in mean flush length between diploid and tetraploid partners.
(iii) Number ofinternodes. The number of internodes showed a high positive correlatio
with flush length (Table 4.3). The diploid of TR produced significantly more nodes pe
shoot than the tetraploid (P<0.01)(Fig 4.2(c)). Conversely, the mean number of
internodes produced by the tetraploids CV and MU exceeded that of their diploid
counterparts (P<0.01 and P<0.05, respectively). PA exhibited a weak similar trend
(P<0.1). The diploid and tetraploid counterparts of the other cultivars did not diffe
significantly in internode number.
(iv) Internode length. Because flush length and internode number were highly
correlated, the mean internode length responses (flush length -4- node n°.) reflect t
of their component factors (Fig. 4.2(d)).
Ploidy and Growth 4.16
section 4.3c)(iv)
(b) FLUSH LENGTH
(a) FLUSH G R O W T H DURATION
TR BE S W CV M U RL PA VO
TR BE S W CV M U RL PA V O
cultivar
cultivar
(d) INTERNODE LENGTH
(c) NUMBER OF INTERNODES
10
2X>4X
c
m
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b
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TR BE S W CV M U RL PA VO
cultivar
-1
TR BE S W CV M U RL PA VO
cultivar
Fig. 4.2 (a-d) Differences in growth characteristics between
diploid and tetraploid counterparts of eight cultivars
Ploidy and Growth 4.17
section 4.3c)(iv)
(e) TOTAL DRY MATTER PRODUCED
(f) DRY MATTER ACCUMULATION RATE
2X>4X
^
ca
u
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cI
as s -1
-^
E o.
o£
- IsdO.OS
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TR BE SW CV MU RL PA VO
cultivar
(g) SHOOT EXTENSION RATE
TR BE SW CV MU RL PA VO
cultivar
(h) INTERNODE PRODUCTION RATE
2x>4X
<- lsd0.05
TR BE SW CV MU RL PA VO
cultivar
cz
TR BE SW CV MU RL PA VO
cultivar
Fig. 4.2 (e-h) Differences in growth characteristics between
diploid and tetraploid counterparts of eight cultivars
section 4.3c)(v)
Ploidy and Growth 4.18
Table 4.3
Correlation coefficients (r) of differences between diploid and tetraploid counterparts
of eight cultivars for various growth characteristics
drv weight
flush length
growth duration
flush length
0.9001
growth duration
0.4958
0.5910
internode length
0.7528
0.9389
0.5272
internode number
0.9132
0.9958
0.5896
internode
length
0.9259
(v) Dry matter. The m e a n total dry matter production of shoots of the tetraploids of C V
and PA was significantly greater (P<0.01) than that of their diploid counterparts (Fig.
4.2(e)). The tetraploid VO also produced significantly (P<0.05) more dry matter than
its diploid partner, and the RL pair exhibited a similar trend (P<0.1). The other
cultivars showed no significant differences in dry weight between ploidy levels. Dry
weights were, not surprisingly, highly correlated with flush lengths and number of
internodes (r=0.9001 and r=0.9132, respectively)(Table 4.3).
The mean rates of dry matter accumulation (dry matter -4- growth duration) were
significantly higher (P<0.01) in the tetraploids of SW, CV, PA and VO than in their
diploid partners (Fig. 4.2(f)). Mean shoot extension rate (flush length -4- growth
duration) was significantly greater (P< 0.01) in the diploid VO than in the diploid (Fi
4.2(g)). Conversely, the tetraploid of CV had a significantly higher (P<0.01) mean
shoot extension rate than its diploid partner, and SW exhibited a similar trend (P<0.1)
section 4.3c)(vi)
Ploidy and Growth 4.19
Internode production rates (internode number -4- growth duration) were significantly
(P<0.01) higher in the tetraploids of BE and CV than in their respective diploid
counterparts (Fig. 4.2(h)). The converse relationship applied to the internode productio
rates of the diploid and tetraploid of VO (P<0.05).
(vi) Interpretation. Because it was not possible to undertake such a study using more
than one tree of each cultivar (at least for the tetraploid accessions - refer section 4
it is conceivable that the data may have been influenced by differences between some
diploid/tetraploid pairs that were not exclusively the result of their ploidy. For examp
although there was no evidence, one tree of a diploid/tetraploid pair may have been in
a poorer state of health than its partner. Accordingly, a degree of prudence is needed i
interpreting the data. It was considered judicious to not place emphasis on the degree o
the response, but to focus on their kind.
The most important finding arising from the investigation was that, for different cultiv
there were clearly different responses in growth characteristics between diploid and
tetraploid forms. A striking example was the longer flush duration in the trifoliate
diploids compared to their tetraploid counterparts, whereas the unifoliate cultivars
generally showed the converse response (Fig. 4.2(a)). In the work reported in Chapter
3, there was also differentiation between the trifoliates and unifoliates, which constit
distinct genetic groups.
As was apparent in the preceding experiment, genome duplication can again be seen to
affect plant physiology. Whereas the differences in internode and flush lengths could
section 4.3c)(vi)
Ploidy and Growth 4.20
arise from the effects of greater cell size in the tetraploids, this explanation would no
account for differences in flush growth duration between diploids and their tetraploid
counterparts. Furthermore, because greater cell size is characteristic of all the tetrap
regardless of genotype, similar growth response differences between ploidy levels would
be expected in all cultivars, if the effect was the result of cell size differences alone
this is not the case, it is concluded that genome duplication results in a degree of
physiological disruption.
Regarding the differences in diploid:tetraploid isozyme band density relationships betwe
the eight cultivars reported in Chapter 3, some speculative observations can be made
from the data of this experiment. The cultivars TR and VO exhibited the greatest
differences in isozyme activity between diploids and tetraploids (the response direction
were, however, conflicting; viz. TR2X<TR4X cf. V02X>V04X (see Table 3.4, and
section 3.3b)(iv)). Here these two cultivars were the only ones that exhibited
significantly greater diploid flush lengths and internode lengths (Fig. 4.2 (b) and (d))
their tetraploid counterparts. Similar relationships were observed in the number of
internodes, although the effect was not significant for VO (Fig. 4.2(c)). However, CV,
which exhibited no distinct difference between isozyme activities of diploid compared to
tetraploid, displayed the opposite growth relationship, i.e. the tetraploid produced
significantly greater flush length, number of internodes and internode length than the
diploid. Moreover, MU, which like CV showed no band density differences between
diploid and tetraploid, also produced significantly more internodes in the tetraploid, a
apparently similar trends in flush length and internode length.
section 4.3c)(vi)
Ploidy and Growth 4.21
The findings point to the possibility that, where tetraploidy results in a disruption of
normal quantitative gene expression of a Citrus plant, as evidenced by a shift (in either
direction) in the general amount of enzyme activity per unit DNA, growth (and
ultimately, tree vigour) becomes impaired. On the other hand, in Citrus cultivars where
there is no distinct alteration to quantitative gene expression as a result of the dupli
genome in tetraploids, growth may be enhanced, in accordance with the gigas
characteristics often reported in polyploids (see section 1.4).
In terms of vigour and tree canopy size, parameters such as flush length and internode
number are more important than dry matter production. Nevertheless, in several
cultivars, the tetraploid produced the greater amount of dry matter (Fig. 4.2(e)), and al
cultivars exhibited faster dry matter accumulation rates in the tetraploids than the dip
(albeit only four were significantly different)(Fig. 4.2(f)). Whereas this gives the
superficial impression that tetraploids may exhibit greater vigour than diploids, this w
be so only on a single flush basis. No observations have been made regarding the
number of flush events that occur per year in diploids compared to their tetraploid
counterparts.
Shoot extension rates and internode production rates (Fig. 4.2 (g) and (h)) are calculate
values which utilise base parameter data. They exhibit similar responses to those of
internode number and flush length discussed above, although the effects are less
conclusive.
These two experiments clearly demonstrated that somatic tetraploids of Citrus are
section 4.3c)(vi)
Ploidy and Growth 4.22
physiologically different from diploids. More importantly, different cultivars exhibit ver
different kinds of responses. This, plus evidence that different kinds of responses relate
to discrete groups of cultivars, suggests that the different responses may be genetically
determined. These concepts are discussed further in the following chapter.
Conclusion 5.1
Chapter 5
CONCLUSION
In the preceding experimental work, the effects of genome duplication on quantitative gen
expression were shown to differ amongst distinct groups of Citrus cultivars (refer sectio
3. 3b)(iv)). Furthermore, the groups of genotypes which exhibited distinct quantitative g
expression responses to genome duplication coincided with discrete phylogenetic groups.
Cultivar differences in growth responses to genome duplication were also demonstrated.
The findings suggested that where there was a marked difference in quantitative gene
expression between a diploid and its isogenic tetraploid, vigour may be relatively impair
in the tetraploid. On the other hand, no such vigour effect may occur in genotypes where
both ploidy levels exhibit similar quantitative gene expression characteristics (see sect
4.3c)(vi)).
Citrus and related genera are the only group of plants known to regularly produce somatic
tetraploids (via apomictic embryogenesis), and do so in a range of genotypes. Therefore,
they provide a unique model for the investigation of the effects of genome duplication,
without the complication of heterozygosity of the homoeologous genomes of allotetraploids
The effect of genome duplication on quantitative gene expression had not been previously
studied for a range of proteins and a number of related genotypes or species.
It was discovered that many isozyme bands exhibited differing levels of activity in diplo
plants compared to tetraploids of the same cultivar. This effect was repeatable but a ver
complex situation was apparent with respect to individual isozyme bands and cultivars
Conclusion 5.2
(section 3.3b)(iii)). All three possible response types were represented - viz. those isoz
and cultivar combinations where:
(i) tetraploids exhibited reduced isozyme activity relative to their diploid counterparts
(ie. the diploid:tetraploid (2X:4X) ratio of activities was greater than 1),
(ii) tetraploids exhibited enhanced isozyme activity relative to their diploid
counterparts (2X:4X ratio of activities less than 1), and
(iii) similar levels of isozyme activity between ploidy levels (2X:4X ratios
approximating 1).
With small data sets, one isozyme band would exhibit enhanced activity in the tetraploid
of some cultivars yet reduced activity in other cultivars, and similar levels of activity
between tetraploids and diploids in the remainder. For another isozyme, the activity
relationships between ploidy levels of the cultivars would differ from the first. Only whe
a very large set of isozyme activity data was collected did a systematic relationship beco
apparent.
When all of the 2X:4X ratio data for each cultivar was considered separately, it was found
that the majority of the values fell within one of the three groups above. That is, within
each cultivar the data was not randomly distributed. The effect of genome duplication on
quantitative gene expression in each cultivar exhibited a strong tendency to either enhanc
reduced or unaffected activities collectively in the tetraploid relative to the diploid,
irrespective of the particular isozyme. This specificity of the 2X:4X isozyme activity
response of each cultivar suggests that the effect was under the influence of some
physiological or anatomical feature, such as differences in cell morphology between ploidy
Conclusion 5.3
levels, rather than gene-specific mechanisms, such as epistatic regulation or gene dosa
control. Furthermore, three significantly distinct groups of cultivars were defined in
accordance with the response-type groups listed above. The assignment of cultivars to t
three groups coincided exactly with three distinct phylogenetic groups. This strongly
suggests that the non-gene-specific feature above is genetically influenced (section
3.3b)(iv)).
Instances of genetic control of cell volume have been reviewed by Nurse (1985). The
lower ratio of cell surface area to volume in tetraploids, compared to diploids, will h
major ramifications on the balance between cell wall enzymes, such as glycosidases and
acid phosphatases, and the prime metabolic enzymes of the organelles (Levin, 1983), whic
may in turn have physiological repercussions for the whole organism. The ratio of nucle
membrane area to nucleus (and cell) volume is also lower in tetraploids relative to dip
(Levin, 1983), but the number of nuclear pores per unit nuclear membrane area remains
similar (Maul, 1977). This situation could lead to limitations in the rate of RNA transp
to the cytoplasm relative to cell volume, which may determine the rate of cell growth a
length of the cell cycle (Cavalier-Smith, 1978). This proposition requires some
qualification however, as there is considerable evidence that cell growth rates are dep
upon "gene concentration" (Cavalier-Smith, 1985). Near doubling of cell size in tetraplo
Citrus compared to diploids will result in reduction of the membrane surface area to vo
ratios. Irrespective of the causal mechanisms, of which, I believe, there are probably
numerous, it seems plausible to conclude that this difference between ploidy types, sho
have a marked effect on their relative physiologies.
Conclusion 5.4
With regard to quantitative gene expression, gene duplications exhibit several levels of
complexity (refer section 3.1). Firstly, studies of trisomies (see Birchler (1983) for a
review of literature) show that, at the simplest level, quantitative gene expression is
generally proportional to gene dosage. Secondly, however, at the total genome level, the
action of regulatory genes results in dosage compensation effects, epistasis and
enhancements in the expression of certain genes. This will lead to imbalances of some gen
products as a result of genome duplication. The situation is made more complex by the
effect of cell morphology differences between ploidy levels. DeMaggio and Lambrukos
(1974) studied peroxidase isozymes in ferns. They demonstrated that peroxidase activity
per cell increased directly with genome number in a series of In, 2n and An gametophyte
ploidy levels. When gametophytes were compared with sporophytes of the same genotype
and ploidy level, they discovered particular peroxidase isozymes unique to each. They als
observed large differences in activity of many specific peroxidase isozyme bands between
the gametophyte and sporophyte. They concluded ...
"that whatever the regulatory mechanisms influencing isozyme activity may be, they must be
modulated by the extra-genomic environment".
These findings are consistent with those for Citrus in the preceding chapters, which sug
a non-gene-specific or 'extra-genomic' feature influences gene expression.
This thesis has demonstrated that there is probably a third level of complexity, namely,
genotypic differences in the effects of genome duplication on quantitative gene expressi
That is, distinct groups of closely related genotypes show different responses in quanti
gene expression as the result of genome duplication. It is possible that these difference
are related to differences amongst the genotypes in comparative cell morphology between
ploidy levels. Few similar instances of genotypic response differences to polyploidy were
Conclusion 5.5
found in the literature. That is not necessarily surprising given that Citrus is the onl
of plants known which produces somatic tetraploids from a range of cultivars. The only
analogous situation is that of colchicine-induced polyploids. The effect of genome
duplication in colchicine-induced tetraploids of Phlox drummondii, compared with their
diploid progenitors, differed amongst cultivars with respect to photosynthetic rates (Ba
et al., 1982), glycoflavone production (Levy, 1976), and alcohol dehydrogenase activity
(Levin et al, 1979). Nakai (1977) found that differences in esterase isozyme levels of
activity between diploids and colchicine-induced tetraploids, differed amongst cultivars
rice (Oryza sativa). The demonstration in this project of cultivar-dependent responses to
genome duplication in Citrus suggests that the above observations are not the result of
mutations which can result from the colchicine treatments (Lapins, 1983).
Genome duplication causes alterations in plant physiology as was demonstrated by
significant differences in flush growth duration and growth rate between the diploid and
tetraploid of certain genotypes (section 4.3c)). Furthermore, growth retardation induced
in diploid scions on tetraploid rootstocks compared with identical scions on diploid
rootstocks (of the same genotype as the tetraploid) indicated a physiological difference
stemming from genome duplication (section 4.3b)). This may be the result of general
growth retardation in the tetraploid rootstock impairing scion growth, or it may be that
synthesis of growth regulatory compounds produced in the roots but effective in the cano
is modified by genome duplication.
The effects of genome duplication on growth appeared to vary with genotype, thus
supporting the conclusion from the molecular data that some genetically determined facto
Conclusion 5.6
affects physiological responses to doubling of the genome.
It was hoped that a molecular screening method could be developed from this project to
enable the dwarfing potential of any tetraploid cultivar to be predetermined by a laborato
test. To achieve this, molecular characterisation would have to be closely correlated with
growth responses. Because of the difficulties in obtaining growth data, it will be necessa
to conduct longer term field experiments before such a test could be reliably applied.
However, the project has clearly shown that there are considerable differences between
cultivars, both in molecular characteristics and in growth. Therefore, it cannot be assume
that, because the tetraploid of one rootstock cultivar promotes a dwarfing effect, that ot
cultivars will do likewise. This information is important in the planning of future breedi
and field evaluation work. Even more significant is that there are similarities in both
molecular and growth characteristics between groups of closely related cultivars. This
information is particularly important because it suggests that certain varietal groups of
rootstocks may be more suitable for use as dwarfing tetraploids than others. With minimal
field testing of representatives of each group it will be possible to ascertain those with
greatest dwarfing potential. In this way breeding programs can be focused on the most
useful cultivar groups, and the least responsive can be largely discounted. Such an
approach will result in greater efficiency in the breeding program, and improved prospects
of producing the best possible dwarfing varieties, by preventing resources being expended
on groups of cultivars that are destined never to produce useful dwarfing tetraploids. Suc
experimentation is now under way at Bundaberg Research Station and the research reported
in this thesis is providing a sound foundation in setting directions for that work.
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Appendix 1
Appendix 1
EXTRACTION BUFFER FORMULAE
CSIRO formula 140 mg sodium borate, 35 mg diethyldithiocarbamic acid, 38 mg sodium
bisulphate, 500 mg PVP40, 500 mg L-ascorbic acid, 130 ul 2mercaptoethanol, 12.5 ml 0.16mM potassium phosphate buffer pH 7.0
(pers. comm. Dr. Steve Sykes, CSIRO, Div. of Horticulture, Merbein)
Peakall formula 8 ml 0.1M Tris-HCl pH 7.5, 0.4 g PVP40, 1 g sucrose, 20 mg EDTA, 10 mg
bovine serum albumin, 5 mg NAD, 5 mg NADP, 10 mg dithiothreitol
(pers. comm. Dr. Rod Peakall, Macquarie Univ., Sydney)
Soltis formula 0.1% v/v [25 ul] 2-mercaptoethanol, l.OmM [0.01 g] EDTA [tetrasodium],
lO.OmM [0.019 g] KC1, lO.OmM [0.05 g] MgCl2.6H20, 4% [1 g] PVP40, 25 ml
0.1M Tris-HCl pH 7.5
(Soltis et al, 1983)
Soltis+ formula based on Soltis formula above, but using 0.2M Tris-HCl plus 2 mg/ml bovine
serum albumin, 667 ug/ml NAD and 333 ug/ml NADP.
Appendix 2.1
Appendix 2
ISOZYME STAIN PROTOCOLS
The isozyme stain protocols used here are based on those presented by Richardson
(1986) with occasional minor modifications in accordance with the methods of Dr.
Peakall, Macquarie University, Sydney (pers. comm.).
In most cases, the agar overlay method of reagent application was used. This was
to give superior band resolution on the rigid Titan III gels compared to the liqu
application method of Richardson et al. (1986). For 60 mm x 76 mm gels, a total of
5 ml of stain comprising 2.5 ml of reagent solution plus 2.5 ml of hot 2% agar (60
was poured over each gel and allowed to solidify briefly before placing in a dark
(37°C) for bands to develop. Bath methods of stain application were used for ribu
1,5-biphosphate carboxylase and cytosol aminopeptidase. These are detailed in the
relevant sections below.
The following abbreviations are used -
NAD - ^-nicotinamide adenine dinucleotide
NADP - /^-nicotinamide adenine dinucleotide phosphate
MTT - methyl thiazolyl tetrazolium blue
PMS - phenazine methosulphate
Cytosol aminopeptidase - C A P
E.C. 3.4.11.1
L-leucine-j8-naphthylamide 2.5 mg
MgCl2 4.76 mg (250 p\ @ 0.2M)
0.1M Tris-maleate pH 5.5 2.25 ml
mix with agar and pour on gel; after agar sets incubate in bath of Fast black K salt 10 mg/ml in 0.1M Tris-maleic pH 5.5
Glucose-6-phosphate dehydrogenase - G 6 P D
E.C. 1.1.1.49
glucose-6-phosphate
6mg
NADP
200 /xg (50 ix\ @ 4 mg/ml)
MTT
600 ptg (100 y\ @ 6 mg/ml)
PMS
100 fig (50 [A @ 2 mg/ml)
MgCl2
9.5 mg (100 p\ @ 1.0M)
0.1M Tris-HCl p H 8.0
2.2 ml
Isocitrate dehydrogenase (NADP + ) - I D H
E.C. 1.1.1.42
DL-isocitric acid
20 m g
NADP
200 pg (50 ix\ @ 4 mg/ml)
MTT
600 fxg (100 fx\ @ 6 mg/ml)
PMS
100 ^g (50 fil @ 2 mg/ml)
MgCl2
1.9mg([email protected])
0.1M Tris-HCl p H 8.0
2.2 ml
Appendix 2.3
Malate dehydrogenase (NAD) - M D H
E.C. 1.1.1.37
DL-malic acid 62.5 mg (1.25 ml @ 50 mg/ml pH 8.0)
NAD 250 fig (50 /xl @ 5 mg/ml)
MTT 600 fig (100 /ii @ 6 mg/ml)
PMS 100 fig (50 /xl @ 2 mg/ml)
0.1M Tris-HCl pH 8.0 1.05 ml
6-Phosphogluconate dehydrogenase - 6 P G D
E.C. 1.1.1.44
6-phosphogluconic acid 2.5 mg
NADP 200 /xg (50 /xl @ 4 mg/ml)
MTT 600 /xg (100 /xl @ 6 mg/ml)
PMS 100 fig (50 /xl @ 2 mg/ml)
MgCl2 1.9mg(100/[email protected])
0.1M Tris-HCl pH 8.0 2.2 ml
Ribulose 1,5-biphosphate carboxylase - R B C
E.C. 4.1.1.39
amido black 2 mg/ml in 45% methanol : 9% acetic acid : 46% water
stain in bath for 15 minutes then destain in 3 washes of the same solve
Shikimate dehydrogenase - SkDH E.C. 1.1.1.25
shikimic acid 10 mg
NADP 200 fig (50 /xl @ 4 mg/ml)
MTT 600 fig (100 /xl @ 6 mg/ml)
PMS 100 /xg (50 /xl @ 2 mg/ml)
MgCl2 1.9 mg (100 /xl @ 0.2M)
0.1M Tris-HCl pH 8.0 2.2 ml
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Appendix 4.1
Appendix 4
ORIGINS OF THE ACCESSIONS
The cultivars used were selected to cover a range of genetic types. Sixteen accessions
were studied; diploid and tetraploid counterparts of eight different Citrus and Citrus
hybrid cultivars. The tetraploids were all derived as part of m y polyploid breeding
program at Bundaberg Research Station. All arose directly as spontaneous nucellar
somatic tetraploids (refer section 1.3), and were confirmed as such by chromosome
microscopy (Lee et al, 1990). The Citrus are known to be prone to spontaneous
mutation. T o avoid the remote possibility of genomic differences between diploid and
tetraploid counterparts due to mutation, the same parent gave rise to both the diploid and
tetraploid of each accession.
Plants were propagated from cuttings to provide sufficient leaf material for the
experiments. Occasionally, two or more advanced plants from such cuttings were
subsequently used as the source material for further cuttings. In such cases, the source
plants were given discrete identifying numbers (eg. S W 2 X 2 - where S W denotes the
cultivar, 2 X denotes the ploidy, and 2 denotes the particular source plant), and only one
of such source plants was used to provide the plant material. A brief history of the
parental accessions follows.
PA
The Parramatta sweet orange (C. sinensis) arose as a selection on the orchard of Mr.
Hawison, Northmead, N S W , in 1863.
It was used as a rootstock and therefore
disseminated as seed. The bud line used originated from seed collected on the Armstrong
Appendix 4.2
Bros, orchard, Grantham, Qld.. Two trees were grown at Maroochy Horticultural
Research Station, Nambour, Qld.. Seed from one of these trees gave rise to the nucella
tetraploid and the diploid accessions. The parent trees have since been removed.
VO
The Volkameriana lemon is believed to have arisen in Italy as a hybrid of Citrus medic
x C. limon. Seed was imported from USDA Orlando, Florida in 1976, by NSW Dept.
of Agriculture. Surface disinfestation with fungicide was the only quarantine treatmen
applied, obviating any danger of induced mutation. Some of this seed was sent to
Queensland and two resultant trees were grown at Maroochy Horticultural Research
Station, Nambour, Qld.. Seed from one of these trees gave rise to the nucellar tetrapl
and the diploid accessions. The parent tree has since been removed.
SW
The Swingle citrumelo is a C. grandis x Poncirus trifoliata hybrid produced at Glen
St. Mary, Florida and selected in the early 1950's. The accession used here was
imported as seed in 1975 from USDA, Orlando, Florida. Surface disinfestation with
fungicide was the only quarantine treatment applied. Two resultant trees were planted
at Maroochy Horticultural Research Station, Nambour, Qld.. One of these was the
source of the seeds which gave rise to both the diploid and tetraploid accessions used
this project. The parent tree has since been removed.
TRO
Troyer citrange arose as a hybrid of Navel orange C. sinensis X P. trifoliata from a
Ar^f-r maidr?. M USDA, Riverside, California in 1909. Seed was imported by the Victori
Appendix 4.3
Dept. of Agric. in 1952. Seed from this source was planted by the N.S.W. Dept of
Agric. from where seed of N.S.W. Accession No. 3254 was sent to Maroochy
Horticultural Research Station and two resultant trees were planted out in 1965. Seed
from one of the these two gave rise to both the diploid and tetraploid accessions used
this project. The parent tree has since been removed.
MU
The origin of Murcott is obscure but it is believed to have arisen in a USDA breeding
program in Florida from a cross made late last century. It is known to be a tangor
(C. reticulata X C. sinensis). Murcott first appeared in Queensland in the 1960's
apparently as an illegal budwood importation from U.S.A.. Legal seed imports were
subsequently made but the earlier introduction has become universally accepted in
Australia. A tree budded from this source gave rise to the tetraploid accession and the
diploid was propagated from the same bud line.
CV
Cleopatra is an ancient variety believed to be a native of India where it is also known
Chota or Billi Kichilli. It was referred to as C. reshni in the now superceded Tanaka
taxonomy, but is now accepted as a variety of C. reticulata. The accession used here is
of the so-called Victoria strain which was probably imported into Victoria early this
century from U.S.A. The U.S. source arose from seed imported into Florida from
Jamaica prior to 1888. A further importation from Florida was made into Queensland
in 1960 - the so-called Florida strain - but the Victoria strain is considered the bett
rootstock. Seed of the Victoria strain was introduced into Queensland in 1951 from
Victoria and a resultant tree was planted at Maroochy Horticultural Research Station,
Appendix 4.4
Nambour, Qld.. This tree provided buds from which further trees were propagated and
planted in 1965 and again in 1980 at Maroochy. One of these was the source of the
seeds which gave rise to both the diploid and tetraploid accessions used in this proje
The parent tree has since been removed.
RL
Rough lemon, C. Union (previously C. jambhiri), is native to India. There are numerous
strains grown in Australia, indeed it grows wild in Queensland and N.S.W., and its
origin in Australia is unknown and probably resulted from numerous seed imports last
century. The Lockyer strain, which is a superior rootstock selection, arose from root
cuttings taken from a tree at Gatton, Qld., in 1954. Plants grown from the cuttings
provided buds that were propagated and two resultant trees were planted at Maroochy
Horticultural Research Station in 1965. One of these was the source of the seeds which
gave rise to both the diploid and tetraploid accessions used in this project. The pare
tree has since been removed.
BE
Benton citrange, C. sinensis X P. trifoliata, originated from crosses conducted by
N.S.W. Dept. of Agriculture. The N.S.W. Accession No. 3697 was the source of seed
brought to Queensland from which two resultant trees were planted in 1975 at Maroochy
Horticultural Research Station in 1965. One of these was the source of the seeds which
gave rise to both the diploid and tetraploid accessions used in this project. The pare
tree has since been removed.
Appendix 5
Appendix 5
DNA QUANTIFICATION BY FLUOROMETRY
The following document is a reprint of our paper detailing the fiuorometric quanti
method for DNA in crude extracts using DAPI fluorometry.
L. Slade LEE and Helen M. GARNETT, (1993)
Estimation of Total DNA in Crude Extracts of Plant Leaf Tissue
Using 4',6-diamidino-2-phenylindole (DAPI) Fluorometry.
Journal of Biochemical and Biophysical Methods 26, 249-260
ATTACHMENT
Additional background information on polyploidy in Citrus is provided in the attac
review paper -
L. Slade LEE, (1988)
Citrus Polyploidy - Origins and Potential for Cultivar Improvement.
Australian Journal of Agricultural Research 39, 735-747.
Articles above removed for copyright reasons