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IHHHWH’\HiHWilii

This is to certify that the

dissertation entitled
The Genetics of Plastid Transmission after

Somatic Hybridization in Higher Plats: Plastid Transmission
in Somatic Hybrids of Solanum lycopersicoides and
Lycopersicon esculentum

presented by

Brent Lee Ridley

has been accepted towards fulﬁllment
of the requirements for

Master—of—Sciencedegl‘ee in M and Genetics

Gate

Major proéscif/r

Date Qctober 22, 1990

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THE GENETICS OF PLASTID TRANSMISSION AFTER SOMATIC HYBRIDIZATION
IN HIGHER PLANTS: PLASTID TRANSMISSION IN SOMATIC HYBRIDS OF
Solanum btcopemicoides AND Lycopersicon esculentum

By

Brent Lee Ridley

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1990

ABSTRACT
THE GENETICS OF PLASTID TRANSMISSION AFTER SOMATIC HYBRIDIZATION

IN HIGHER PLANTS: PLASTID TRANSMISSION IN SOMATIC HYBRIDS OF
Solanum chopersicoides AND Lycopersicon aradentum

By

Brent Lee Ridley

Restriction fragment length polymorphisms were used to examine the plastid transmission of
somatic hybrids of tomato with Solanum chopelsicoides. The plastids in 68 of ‘70 hybrids were from
tomato. One hybrid, plant 240 had S. chopersicoides plastids and another, plant 63 had a minute of
parental plastids. Plastid DNA from several branches was analyzed to follow plastid segregation in
hybrid 63. This hybrid had sorted into branches with pure plastids, although one branch may have
had a plastid mixture.

Plastid transmission after somatic hybridization is reviewed in consideration of plastid
evolution and genetics. Plastid genomes are highly conserved during evolution, and highly resistant to
genetic changes during somatic hybridization. Plastid fusion and plastid DNA recombination are very
rare after either somatic or sexual hybridization. Plastids segregate rapidly, and often stochastically in
both sexual and somatic hybrids. When plastid transmission deviates from random behavior it is

probably for similar reasons in both somatic and sexual hybrids.

Cepyﬁght by
Brent Lee Ridley
1990

I would like to dedicate this thesis to my parents whose love and support has
provided me with so many possibilities.

iv

ACKNOWLEDGMENTS

I am very grateful to Dr. David Dilley and Dr. James Hancock for their guidance. I would like
to thank Dr. Kenneth Sink for making this research possible. I would especially like to thank my
major professor Dr. John Kelly for his support. I am grateful to Dr. Barbara Sears for her expert
advice and critical review of my manuscripts. I also appreciate the eﬂons of Dr. Thomas Friedman as
a member of my guidance committee. I appreciate the technical advice I received from Dr. Jeff
Palmer. I am grateful to Dr. A. L. Phillips for the tomato chNA library, Dr. C. M. Rick for seed of
LA 1990, Dr. Patrick Moore for parental nuclear DNA samples, and Mr. Amnom Levi for parental
mitochondrial DNA samples. I am also grateful to Mrs. Marlene Camron and Mr. Amnom Levi for

assistance with the plant propagation

TABLE OF CONTENTS

PAGE
LIST OF TABLES ................................................................. vii
LIST OF FIGURES ................................................................ viii
CHAPTER 1. literature Review ..................................................... 1
Introduction ................................................................. 1
Plastid Biochemistry, Evolution and Genetics ..................................... 2
Plastid Transmission in Somatic Hybrids ......................................... 24
CHAPTER 2. Non-Random Plastid Transmission in Somatic Hybrids of
Lycopem'con esculentum and Solanum Mopem'ciodes ........................ 40
Introduction ................................................................. 40
Materials and Methods ........................................................ 42
Results ..................................................................... 43
Discussion .................................................................. 46
CHAPTER 3. Conclusion ........................................................... 50
APPENDIX. Non-Random Organelle Transmission in Somatic Hybrids of
Lycopem'con eswlenlum and Solanum lycopelsiciodes ........................ 57
Summary ................................................................... 58
Introduction ................................................................. 59
Materials and Methods ....................................................... 60
Results ..................................................................... 61
Discussion .................................................................. 66
LIST OF REFERENCES ........................................................... 71

LIST OF TABLES

PAGE
CHAPTER 1.
Table 1. Comparison of the deduced amino acid homologies of
some plastid and E. coli genes .......................................... 8
Table 2. Plastid competitive ability in Nicotiana somatic hybrids ...................... 27
Table 3. Chloroplast transmission after somatic fusion .............................. 33
APPENDDL
Table 1. Restriction fragment lengths of L. esculentum and
S. chopasicoides mitochondrial DNA .................................... 63

LIST OF FIGURES

PAGE
CHAPTER 1.
Figure 1. The gene map of spinach plastid DNA ................................... 4
Figure 2. Ribosomal RNA operons of plastids and eubacteria ........................ 6
Figure 3. Diagram of the chromosomal pairing of a Denothem ring
chromosome ........................................................ 21
Figure 4. Compatibility relations between different nuclear genome
complements and plastomes of Euoenothera .............................. 22
Figure 5. Distribution of the natural species among the compatible
combinations of Euoenothem ........................................... 23
CHAPTER 2
Figure 1. Southern analysis of plastid DNA from the parental lines
and thirteen somatic hybrids ........................................... 44
Figure 2. Southern analysis of plastid DNA from the parental lines
and eleven samples of hybrids 63 and 240 ................................ 45
APPENDDL
Figure 1. Southern analysis of plastid DNA from the parental lines
and thirteen somatic hybrids ........................................... 62
Figure 2. Southern analysis of puriﬁed mtDNA from L. esadauum,
S. Iyeopem‘coide: and five somatic hybrids ................................ 64
Figure 3. Southern analysis of puriﬁed mtDNA from L. esculentum,
S. chopelsicoides and somatic hybrid 69 .................................. 65

CHAPTER 1

Literature Review

Introduction

About two-thirds of the angiosperms investigated have maternal inheritance of plastids during
sexual reproduction, and most of the remaining species have bi-parental inheritance (Kirk and Tilney-
Bassett, 1978; Sears, 1980b). Maternal inheritance precludes the independent assortment of
agronomically important cytoplasmic traits in almost all crop plants. Independent assortment of
cytoplasmic genomes would facilitate the breeding of improved crop plants. Agronomically important
mitochondrial traits include cytoplasmic male sterility and resistance to some diseases (Pring and
Lonsdale, 1985). Plastomes (plastid chromosomes) encode atrazine resistance (Robertson, 1985),
resistance to certain diseases (Vaughn and Duke, 1984), and control of the rate of photorespiration
(Ogren, 1984).

Artificially induced fusion of protoplasts from plants with diﬁerent cytoplasmic genomes
produces heteroplasmic cells. Subsequent sorting of the organelles during cell division often results in
novel combinations of cytoplasmic genomes. The plastomes in somatic hybrid plants are almost
always transmitted without recombination and often are inherited randomly from either parental
protoplast ('Fluhr 1983; Kumar and Cocking, 1987). Shoots with mixed plastids are rare because
sorting out usually is completed before shoot regeneration can occur. This is a consequence of the
rarity of plastid fusion, which precludes chNA (chloroplast DNA) recombination (Possingham and
Lawrence, 1983), and rapid sorting out of plastids during cell division (Akada and Hirai, 1986). In
contrast, mitochondrial genomes almost always undergo recombination or rearrangement in somatic
hybridization experiments (Hanson, 1984). Mitochondria probably sort more slowly than plastids in
somatic hybrids, and this may stem from the prevalence of mitochondrial fusion (Bendich and
Gauriloff, 1984) and mtDNA (mitochondrial DNA) recombination (Izhar et al., 1983; Aviv and

Galun, 1986; Aviv and Galun, 1987).

Plastid Biochemistry, Evolution, and Genetics

Photosynthesis is the primary function of plastids, but they have other important metabolic
duties as well. Plastids participate in chlorophyll, amino acid and fatty acid biosynthesis; nitrate and
sulfate reduction; and starch metabolism (Kirk and Tilney-Bassett, 1978). About eighty to ninety
percent of the proteins found in plastids are encoded in the nucleus, translated on cytosolic
ribosomes, and transported into the organelles. The remaining proteins are plastome encoded, and
most function in the plastid transcription and translation systems, photosynthetic complexes, and
chlororespiration system (Ohyama et al., 1988; Wu and Yang, 1989). Plastome genes are transcribed
and translated in the plastid by enzymes which are distinct from those found in the nucleus and
cytosol.

The evolutionary origin of plastids and the physiological processes involved in the transmission
of their genetic information have impacted the structure of modern plastomes. Careful examination
of the plastomes in this context provides information regarding the mechanisms controlling their
inheritance. Consequently, review of the relevant issues of plastome evolution, sexual inheritance and
somatic transmission is useful in understanding plastid transmission in cell fusion hybrids.

From an overall perspective the plastid genome is more evolutionarily conserved than other
organelle genome (Palmer, 1985d). Plastids probably originated as photosynthetic procaryotes which
became endosymbiotic after they were engulfed by the predecessors of modern eucaryotes (Wallace,
1982; Gray, 1983). These organelles have evolved extensively from their procaryotic ancestors, mainly
through the transfer of most of their genetic functions to the nucleus (Palmer, 1985d; Brinkmann et
al., 1987). Plastids and cyanobacteria, their putative ancestors, have a very similar arrangement of
some their genes (Palmer, 1985a). Furthermore, the sequences of plastid genes have considerable
homology to cyanobacterial genes having similar functions. However, almost all chNAs examined

from diverse groups of plants and algae are consistently 20-30 times smaller than their presumed

3
ancestors. Nearly all chNAs analyzed are circular molecules between 120 and 217 kilobase pairs
(kb) in size (Palmer, 1985b).

This rather tight conservation of size is observed in all of the over 200 angiosperms examined,
in representatives of several groups of land plants outside the angiosperms, and in representatives of
several groups of algae. In contrast, plant mitochondrial genomes vary greatly in size, ranging in
kinetic complexity from 200 to 2000 kb in the angiosperms (Palmer, 1987). The difference in the
conservation of size between the genomes of different organelles suggests that selection may restrict
plastid genome size. The uniformity of plastid genome size suggests that the reduction occurred soon
after the beginning of endosymbiosis, and the genome remained relatively stable thereafter (Palmer,
1985a).

The angiosperm plastome architecture and genie content has been studied intensively and such
analyses provide interesting evolutionary insights (Palmer, 1985c). Plastomes are amenable to
molecular analysis of this sort because they are relatively small. Unfortunately, chNA analysis of
land plants outside the angiosperms has been limited to a few observations in the gymnosperms, ferns
and liverworts. Tight conservation of architecture and coding complement are the most striking
features of plastomes. The chNA of spinach was the ﬁrst to be investigated thoroughly and it has
the putative angiosperm ancestral gene arrangement (Palmer, 1987; Figure 1).

The most distinctive features of the plastome map are the large inverted repeats (Figure 1). In
land plants these repeats always contain the rRNA (ribosomal RNA) genes and are always located
asymmetrically, dividing the genome into small and large single-copy regions. Plastid genomes with
large inverted repeats are often devoid of any other repeated sequences (Palmer, 1985b). However,
direct repeats are found in the green algae Orlamydomouas (Gelvin and Howell, 1979), and tandom
repeats are found in the angiosperm Oenothera (Blasko et al., 1988) in addition to the large inverted
repeats. Plastid DNAs rapidly undergo homologous recombination across the large inverted repeats
(Palmer, 1983). Hence chNAs exist as an equal proportion of two isomers (ﬂip-ﬂop isomers), each

having the small single-copy region in an opposite orientation relative to the large single-copy region.

SPINACH
chNA

 

Figure 1. The gene map of spinach plastid DNA. The abbreviations for the genes are standard
(Hallick and Bottomley, 1983). Redrawn from Palmer, (1985a).

The size of the inverted repeat varies from 10 kb to 76 kb in different angiosperms; most differences
in overall plastome size result from differences in the size of the inverted repeat (Palmer, 1985c). In
fact, the range of plastome size for all land plants examined is only 110-150 kb when variation due to
the inverted repeat is removed (Palmer, 1987). The inverted repeats are a general feature of
angiosperm chNAs, and probably the chNAs of all land plants. However, this conclusion is based
on limited observations in a small number of groups outside the angiosperms.

The arrangement of genes encoded by the chNA is highly conserved among the land plants
which have been examined (Palmer, 1985d). The order of chNA sequences is essentially the same
between the angiosperm spinach, the gymnosperm Ginkgo biloba, and the fern Osmunda
cinnamomea (Palmer and Stein, 1986). The entire plastome of the angiosperm Nicotiana tabacum
(Shinozaki et al., 1986b) and the liverwort Mambantia polymorpha (Ohyama et al., 1986) has been
sequenced, and the genes and open reading frames are arranged in nearly the same order, except for
a large inversion in the large single-copy region. When the orders of all available chNA sequences
from land plants are compared, many of the differences between them can be attributed to single

inversions within the single-copy regions (Palmer and Stein, 1986). This contrasts with those plants

5
lacking the large inverted repeat, which have incurred many chNA rearrangements (Palmer et al.,
1987; Strauss et al., 1988).

With just a few exceptions the same set of genes is encoded by the plastid genomes of all land
plants and two algae, Orlamydomonas reinhaldtii and England gmcillis (Palmer, 1985a).
Consequently, the nuclear genomes of diverse organisms probably encode a similar set of plastid
proteins. The plastome is large enough to encode more than 100 proteins and stable RNAs.
Measurements of the proportion of the plastome transcribed into RNA suggest that most of this
coding potential is utilized in diverse organisms, including E. gmcilli: (Koller and Delius, 1984), C.
reinhatdtii (Howell and Walker, 19'”), and angiosperms (Oishi et al., 1981; Poulsen, 1983). Nucleic
acids have not been shown to cross the plastid membrane so chNAs probably encode all the stable
RNAs of the organelle and any proteins synthesized on plastid ribosomes (Ellis, 1981). Isolated pea
chloroplasts synthesize up to 80 soluble peptides in viva (Ellis et al., 1980). The gene content
encoded by the chNA sequences of N. tabocum and M. polymorpha is strikingly similar (Shinozaki et
al., 1986b; Ohyama er al., 1986). Tobacco has 122 and M. polyrnapha has 121 different genes and
unidentiﬁed opened reading frames. About 100 of these have been identiﬁed either functionally or
based on sequence homology with genes from other organisms. The coding complement is essentially
conserved between these distantly related plants (Weil, 1987).

Convincing evidence for the endosymbiotic origin of plastids is based on comparison of the
sequence and organization of plastid and bacterial rRNA genes. The rRNA genes are always plastid-
encoded, without exception, in numerous plants examined thus far (Palmer, 1985a). The 16S rRNA,
238 rRNA and the intergenic spacer are oriented the same way and cotranscribed similarly in _
eubacteria and plastids (Figure 2). This large transcript also includes tRNA genes for isoleucine and
alanine within the intergenic spacer, and these are arranged in the same orientation relative to the
rRNA genes in eubacteria and plastids. This precursor is cleaved to yield the individual RNA
products in both cases (Crouse er al., 1984). The SS rRNA gene is part of this transcript in

eubacteria, but probably is transcribed independently in angiosperm plastids (Bohnert er al., 1982).

: s
conu
,3, ‘f‘ rosacco

16s A A
E I] ll ll V] *5a

Intron Int ron

 

E. Coll

1e. 5% 23. 5, Anacystls

I ”Ir 1]

 

Figure 2. Ribosomal RNA operons of plastids and eubacteria. Abbreviations for the gene
products are standard. Redrawn from Palmer (1985a).

Comparison of the 16S and 238 rRNA gene sequences from the cyanobacteria Anacystis
nidulans, to the sequences from angiosperm plastids reveals remarkable conservation (Gillham et al.,
1985). There is 83% homology between the 168 rDNAs and 78% homology between the 238 rDNAs
of N. tabacum and A. nidulans (Palmer, 1985a). To add perspective to this, the 16S and 238 rDNAs of
the monocot Zea maize, and the dicot N. tabacum are 96% and 92% homologous, respectively
(Tomioka and Sugiura, 1983; Kumano et al., 1983; Douglas and Doolittle, 1984; Palmer 1985a). The
secondary structure of rRNAs are also highly conserved between eubacteria and plastids. Glotz et al.
(1981) observed over 450 compensating base changes between Zea maize and Escherichia coli in rRNA
regions that can form stem-loop structures. Many of the regions that are highly conserved in plastids
are those that are functionally important in eubacteria, such as the region in the 3’ end of the 168
rRNA, which is complementary to the Shine-Delgarno sequence (involved in ribosome binding) found
on all procaryotic mRNAs. Most chloroplast mRNAs can be translated efﬁciently on E. coli
ribosomes, but not on plant cytoplasmic ribosomes (Bottomley and Whitﬁeld, 1979). This
demonstrated the functional similarity of the plastid and eubacteria! translational apparatus.

Studies of several groups of land plants and algae indicate that all plastomes probably encode
a complete set of tRNA genes (Crouse et al., 1984). The chNA of M. polymorpha encodes 32

species of tRNA (Ohyama et al., 1986), and that of N. tabacum encodes 30 (Shinozaki et al., 1986b).

7

These tRNAs are sufﬁcient to read all 61 codons which signify amino acids, when the wobble rules
and anticodon base modiﬁcations are considered (Weil, 1987). The Euglena plastome tRNAs are
encoded polycistronically, in clusters of two to six genes. In contrast, the plastid tRNA genes of land
plants are not clustered signiﬁcantly and are transcribed independently, with the exception of the two
tRNAs in the rRNA intergenic spacer (Palmer, 1985a). The sequence of plastid tRNAs is highly
conserved between plants and algae (Gillham et al., 1985). In fact, the tRNAs of plastids have much
more sequence homology to their eubacterial tRNA analogues than to cytoplasmic analogues from
the same plant. The secondary structure of most domains of the tRNAs are also highly conserved
between plastids and eubacteria.

About 20 of the 60—65 plastid ribosomal proteins are encoded by chNAs, and the remainder
are nuclear-encoded (Palmer, 1985a). Nineteen putative plastid ribosomal protein genes have been
identiﬁed in both N. tabacum and M. polymorpha by comparison of the amino acid sequence
(deduced from the DNA sequence) with that of the homologous genes from E. coli (Ohyama et al.,
1986; Shinozaki et al., 1986b). About half of these are organized in clusters reminiscent of those in E.
coli, and the others are scattered throughout the genome. Furthermore, some plastid ribosomal
proteins have considerable immunological homology with their eubacterial homologues (Dorne et al.,
1984). Plastome-encoded plastid ribosomal proteins from C. reinhardtii have considerably more
antigenic, size and charge similarities to eubacterial ribosomal proteins than do those that are
nuclear-encoded and imported from the cyt0plasm (Schmidt et al., 1984). This may indicate slower
evolution of plastid-encoded genes relative to nuclear ones and/ or selective transfer of genes which
are less functionally constrained to the nucleus (Palmer, 1985a).

Plastid genomes encode about 16 of the polypeptides which form the four supramolecular
complexes (the ATPase complex, the cytochrome b6-f complex, and the photosystem I and II
complexes) essential for the light reactions of photosynthesis (Weil, 1987). The arrangements and
transcription patterns of several of these genes also resemble their procaryotic equivalents (Palmer,

1985a).

8

Table 1. Comparison of the deduced amino acid sequence homologies of some plastid and E.
coli genes (Palmer, 1985a). The abbreviations for the genes are standard (Hallick and Bottomley,

1985).

 

rbcL ath atpE atpH atpA

 

Dicot-dicot
Pea-spinach 94 92 82 -- --
Tobacco-spinach 93 96 87 -- --
Monocot-dicot
Maize-tobacco 90 91 70 -- --
Wheat-spinach -- -- -- 100 --
Monocot-monocot
Maize-barley 95 97 98 -- --
Angiosperm-E. coli
Tobacco-E. coli -- 62 27 -- 54
Spinach-E. coli -- 67 26 30 --
Maize-E. coli -- 58 ZS -- --

 

Six of the eight subunits of the chloroplast ATPase complex are encoded by chNAs (Ohyama et al.,
1986; Shinozaki et al., 1986b), and the sequences of four of these (atpA, ath, atpE, and atpH) have
been compared (Table 1). These genes have 82-100% amino acid sequence homology among diverse
angiosperms and 30-67% homology between the angiosperms and E. coli (Palmer, 1985a). Five genes
from photosystem II are encoded by angiosperm chNAs. The gene for the 32 kilodalton (kd)
photosystem II thylakoid membrane protein which binds the herbicide atrazine, has been sequenced
in several organisms. This gene is extremely conserved among diverse groups of organisms. The
conservation of amino acid sequence of the 32 kd protein among five distantly related angiosperms
approaches 100%, and is about 87% between Euglena and the angiosperms (Palmer, 1985a).

Plastid genomes also encode a component of one enzyme of the Calvin cycle, the large subunit
of ribulose- 1-5-bisphosphate carboxylase-oxygenase (RuBisCO). The sequence of the gene for the
large subunit of RuBisCO (rbcL) has been compared among several angiosperms, the green algae
Euglena gracilis, two species of cyanobacteria, and a nonsulfur purple bacterium (Palmer, 1985a).

The amino acid sequence of this gene is highly conserved in every case except between the

9
cyanobacteria-plastid group and the nonsulfur purple bacteria (Table 1), attesting the close
evolutionary relationship of plastids and cyanobacteria.

The regulatory sequences of plastid genes parallel those of procaryotes. Plastid and E. coli
promoter consensus sequences have nearly perfect homology in both the -10 and -35 regions (Briat et
al., 1986; Kung et al., 1986; Zurawski and Clegg, 1987). Furthermore, at least some plastid promoters
function with the E. coli transcriptional machinery in cell-free systems (Bottomley and Whitﬁeld,
1979) or when chNA is cloned randomly into a plasmid vector designed to select promoter
sequences (Kong et al., 1984; Kung and Lin, 1985). Plastid termination sequences also resemble
procaryotic sequences (Palmer, 1985a). Short inverted repeats which can form hairpin loops are
characteristic of E. coli rho-independent terminators and are found downstream from the coding
regions of many plastid genes (Crouse et al., 1984). However, evidence for rho-dependent
termination in plastids is lacking (Zurawski and Clegg, 1987). Most plastid mRNAs have a highly
conserved ribosome-binding site (Shine-Delgarno sequence) which is complementary to the 3’ end of
the plastid 16S rRNA (Kozak, 1983). It is clear from this and other evidence that plastid transcription
and translation strikingly resemble that of procaryotic organisms.

Nevertheless, in plastid genomes the polycistronic expression of related genes found in
procaryotes has often been dismantled (Palmer, 1985a). This is probably the result of the transfer of
many genes to the nucleus (Brinkmann et al., 1987). In addition, rearrangements have dispersed
genes more randomly throughout the plastome (Palmer, 1985b). Many genes belong to smaller
transcriptional units or are transcribed independently, although some of the ancient operons, such as
the rDNA operon, remain virtually intact (Whitfield and Bottomley, 1983). Most of these changes
probably occurred very early in the evolution of land plants since most plastid genomes have nearly
the same complement of genes in nearly the same arrangement.

The plastome of the alga, EugIena gmcilis has at least 50 introns (Koller and Deluis, 1984). In
contrast, the plastid genomes of land plants have few introns. The tobacco plastome has 15 introns
and the M. polymorpha plastome has 18 introns, in both proteinocoding and tRNA genes (Shinozaki et

al., 1986b; Ohyama er al., 1986). In addition, the mRNA from the N. tabacum plastid gene encoding

10

the ribosomal protein 812 is trans-spliced from two independent transcriptional units (Hildebrand et
al., 1988; Plant and Gray, 1988). TWO groups of introns with different excision mechanisms have been
detected in plastomes (Shinozaki et al., 1986a; Weil, 1987; Plant and Gray, 1988) using sequence or
structural similarities to well characterized examples from other genetic systems (Sharp, 1985).
Group I introns, which splice autocatalytically in vitro, are structurally homologous to introns in the
rRNA gene Tetrahymena and some mitochondrial introns of fungi (Shinozaki et al., 1986a; Plant and
Gray, 1988). Most of the introns of land plant plastid genomes belong to group II (Plant and Gray,
1988). Group II introns sometimes splice autocatalytically in vine and can be folded into secondary
structures similar to those postulated for the introns in maize mitochondrial cytochrome oxidase,
yeast cytochrome oxidase and yeast cytochrome b (Michael and Dujon, 1983).

The only angiosperms lacking the large inverted repeat, of 200 or more species examined,
belong to eight related genera of the legume family; Pirum, Vicia, Lathytus, Cicer, Medicago,
Tn'folium, Melilotus, and Wisteria (Palmer, 1985a). Interestingly, the chNA sequences of most
members of this group are highly rearranged relative to the ancestral order and these changes have
occurred after the loss of the inverted repeat (Palmer et al., 1987). Two gymnosperms; the Pinaceae,
Pseutosuga marsiesii and Firm: radiata, also are missing the inverted repeat and have incurred many
chNA rearrangements (Strauss et al., 1988). The extensive rearrangements occurring in the
chNAs of those species having lost the large inverted repeat suggest the repeat has a stabilizing
inﬂuence (Palmer et al., 1985; Strauss et al., 1988).

It is readily apparent from the above discussion that plastid genomes have evolved slowly.
However, the physiological and/or genetic causes of this are more difﬁth to ascertain. Perhaps the
dense arrangement of genes in chNAs inhibits rearrangement since most rearrangements would
disrupt functional sequences (Palmer, 1985b). However, rearrangement is common in the smaller
plastid genomes of species having lost a large inverted repeat suggesting that other mechanisms are
involved (Palmer et al., 1987; Strauss et al., 1988).

It has been difﬁcult to demonstrate intermolecular recombination of chNAs in higher plants

(Chiu and Sears, 1985), and genetic or molecular evidence of it is available only from two experiments

11
with higher plants (Medgyesy et al., 1986; Thanh and Medgyesy, 1989), and from species of
Orlamydomonas (Sears, 1980a; Mets and Geist, 1983; Lemieux et al., 1984). In both cases, the
recombinant molecules have crossover points at frequent intervals, as determined by genetic and
restriction enzyme analysis. The presence of many crossover points is evidence for a mechanism
involving gene conversion (non- reciprocal recombination).

If gene conversion is the major mode of intermolecular recombination in plastids, it probably
occurs intramolecularly as well. Accordingly, both large inverted repeats from several organisms have
been sequenced, and they are identical (Kato et al., 1981; Speilmann and Stutz, 1983; Sugita et al.,
1984; Zurawski et al., 1984; Ohyama et al., 1986; Shinozaki et al., 1986b). Furthermore, mutations
have been recovered in the large inverted repeat of Chlamydomonas, and both copies are identically
altered (Dron et al., 1983; Erickson et al., 1985). This implies that there is a mechanism for copy
correction (gene conversion) between the repeats. It seems plausible that copy correction would also
occur between the multiple copies of the chNA within each plastid. With such a mechanism in
operation, a mutant molecule within the population of chNAs in a plastid could be either eliminated
or ﬁxed through successive rounds of gene conversion (Birky, 1978). A mutation would occur ﬁrst in
one molecule in a population of 20-200 wild type molecules per plastid (Possingham and Lawrence,
1983; Steel-Scott et al., 1984; Bendich, 1987), so there would be a greater chance that the mutation
would be eliminated rather than ﬁxed (Sears, 1983). Once established within a single plastid the
mutation must also compete in a population of perhaps 10-200 plastids per cell (Possingham and
Lawrence, 1983). Cells with the mutant plastids must also segregate into the germ line if they are to
be carried to subsequent generations (Kirk and Tilney-Bassett, 1978). It is likely that these are
stochastic processes, so the chances for successful establishment of a mutation are low, because of its
initially low abundance (Birky, 1983). Thus the high ploidy of the plastome and the large number of
plastids per cell may contribute signiﬁcantly to the conservation of chNA.

There is abundant evolutionary evidence pertaining to mutations in the chNAs of
angiosperms (Palmer, 1987). Sequence inversions in plastid genomes having the large inverted

repeats are quite rare evolutionarily. All of the inversions discovered upon examination of about 160

12

species of ﬂowering plants having large inverted repeats are contained within the single-copy regions
(Palmer, 1985a). Insertion and deletion mutations are more common. The great majority of these
mutations are 1- 10 base pairs (bp), and those of about 50-1000 bp are somewhat infrequent (palmer,
1985a). Large length mutations (greater than 1-2 kb) are extremely rare in chNA of land plants
(Palmer, 1987). Gain or loss of restriction sites due to small length mutations and base substitutions
is responsible for most of the interspeciﬁc changes in chNA restriction pattern that are observed
(Palmer, 1985b).

The rate of silent substitutions in chNAs is comparatively low; about 1.0 - 3.0 x 10'9 per year
for several genes in the single-copy regions, and at least three times lower for sequences in the
inverted repeats (Zurawski et al., 1984; Wolfe et al., 1987). In comparison, the silent substitution rate
is estimated at 0.1 - 11 x 10'9 per year for mammalian nuclear genes, 5 - 30 x 10'9 per year for plant
nuclear genes, 18 - 54 x 10'9 per year for mammalian mitochondrial genes and 0.2 - 1.0 x 10'9 per year
for plant mitochondrial genes (Wolfe et al., 1987). The estimate for the plant nuclear genome is
tentative since few sequences for the same gene are available from different species. Additionally,
many of the dates of lineage divergence used to calculate substitution rates in plant genomes are
unreliable because fossil evidence of plant evolution is often scarce.

Although intraspeciﬁc variation in chNA restriction pattern is somewhat rare among
angiosperms it has been detected, particularly in larger samples. Such variation has been observed
between populations (Palmer et al.,1985; Teeri et al., 1985; Banks and Birky, 1985), among individuals
within the same population (Robertson, 1985; Wagner et al., 1987), and even within individual plants.
Moon et a1. (1987) sequenced two chNA fragments derived from an individual rice plant, which
encode the same region of the genome. One of the fragments had a truncated rbcL gene and a
frame-shifted ath gene and the other had functional genes. The defective fragment was always 10
times less abundant than the normal one in bulk DNA isolations from many plants or isolations from
individual plants. The authors suggested that the polymorphic chNAs are maintained stably by
genetic complementation of different defects in the different types of molecules. Rose et a1. (1986)

examined the restriction pattern of chNA from 23 Medicago sativa protoclones derived from two

13
clonally propagated plants. A novel restriction pattern was detected in several of the protoclones, but
the novel pattern was exactly the same in each protoclone. The chNA of selfed progeny of one of
the plants used to derive the protoclones segregated, giving both the original pattern and the novel
one. These results indicate chNA variation can occasionally be maintained within a population or
by an individual plant, and this variation may be difﬁcult to detect.

The inheritance of plastids in the plant kingdom is usually maternal, but many taxa inherit
plastids biparentally or paternally (Kirk and Tilney-Bassett, 1978; Tilney-Bassett and Abdel-Wahab,
1979; Sears, 1980b; Whatley, 1982). Cytoplasmically inherited traits, such as restriction fragment
length polymorphisms (RFLPs), pigment mutations, and antibiotic resistance must be used to follow
organelle inheritance plants. Three genetic criteria that may be used to establish the cytoplasmic
inheritance of a trait are: 1) non-Mendelian inheritance such an reciprocal differences after
reciprocal crosses, and 2) the sorting out of genetic entities during development 3) molecular genetic
evidence that the trait is cytoplasmically encoded (Kirk and Tilney-Bassett, 1978). Genetic analysis is
necessary to establish the mode of organelle inheritance deﬁnitively, since cytological observation of
the organelles during the reproductive process is sometimes misleading (Sears, 1980b). In higher
plants, cytoplasmically inherited traits must be assigned to either plastids or mitochondria by
morphological, cytological or biochemical analysis (Michaelis, 1966; Kutzelnigg and Stubbe, 1974;
Fluhr et al., 1985; Borner and Sears, 1986).

By far the largest body of evidence concerning the mode of plastid inheritance is available for
the angiosperms. Both maternal and biparental inheritance are found in both dicot and monocot
species, and strictly paternal inheritance is very exceptional (Tilney-Bassett and Birky, 1981). It is
interesting that some genera, such as Epilobium (Schmitz and Kowallik, 1986) and Solanum
(Simmonds, 1969), initially thought to have strictly maternal plastid inheritance transmit paternal
plastids very infrequently. Similarly, Medgyesy et al. (1986) discovered very minimal paternal chNA
inheritance in Nicotiana, which was previously considered to have strictly maternal inheritance. They
accomplished this by fertilizing N. plumbagimfolia plants using pollen from a line with plastome-

encoded streptomycin resistance. The seedlings from these crosses were induced to form callus on

l4
streptomycin-containing media. Resistant sectors were green on a background of white cells, since
streptomycin bleaches sensitive cells, but does not kill them. Thus, tiny sectors of paternal plastids
were found in 0.07% to 2.5% of these calli. Earlier studies would not have discovered paternal plastids
in Nicotiana because the plants having them had so few that they were undetectable by conventional
methods. This suggests that other species thought to have strictly maternal plastid inheritance might
likewise inherit some paternal plastids.

largely paternal plastid inheritance seems to be the general rule in the gymnospcrms although
data is available for only a few groups. Genetic evidence for paternal inheritance is available for
species of Picea, Pinus, Pseudotsuga and Cryptomen’a (Stein and Keathley, personal communication;
Neale et al., 1986; Wagner et al., 1987). However, Ohba et al. (1971) observed 0% to 3% biparental
plastid transmission in crosses of Cryptomen'a japarica. This points to the possibility of traces of
maternal plastid inheritance in the other gymnospcrms since this study examined a much larger
population than the others. Cytological examination of spermatogenesis and fertilization suggests that
plastids are inherited through a variety of mechanisms in the gymnospcrms but genetic and
biochemical evidence is needed to conﬁrm this (Sears, 1980b).

Different methods of plastid inheritance have arisen on several independent occasions in the
angiosperms, since more than one method of plastid transmission exists within several orders and even
within some families (Tilney-Bassett and Abdel-Wahab, 1979; Sears, 1980b). Most higher plants
transmit plastids uniparentally and this could contribute to the conservation of the plastome. However,
the plastome is also conserved in biparental species such as the Euoenothera (Gordon et al., 1982)
suggesting that other factors are involved. It is also possible that the high ploidy and frequent gene
conversion of plastid genomes have restrained them from genetic alteration by eliminating most
recombinant molecules (Birky, 1978; Birky, 1983; Sears, 1983). Moreover, the genomes from different
plastids probably seldom interact since plastids rarely fuse (Esau, 1972, Vaughn, 1981, Sears, 1983).
Thus plants would be released from selection for biparental exchange of plastomes since advantageous
recombinant genotypes would be produced extremely rarely (Medgyesy, et al., 1985; Chin and Sears,

1985; Thanh and Medgyesy, 1989). Hence the mechanisms of plastid inheritance would be free to

15
evolve in response to other pressures (Sears, 1983; Birky, 1983). This may explain the heterogeneity of
plastid transmission since different mechanisms may have arisen in response to a variety of selection
pressures on a variety of occasions.

Sears (1980b) and Whatley (1982) reviewed the voluminous cytological literature on sexual
reproduction in plants and noted that the male or female plastids are eliminated during a variety of
reproductive phases. In angiosperms the plastids can be eliminated from the male gametes by
exclusion from the generative cell. Those which are not excluded may disintegrate or be genetically
incapacitated as the pollen grain matures (Whatley, 1982; Day and Ellis, 1984). In addition the male
plastids of pollen-bearing plants can be excluded from the egg during the process of fertilization. In
several species of gymnospcrms the female plastids appear to degenerate within the egg cell following
fertilization. In the lower land plants, which have motile sperm, the plastids are often collected into a
cytoplasmic vesicle and ejected from the male gamete prior to fertilization (Sears, 1980b; Whatley,
1982).

More cytological evidence is available regarding the mechanisms of cytoplasmic inheritance in
angiosperms than for any other group. During angiosperm pollen production haploid microspores
divide asymmetrically to produce a smaller generative cell and a larger vegetative cell. All or most of
the plastids partition into the vegetative cell during this division in most species (Reviewed by: Sears,
1980b; Whatley, 1982; Connett, 1987; e.g. Russell, 1983; Russell, 1984; Schroder, 1985). The plastids
within the generative cell are probably the only pollen plastids which can enter the egg (Whatley,
1982). Complete elimination of plastids from generative cells is difﬁcult to prove using light or
electron microscopy, and some species in which no generative cell plastids were detected inherit
paternal plastids (Connett, 1987). This may stem from the difﬁculty in distinguishing small
proplastids from mitochondria in the pollen of many species (Connett, 1987). Additionally, complete
serial sectioning of a number of cells is needed to be sure that a few plastids do not remain
undetected (Sears, 1980b). This requires a large amount of work and has seldom been done.

Plastomes probably are altered during pollen production in some angiosperms.

Ultrastructural evidence indicates that plastids disintegrate in the generative cell before or after it

16

divides mitotically to form the sperm cells in species of Nicotiana (Clauhs and Grun, 1977) and
Epilobium (Schmitz and Kowallik, 1987). The haploid plants regenerated from anther culture of
certain species are frequently albino. Albino rice plants derived from anther culture have plastids
with highly abnormal ultrastructure (Vaughn et al., 1980). Day and Ellis (1985) examined the chNA
from anther-culture-derived albino wheat and barley plants using Southern blotting. In some of the
plants up to 70% of the genome had been deleted. They propose that a mechanism involved in
maternal plastid inheritance is responsible for these deletions (Day and Ellis, 1984). Plastid DNA
degradation probably begins prior to the microspore mitosis since the haploid plants studied only
regenerated from immature pollen, prior to formation of the sperm cells (Vaughn et al., 1980), and
cultured plant cells probably are inviable without plastids. In support of this, Mogensen and Rusche
(1985) did not ﬁnd any recognizable plastids in immature sperm cells of barley after examining
electron micrographs of complete serial sections from several pollen grains.

Fertilization also provides an opportunity for the elimination of paternal plastids in
angiosperms (Sears, 1980b; Whatley, 1982; Connett, 1987). In spinach the cytoplasms of the pollen
tube (derived from the vegetative cell) and the sperm cells (derived from the generative cell) have
plastids when the pollen tube grows through the ﬁliform apparatus into the degenerating synergid
(Wilms, 1981a; Wilms, 1981b). After the pollen tube discharges, the tube plastids remain in the
degenerating synergid where they disintegrate. The nucleus of one of the sperm cells enters the eg
for fertilization, leaving its organelle-containing cytoplasm in the degenerating synergid, thus
excluding the male plastids. In contrast, Plumbago zeylanica lacks synergids and the pollen tube
discharges into the intracellular space between the egg and central cells (Russell, 1983). When the
sperm cell and egg cell membranes fuse during fertilization the sperm cytoplasm, with its organelles,
enters the egg along with the nucleus.

Plants with biparental plastid inheritance allow the study of some unique features of
cytoplasmic inheritance. Such plants produce embryos with either maternal, paternal, or mixed plastid
complements (Kirk and Tilney-Bassett, 1978). In plants derived from mixed embryos the maternal and

paternal plastids segregate during vegetative development, ultimately producing pure or chimeric

17

shoots. Chimeric shoots have different plastid types in the different concentric tissue layers of their
apical meristems (Kirk and Tilney-Bassett, 1978; Tilney-Bassett, 1986). Plastid chimeras can be
relatively stable during plant growth. Plastome-encoded, pigment-deﬁcient mutants can be used to
follow the progress of this segregation during plant growth (Kirk and Tilney-Bassett, 1978). There are
four predominant phases in the process: 1) the initial phase with mixed cells only and no visible
manifestation, 2) a ﬁne mosaic phase during which the mixed cells sort-out as they divide producing
pure white and pure green cells, 3) a coarse mosaic phase with few remaining mixed cells during which
pure tissues sort-out from a mosaic of pure cells, and 4) a ﬁnal phase during which pure white, pure
green, or chimeric shoots segregate from pure tissues.

Plastid segregation is a stochastic process if the different plastids replicate with equal efficiency,
partition randomly between progeny cells, and do not have a differential effect on cell division or tissue
development (Michaelis, 1967a). If plastid segregation is a stochastic process, statistical models can be
created to predict its features. Michaelis (1967a) pr0posed such a model, and described the
mathematical details. This model requires the following additional assumptions: 1) Prior to division
the number of plastids per cell is a constant (2N). 2) Plastids always partition equally between the two
daughter cells (1N per cell). 3) Each plastid replicates once between each cell division.

The model predicts several general features of plastid somatic segregation (Kirk and Tilney-
Bassett, 1978). A cell must divide approximately 10 x N times to achieve almost complete (>99%)
sorting-out. Furthermore, the most abundant class of mixed cells always has the same proportion of
plastid types as the initial cell had. After sorting out is complete, the proportion of cells with each
plastid type is the same as the proportion of each plastid type in the initial cell. This model provides
an excellent basis for an understanding of plastid somatic segregation; most plastid traits sort out as
predicted, although deviations are fairly common (Michaelis, 1967b; Kirk and Tilney-Bassett, 1978;
Birky, 1978; Kumar and Cocking, 1987).

I have, until this point, assumed that individual plastids are the units of plastid segregation.
However, it is logical that chNA molecules should be the segregation units, since they encode the

plastid genetic information. Higher plants have about 500-50,000 copies of the plastome per cell

18

(Possingham and Lawrence, 1983; Bendich, 1987), so it would take at least 2500 (10 x N) cell divisions
for complete sorting-out if chNA molecules were the segregation units. An annual plant would
need several reproductive generations to complete 2500 cell divisions. However, annuals usually sort
out completely in only one or two generations (Michaelis, 1967a). This suggests that the predominant
segregation units must be plastids. There are approximately 10-30 plastids in the meristematic cells of
higher plants (Possingham and Lawrence, 1983; Steele-Scott et al., 1983) so complete sorting-out
should take about 50-150 cell divisions. This number corresponds well with the cell division
requirements for one to two generations in annual species (Michaelis, 1967b).

Unequal plastid partitioning into daughter cells, incomplete plastid mixing between divisions
and plastid selection will always reduce the eﬂ‘ective number of segregation units, so calculations using
the number of plastids per cell give the maximum number of divisions needed for segregation
(Michaelis, 1967a). For example, the ﬁrst division of angiosperm zygotes is unequal and only the
smaller of the cells contributes to the adult plant, thus reducing the effective number of plastids per
cell (Whatley, 1982). Plastids have been observed to mix more slowly than most other cytoplasmic
particles (Honda et al., 1964), so sister plastids may partition into the same daughter cell more
frequently than expected (Michaelis, 1967b; Kirk and Tilney- Bassett, 1978). Electron microscopic
observations in Oenothem suggest that plastid mixing in the zygote is particularly slow (Meyer and
Stubbe, 1974).

It is implicit that plastids do not exchange genetic information regularly since they, not chNA
molecules, are the predominant units of vegetative segregation. Cytological observations conﬁrm that
plastid fusion occurs very rarely (Esau, 1972; Vaughn, 1981), if at all (Sears, 1983) in angiosperms.
Chiu and Sears ( 1985) exploited the biparental plastid inheritance and available pigment mutations of
Oenothem to look for plastid recombination. No green recombinant sectors were detected in an
estimated 1,000 to 3,700 seedlings with mixed plastids. They suggested that rare recombinant
molecules may have remained undetected if there had been insufﬁcient cell divisions for complete
sorting-out in these seedlings. Medgyesy et al. (1985) successfully selected a recombinant Nicotiana

somatic hybrid in callus culture using a clever scheme. One cell line was green, streptomycin-sensitive

19

and lincomycin-resistant. The other line had two chNA mutations; one conferring pigment
deﬁciency and the other conferring streptomycin-resistance. Streptomycin-sensitive cells bleach, but
continue to grow slowly on streptomycin-containing media. Since the plastids of the other cell line
were also white due to the chNA mutation, only the cells with fused and/ or recombinant plastids
could turn green on streptomycin-containing media. The scheme maintained heteroplasmic cells, but
selected green recombinant cells which grew faster than the white cells. Later, growth on lincomycin
media was used to distinguish the rare recombinants from pigment deﬁciency revertants. The authors
tentatively estimate that 2000 to 20,000 heteroplasmic cells were screened to obtain one recombinant.
The extreme rarity of chNA recombination in angiosperms (Kutzclnigg and Stubbe, 1974;
Medgyesy, et al., 1985; Chiu and Sears, 1985) is in accord with the rarity of plastid fusion and the
rapid pace of plastid vegetative segregation.

Carefully designed experiments attempting to correlate cytological observation of the plastid
input with genetic analysis of the output, using either sexual or somatic hybridization have not been
reported. There is, however, some evidence suggesting that plastid input and output can be correlated
as predicted by the Michaelis model if neither plastid genotype has a selective advantage. Electron
microscopic observations reveal that Oenothera egg cells contribute about 3—4 times more plastids to
the zygote than sperm cells do, and this roughly correlates with the general maternal bias of plastid
inheritance in the genus (Meyer and Stubbe, 1974; Kirk and Tilney Bassett, 1978). Akada and Hirai
(1986) conducted more convincing experiments using somatic hybridization of wild Nicotiana species.
They used isoelectric-focusing of the large subunit of RuBisCO to analyze the plastids of 23 calli
presumably derived from single fusions. They assumed each of the mesOphyll-derived fusion partners
contributed the same number of plastids and neither plastid type had a selective advantage. They
found balanced plastid transmission as predicted by the Michaelis model. Furthermore, the degree of
sorting—out corresponded to that expected after 40 divisions of cells having 20 plastids. Plastid counts
of the material they used are lacking. However, roughly 20 plastids per cell have been observed after

three or more divisions of mesophyll-derived protoplasts of N. tabacum (Thomas and Rose, 1983).

20

More rapid division of the plastids from one parent can increase their frequency in subsequent
cell generations. Contrary to an assumption of the Michaelis model, each plastid probably does not
replicate exactly once per cell cycle. Plastid division can occur continuously during the cell cycle but
the maximum number of plastids per cell is limited, and this limit varies with the developmental
regime of the tissue (Possingham and Lawrence, 1983; Thomas and Rose, 1983). When the number
of plastids per cell is below the limit, those that divide more rapidly can reproduce more frequently
and predominate in subsequent generations.

Oenothera has yielded genetic evidence supporting this concept. Schiitz (1954) discovered
differences in competition among the ﬁve different plastomes of Oenothera subsection Euoenothera
when they were brought together by sexual matings. Each of the ﬁve plastome types was assayed using
reciprocal crosses in which one parent carried a plastome-encoded albino mutant, and the other
carried the wild-type plastome to be tested (Cleland, 1972, Hagemann, 1976; Kirk and Tilney-Bassett,
1978). The germinating seeds from these crosses were scored by measuring the relative size of the
green sectors on their cotyledons, giving a quantitative estimate of the relative competitiveness of each
plastome. This “variegation rating" ranked the ﬁve plastomes in nearly the same order regardless of
the direction of the cross or the nuclear background (Kirk and Tilney-Bassett, 1978; Chiu et al., 1988).
Schotz (1968) attributed this phenomenon to differences in the rate or onset of plastid division. The
nuclear genome also influences the variegation rating, but not to the same extent as the plastome.

After certain interspeciﬁc hybridizations of Oenothem the plastome cannot function properly
with the hybrid nuclear genome, causing several types of physiological disturbances (Cleland, 1972;
Kutzelnigg and Stubbe, 1974; Kirk and Tilney-Bassett, 1978). Some manifestations of this
phenomenon are: periodic chlorophyll deﬁciency and re-greening, complete chlorOphyll deﬁciency,
impaired growth, deformed leaves, inhibition of embryo development, and lethality (Kutzelnigg and
Stubbe, 1974; Kirk and Tilney-Bassett, 1978). Nuclear control of these "plastome- genome"
incompatibilities is usually polygenic (Kirk and Tilney-Bassett, 1978). The phenomenon is called

hybrid variegation when the affected plastids are deﬁcient in chlorophyll development.

21

ABC DE FGHIJKLMN
—e——o——e——o——e——e——O—
—e——e——e——e——e——e—-e—
BCDEFGHIJKLMNA

Figure 3. Diagram of the chromosomal pairing of an Oenothera ring chromosome. The
chromosomal arms marked "A” pair to form a continuous chain or ”ring chromosome”. Redrawn
from Kirk and Tilney-Bassett (1978).

Such plastids behave similarly to plastids with pigment mutations except they green when reunited
with a compatible nuclear genome since the plastome is not altered (Schotz, 1962).

Hybrid variegation has been demonstrated in Pelalgonium (Metzlaff et al., 1982; Pohlheim,
1986) and other genera (Menacl et al.,l987), but has been studied most extensively in Oenothera
(Kirk and Tilney-Bassett, 1978). Oenothera subsection Euoenothera is well suited for such
experiments since evolutionarily distinct haploid nuclear complements occupying the same nucleus
can form ring chromosomessuppressing genetic reassortment (Cleland, 1972). Ring chromosomes
result from multiple reciprocal translocations of chromosomal arms within the haploid complements.
During meiosis chromosomal complements with different translocations pair in a zig-zag
arrangement (called a ring chromosome) since homologous arms are displaced to different
chromosomes (Figure 3). When the entire genome forms one ring each haploid complement
segregates as a unit and recombination is greatly suppressed.

Because of the prevalence of ring chromosomes in Euoenothera cytoplasmic substitution is
possible without repeated back-crossing. Stubbe (1960) studied the reaction of the ﬁve different
plastid types of subsection Euoenothera and found profound differences in their ability to green with
different combinations of the three basic haploid nuclear complements (Figure 4). The manifestation
of this plastome-genome incompatibility ranged from small changes in chloroplast pigmentation to

complete lethality.

 

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Figure 4. Compatibility relations between different nuclear genome complements and
plastomes of Euoenothera (from Stubbe, 1959). The use of more than one sign in some squares
depends on slight differences between different A-complexes.

Not surprisingly, each plastome type is more compatible when associated with genomes closely
related to the one with which it co- evolved (Cleland, 1972; Kirk and Tilney-Bassett, 1978).
Replication speed might also be important in the evolution of the subsection, as depicted in
Figure 5 (Stubbe, 1964). The heavy line in the ﬁgure encloses all the nuclear genome- plastome
combinations which green normally, and the species are written in the boxes corresponding to their
genetic complements. Whenever a functional complement is not represented by a species there is a
modern species with same nuclear complement and a more rapidly replicating plastid. On this basis
Stubbe (1964) has suggested that more rapidly replicating plastids have replaced slower ones during
the evolution of some species. Restriction analysis of the ﬁve plastomes is consistent with this
hypothesis; the slowest plastid might be the most evolutionarily primitive, and the most rapid plastid
types might be the most modern (Gordon et al., 1982). However, cladistic of analysis a larger number

of evolutionarily informative restriction site differences is needed to verify this conclusion.

 

 

 

 

 

 

 

 

 

 

 

 

 

wtgminilviv
genome

AB bierw's-I Dim-2

BB was

BC WI

CC wgr'llicola
AC WW L

 

 

 

 

Figure 5. Distribution of the natural species among the compatible combinations (framed
squares) of Euoenothera. The signs between the plastomes I-V refer to the relative strength
(multiplication rate) of the plastids. Adapted from Stubbe (1964) as modiﬁed by Chiu et al., (1988).

The genetics of biparental plastid inheritance in the cultivated geranium, Pelalgonium x
Hortorum Bailey has been studied extensively (Kirk and Tilney-Bassett, 1978; Kirk and Tilney-Bassett
1978). A single nuclear locus in conjunction with some modifying genes controls the mode of plastid
inheritance in Pelatgonium (Tilney-Bassett, 1976). The alleles at this locus switch the plastid
inheritance pattern between type I; maternal zygotes (MZ) > biparental zygotes (BPZ) > paternal
zygotes (P2), and type 11; M2 > BPZ < PZ. Type I plants (PrlPrl) breed true, and Type 11 plants
(PrlPrﬂ always segregate, usually with an ”unexpected" 1:1 ratio of type I to type II. Tilney-Bassett
and Abdel-Wahab (1982) found no homozygous (Pr2Pr2) plants after extensive analysis of many
crosses. To explain their perplexing data they propose that this locus encodes a rather complex self-
incompatibility system and that the same or a tightly linked locus also controls the patterns of plastid
inheritance.

Neither type of plastid segregation pattern in Pelalgom'um is consistent with the Michaelis

model, because the distribution of maternal and paternal plastids in the biparental plants from

24
particular crosses has no mode corresponding to the average plastid composition of the zygotes
(Tilney-Bassett and Birky, 1981). Furthermore, the degree of sorting-out is extremely variable in both
type I and type II crosses. These inconsistencies can be explained in terms of another model which
was originally proposed to describe Chlamydomonas plastid and yeast mitochondrial inheritance
(Birky, 1975; Birky, 1978; Birky; 1983; Sears, 1980; Thraikill et al., 1980; Birky et al., 1981; Galloway
and Holden, 1985).

According to this model the organelles within a cell constitute a population which follows the
rules of population geneties. In contrast to the Michaelis model wherein each organelle replicates
exactly once, a smaller subsample of the intracellular population is selected at random to form the
next generation. The model does not stipulate how the subsample is selected; it could involve
differential organelle replication, differential organelle degradation, or other mechanisms. The
subsample is small so there is random drift in the frequency of organelle types. Tilney-Bassett and
Birky (1981) suggest that genes at the Pr locus act in the zygote to control the selection of a
subpopulation of plastids to be transmitted. Type 1 (M2 > BPZ > PZ) plants preferentially select
maternal plastids, but often include a few paternal ones. Type. 11 (M2 > BPZ < PZ) plants may
select either maternal of paternal plastids, but often completely exclude all the plastids from the other
parent. Careful cytological observation with precise plastid counts during the Pelargonium
fertilization, embryo development and germination processes would enhance our understanding of its

organelle inheritance.

Plastid Transmission in Somatic Hybrids

Plastid evolution and sexual reproduction has interesting parallels with plastid behavior during
somatic hybridization experiments, despite the artiﬁcial conditions created through somatic
hybridization (for reviews of plastid transmission following somatic hybridization see: Fluhr, 1983;
Harms, 1983: Gleba and Syntnik, 1984; Pelletier and Chupeau, 1984; Pelletier, 1986; Maliga and

Menczel, 1986; Grun, 1986; Kumar and Cooper-Bland, 1986; Kumar and Cocking, 1987). The

25
striking evolutionary conservation of the plastome gene complement and arrangement implies that
plastomes are highly resistant to genetic changes. Indeed, the chNA-coding complement and
arrangement is stable during somatic hybridization (Medgyesy et al., 1985; Thanh and Medgyesy,
1989). Plastome recombination is extremely rare in both sexual and somatic crosses (Kutzelnigg and
Stubbe, 1974; Chin and Sears, 1985; Medgyesy et al., 1985) probably due to the rarity of plastid fusion.
Plastid fusion has not been observed in viable angiosperm cells after fusion (Fowke and Rennie, 1976;
Fowke et al., 1977; Rennie er al., 1980; Hodgson and Rose, 1984). However, plastid fusion may be
required for plastome recombination and recombination does occur rarely after somatic hybridization
(Medgyesy et al., 1985; Thanh and Medgyesy, 1989).

The rapid pace and stochastic characteristies of plastid segregation in somatic hybrid cells
(Akada and Hirai, 1986) are consistent with observations of the process in sexual hybrids (Michaelis,
1967a; Birky, 1978). Michaelis’ (1967a) mathematical model of plastid segregation predicts that when
plastid selection causes unbalanced transmission, mixed cells will be maintained for fewer cell
divisions than if there were no selection. Mixed shoots may be more unusual in hybridization
experiments with unbalanced plastid transmission than in experiments with balanced transmission.
There have been at least twenty reports of somatic hybridization experiments where at least 10
hybrids were analyzed and there was non-random plastid transmission. Only ﬁve of these report the
recovery of shoots with mixed plastids. On the other hand, twelve hybridizations where more than 10
plants were analyzed seem to have had balanced plastid transmission and ﬁve of these had shoots with
mixed plastids (Table 3).

Furthermore, plastid transmission in somatic hybridization experiments frequently deviates
from the expectations of purely random behavior (Table 3) and these deviations can usually be
explained using concepts developed to explain similar phenomena in sexual crosses. The possible
causes of unequal transmission of organelles in somatic hybrids are: 1) an initially larger contribution
of organelles from one fusion partner, 2) more efﬁcient replication of the organelles from one

partner, 3) genetic incompatibility of the nuclear genome with the organelles from one partner, or 4)

26
selection of the organelles from one partner in vitm using antibiotics, herbicides or culture conditions
(Kumar and Cocking, 1987).

The relative number of plastids contributed by the donor cells could inﬂuence plastid
transmission in sexual and somatic hybrids similarly (Whatley, 1982; Flick and Evans, 1982; Fluhr,
1983; Connett, 1987). Differential transmission of plastids due to unequal input from the gametes is a
well-documented aspect of sexual reproduction in higher plants (Sears, 1980b; Whatley, 1982;
Connett, 1987). If segregation in somatic hybrids were purely stochastic, the proportion of pure cell
lines after complete sorting-out should reﬂect the original plastid ratio in the heterokaryon
(Michaelis, 1967a; Birky, 1978; Akada and Hiria, 1986). Mesophyll and suspension cells probably
contribute a signiﬁcantly different number of plastids when they are fused. Mesophyll cells have more
plastids than any other tissue examined (Steele-Scott, 1984), about lO-fold more than meristematic
cells (Possingham and lawerence, 1983). No information about the number of plastids found in long
term suspension cells is available, but these rapidly dividing cells are similar to the meristematic cells
of whole plants and probably have relatively few proplastids. Thus, when mesophyll and suspension
cells are fused the plastids from the mesophyll partner should predominate in the heterokaryon.

Akada et al. (1983) examined plastid segregation in Nicotiana glauca + N. Langmorﬁi' hybrid
callus after reciprocal fusions of suspension and mesophyll cells, or fusion of suspension cells from
both species. Samples of 10-13 calli from each of the fusions seem to have had balanced transmission,
suggesting that the tissue source was unimportant. This fusion pair was well chosen since it has had
primarily balanced plastid transmission in other experiments (Table 3), reducing the possibility of
factors other than tissue source confounding the result. However, the sample size they analyzed was
small, particularly since samples with mixed plastids are uninformative without analysis of their

plastid ratio, and the authors list over half the calli they analyzed as mixed without giving the ratio.

 

* hereafter " + " denotes somatic hybridization and an ")6 denotes sexual hybridization

27

Table 2. Plastid competitive ability in Nicotiana somatic hybrids. This table only includes data
from somatic hybridization experiments which did not involve plastid selection using antibiotics
herbicides or other chemicals. The data from all the hybridizations where the parental species pair
has the same isoelectric-focusing pattern for the large subunit of RuBisCO have been combined, and
cumulative ratio is given in the same order as the parental isoelectric-focusing patterns. Statistical
signiﬁcance is based on the conﬁdence limits of the binomial distribution with the null hypothesis of
an equal proportion of each plastid type, and all the categories listed as not signiﬁcant (N.S.) are not
signiﬁcant at the 10% conﬁdence level.

 

Parental Plastid Number Mode of Cumulative Statistical

 

RuBisCO Types of Exps. Transmission Ratio Signiﬁcance
Type II + Type III 10 Unbalanced 28:110 < 1%
Type II + Type III 3 Unbalanced 26:4 < 1%
Type II + Type A 5 Unbalanced 33:79 < 1%
Type II + Type I 6 Balanced 64:55 N.S.
Type II + Type II 4 Balanced 92:102 N.S.
Type III + Type III 1 Balanced 10:12 N.S.

 

There are eight reports of mesophyll-suspension cell fusions of various species where
mesophyll-derived plastids clearly predominated, none where the plastids from the suspension partner
predominated and two reports of relatively large populations having balanced transmission (Table 3).
The cumulative ratio of plastids from mesophyll fusion partners to suspension fusion partners from all
these experiments is 185 to 97 (Table 3). However, the implications of this are uncertain because the
number of comparisons is small and many uncontrolled factors could affect the result.

Examination of the process of plastid segregation as heterokaryons divide to produce callus
exposes some pitfalls inherent in investigations of plastid transmission in somatic hybrids. Akada and
Hirai (1983, 1986) demonstrated that different sectors of hybrid calli have very different plastid ratios.
The largest difference between sectors of the same callus was about 80%. This illustrates the
possibility of inadvertently selecting one type of plastid during routine culturing of small numbers of
hybrid calli, if portions of the calli are discarded.

The relative replicative competitiveness of different plastids is important in determining plastid

sexual transmission in Euoenothera (Schotz, 1954; Kirk and Tilney- Bassett, 1978), and may have had

28

a role in the evolution of this subsection (Stubbe, 1964). Perhaps replicative competitiveness is a
determinant of plastid transmission of somatic hybrids. In fact, there is a relationship between the
behavior of plastids in Nicotiana somatic hybrids and a plastome encoded biochemical marker (Table
2; Table 3). This marker is the isoelectric-focusing pattern of the large subunit of the Calvin cycle
enzyme, ribulose bisphosphate carboxylase-oxygenase (RuBisCO). The enzyme probably is not
functionally related to plastome replicative competitiveness. However, the isoelectric-focusing
pattern of the large subunit of RuBisCo has been highly conserved during evolution, and has been
used to follow evolution within several genera including Nicotiana (Chen and Wildman, 1981). Sixty-
three species of Nicotiana have only four RubisCO large subunit isoelectric-focusing patterns and the
patterns of the remaining two species are unknown. Twenty-eight species have pattern 111, 23 have
pattern A, 7 have pattern 11, and 5 have pattern I (Chen and Wildman, 1981).

All ﬁve somatic hybridizations between different pairs of Nicotiana species having the same
large subunit pattern appear to have balanced plastid transmission (Table 2; Table 3). The three
hybridizations between different species pairs with patterns [I and I also appear to have balanced
transmission. The two hybridizations between different pairs of 1 species with patterns II and A both
have unbalanced transmission, and the type A plastids are always favored. The suggested order of
replicative efﬁciency for these plastid types is type I = type II < type A. This conclusion is tentative
since the number of different species pairs hybridized is small and many uncontrolled factors could
affect the result. The type II plastome may be the evolutionary progenitor of the type A plastome
(Kung et al., 1982). This suggests that relative replicative competitiveness may have a role in the
evolution of Nicotiana species that is similar to its putative role in Euoenothera evolution (Stubbe,
1964; Chiu et al., 1988).

The results of fusions between type II and III species are more complex. There are seven
groups of hybridization experiments having different parental species pairs with these RuBisCO
patterns excluding experiments without enough observations for statistical signiﬁcance (Table 3). In
four of these type II + type III fusions, type III plastids prevail, and in three fusions type II plastids

prevail (Table 2; Table 3, pp. 43-45). Nicotiana tabacum and N. sylvesais are the only type II species

29

used for all the type II + type III fusions. Differences between these species may not be responsible
for the differences between transmission patterns type II + type III fusions, since a fusion between
them has balanced transmission. Furthermore, N. (abacum is thought to have arisen from an
interspeciﬁc hybridization with N. sylvesm's as the cytoplasmic parent (Gray et al., 1974). The
differences in transmission are probably due to differences between the ﬁve type III species used for
the fusions. More species have type III plastids than any of the other types and most of these are
native to South America, the center of origin of the genus (Chen and Wildman, 1981; Goodspeed,
1954). Therefore, it would not be surprising to ﬁnd greater plastid diversity within this group.

Examination of the reaction of these species to the fungal toxin, tentoxin is also relevant.
Tentoxin is another plastome encoded trait which probably has no functional relationship to plastid
replicative competitiveness (Durbin and Uchytil, 1977b). All three type III species used in the fusions
where type II plastids prevailed are sensitive to tentoxin (Table 3). Conversely, three of the four type
II + type III fusions where type 111 species prevailed utilized tentoxin insensitive type III species.

Fusions between N. labacum, N. sylvesmls, and N. undulata, in all three combinations may
demonstrate that plastome-controlled differences in replicative competitiveness are not the only
influence of plastid transmission in somatic hybridizations. The N. tabacum + N. sylvesm's fusion has
balanced transmission (Table 3), yet N. undulata plastids prevail in the N. tabacum + N. undulata
fusion (Table 3) and N. sylvesuis plastids prevail in the N. sylvestris + N. undulata fusion (Table 3).
However, the origin of the cytoplasm of the male sterile line of N. (abacunr used in the N. tabacum +
N. undulata fusion was questionable, and may not have been N. undulata. The identity of this
unknown cytoplasm was surmised using chNA restriction, RuBisCO large subunit isoelectric-
focusing, and the male sterile flower morphology, but the data was not published (Bonnett and
Glimelius, 1983). Of the three methods used only the chNA restriction analysis was potentially
deﬁnitive. However, their identiﬁcation is very questionable since chNA restriction was used only
used to determine that the plastids were not from N. plumbagim’folia as thought originally. Plastid
DNA restriction data distinguishing only four of the twenty- three Nicotiana species, not including N.

undulata, was available when they published (Kung et al., 1982).

30

The inﬂuence of the nuclear genome on plastid transmission in somatic hybrids is almost
certainly signiﬁcant. By far the largest portion of proteins needed for normal plastid function are
nuclear-encoded (Parthier, 1982; Palmer, 1987). However, evidence for a nuclear inﬂuence on
somatic hybrid plastid transmission is lacking, primarily because of the lack of relevant nuclear-
encoded genetic markers. The nuclear genome is much larger and more recombinationally ﬂuid than
the plastid genome, so the vast majority of nuclear—encoded traits would not be genetically linked to
genes that inﬂuence plastid transmission. Only those genes which directly inﬂuence plastid
transmission or closely linked genes could co-segregate with changes in the plastid transmission of
somatic hybrids.

There is no obvious pattern when the of mode plastid transmission in Nicoa'ana somatic
hybrids and various nuclear-encoded traits of their parents are compared (for some examples see
Table 3). The parental traits used for these comparisons are the phylogenetic relationship, which was
determined by extensive morphological and cytogenetic analysis (Goodspeed, 1954; Burbidge, 1960),
the sexual cross compatibility (Pandy, 1979), and various properties of the small subunit of RuBisCO
(Kawashima et al., 1974; Gray, 1977; Chen and Wildman, 1981). In addition, plastid transmission is
not noticeably different when intergeneric and intrageneric fusions, or fusions of sexually compatible
and incompatible species are compared (Table 3).

Hybrid bleaching, which is fairly common following interspeciﬁc sexual hybridization, involves
a chlorophyll deﬁciency resulting from genetic incompatibility between the plastome and the nucleus
(Kirk and Tilney-Bassett, 1978). The only reports of hybrid bleaching after somatic hybridization
involve Bmssica napus cybrids with Raphanus scrim: plastids (Pelletier er al., 1983; Menczel et al.,
1987). However, hybrid bleaching was initially discovered following sexual hybridization of these
species. It is curious that no other instances of hybrid bleaching have been reported after somatic
hybridization. Perhaps the strong plastome— nuclear genome incompatibility which results in hybrid
bleaching reduces the likelihood of obtaining somatic hybrid plants.

Certain aspects of the somatic hybridization process can inﬂuence plastid transmission, but the

role of such inﬂuences is not always completely clear. Clearly, in vino selection of plastome-encoded

31
traits, such as antibiotic resistance (Medgyesy et al., 1980; Menczel et al., 1981; Fluhr et al., 1983;
Pental et al., 1984; Bourgin at al., 1986), herbicide resistance (Robertson at al., 1987; Guri et al., 1988)
or pigment deﬁciency (Nakata and Oshkima, 1982; Aviv et al., 1984; Barsby et al., 1984; Fluhr et al.,
1984; Menczel et al., 1986; Glimelius and Bonnett, 1986) almost always results in exclusive
transmission of the selected plastid (Table 3).

Iodoacetate is a nonspeciﬁc metabolic inhibitor which acts on the sulﬂiydryl groups of proteins
and other biomolecules. Iodoacetate treatment has been used to inhibit cell division of the
prot0plasts from one fusion partner, and the heterokaryons formed after fusions with such prot0plasts
can divide due to metabolic complementation with the biochemical constituents from the untreated
fusion partner (Wright, 1978). Iodoacetate treatment of N. plumbagimfolia protoplasts may have
selected against plastids from the treated fusion partner after a fusion with N. tabacum, since this
fusion pair has balanced transmission when iodoacetate is not used (Sidorov et al., 1981; Table 3).
However, the drug does not seem to affect the plastid transmission of some other fusions (Table 3),
perhaps because it was used at a lower concentration (Fluhr et al., 1983; Aviv and Galun, 1986,
Morgan and Maliga, 1987). It is interesting that both somatic hybridizations of N. Iangsdorﬁi and N.
glauca utilizing electrically induced protoplast fusion only generated hybrids with N. Iangsdorﬁi
plastids (Chapel et al., 1986; Morikawa et al.,1987) In contrast, both fusions with the same partners
utilizing the conventional polyethylene glycol method had balanced plastid plastid transmission
(Akada and Hirai, 1983; Chen et al., 1977). Perhaps the fusion or the subsequent culture conditions
used in the electrofusion experiments inﬂuenced plastid transmission in the hybrids. Changes in the
culture conditions are postulated to have affected the plastid transmission of N. tabacum + N.
lazightiana hybrids, but this conclusion is quite tentative (Maliga er al., 1978; Maliga et al., 1980;
Menczel et al., 1981; Table 3).

Somatic fusion can be used to study plastid genetics in plant species lacking biparental
inheritance. As discussed above, plastids have behaved similarly after sexual and somatic
hybridization. However, somatic hybridization procedures are both difﬁcult and laborious so the

number of experimental observations is restricted. Perhaps as a consequence of this, somatic

32
hybridization experiments designed speciﬁcally to study plastid genetics have been rare. Therefore,
most conclusions about plastid behavior after somatic hybridization are based on comparisons of
several experiments from different laboratories. Such conclusions are subject to error due to the
inﬂuence of many uncontrolled factors. In addition, the in vitno culture conditions used during

somatic hybridization can inﬂuence plastid transmission in ways that are not always readily apparent.

33

Table 3. Chloroplast transmission after somatic fusion. All fusion experiments involving the
same plastid donors are grouped together. ”NO SELECTION‘ indicates that no in vitm selection of
plastids was employed. 'SELECI'ION' indicates that in vimo selection of plastids was employed, and
the plastome encoded trait used for selection follows. “SELECTION?" indicates that in viva selection
of plastids is suspected, and the putative selective agent follows. Species: The species which originally
donated the plastids. The chromosome number of the species is in brackets. Species Origin: The
continent where the species originated. North America (NA), South America (8A.), Australia
(Aus.). Tentoxin: The reaction of the species to the fungal toxin tentoxin. Sensitive (S), Insensitive
(I). Ru BisCO: Isoelectric focusing data for the enzyme ribulose bisphosphate carboxylase. Small
subunit (SSU), Large Subunit (LSU). The nomenclature is from Chen and Wildman (1981). Each of
isoelectric points of the SSU peptides from the entire genus is given a consecutive number starting with
the most alkaline. Species with numbers in common have SSU peptides with the same isoelectric
point. Nuclear Genome: “Hybrid“ indicates that the plants analyzed contain nuclear genomes from
both donors. The ﬁrst three letters of the species donating nuclear genomes appear in parentheses
when these species are not the same as the species donating the plastids. 'Cybrid' indicates that the
plants analyzed contain nuclear genomes from only one donor and the ﬁrst three letters of that species
appear in parentheses. Verification: The method used to conﬁrm that the plants analyzed were
hybrids or cybrids. Chromosome number (C#), Chloroplast transfer (cp-T), Isozyme analysis (IZ),
Microscopic isolation of heterokaryons (Micro-iso), Interspeciﬁc mitochondrial DNA recombination
(mt-R), Induction of male sterility (MS), Complementation of different parental nuclear encoded
genetic markers (NC), Hybrid pattern of the small subunit of RuBisCO (SSU). Tissue Source: The
tissue used for protoplast isolation is listed in the same order as the plastid donor species. Callus
culture (C), Etiolated shoots (ES), Hypocotyl (HC), Leaf mesophyll (M), Suspension culture (S).
Plastid Ratio: The number of plants or calli with plastids of each type is listed in the same order as the
plastid donor species. eg. lst parent/mixed/an parent. Statistical inference: 'Unbalanced' indicates
that the null hypothesis of an equal proportion of plastid types is rejected based on the 95% conﬁdence
limits of the binomial distribution (Steel and Torrie, 1980). Conﬁdence limits other than 95% are
given in parentheses.

Table 3

PARENTAL SPECIES

 

SOMATIC HYBRIDIZATIONS

 

 

 

 

 

 

 

SPECIES (2n) lSU'BGENUS & lSPECIES 2'I'BN- 3Roll-Co NUCLEAR VERI'I- TISSUE PIAS'IID STATISTICAL
Nico . SECTION ORIGIN TOXIN SSU LSU GENOME CA‘HON SOURCE RATIO INFERENCE
N. rabacum (48) Tabacunr SA. I 7.9 II 5 SELECTION: pigment deﬁciency
Genuianae cybrid nit-R M/M 0/47 unbalanced
N. undulara (20) Pearnr‘or’der SA. 5 6,9 III (tab)
Undulatae 6 SELECTION: antibiotic resistance
cybrid cp-T M/M 8/0 unbalanced
(sexual compatibility not available) (tab)
7 NO SELECTION:
cybrid Cl S/M 0/22 unbalanced
,(ub) .
cybrid a Micro- C/M 0/4/1 —
hybrid 150
N. rabacwn (48) Tabacum SA. I 7,9 II 9 NO SELECIION:
Genuianae hybrid NA. S/M 0/2 N.S.
N. orophora (24) Tabacum SA. - 7 III
Tomentosae
(sexually compatible; unilaterally?)4
N. rabacum (48) Tabacum SA. 1 7,9 II 10 NO SELECIION:
Genuianae hybrid Cl S/M 1/4 N.S.
N. glutinosa (24) Tabacum SA. - 7 III
Tomentosae
(sexually compatible)4
N. :abacran (48) Tabacum SA. I 7,9 II 16 NO SELECTION:
Genuianae hybrid CI C/M 18/21 N.S.
N. lau'gha'ana (24) Rusrrca SA. 1 9 III 17
Paniculatae hybrid IZ S/M 0/28 unbalanced
(unilaterally sexually compatible; tab it lmi)4 TOTAL- 18/49 unbalanced
N. tabacum (48) Tabacum SA. l 7.9 II 1 I SELECTION: antibiotic resistance
Genuianae hybrid SSU M/S 11/0 unbalanced
N. rustica (48) Rumba SA. 1 6,9 III 12
Rusticae hybrid cp-T S/M 2000/62/0 unbalanced
4 13 NO SELECTION:
(sexually compatible) hybrid SSU M/M 2/2 N.S.
“hybrid ssu M/S 1/5 N.S.
”hybrid ssu S/S 3/11 unbalanced
(90% level)
TOTAL: 6/ 18 unbalanced
N. sylvean‘s (24) peamr'or'des SA. I 7 II 18 NO SELECTION:
alatae cybrid mt-R M/M 3/7 N.S.
N. rustica (48) an'ca SA. I 6.9 III l9(syl)
Rusticae hybrid SSU S/S 0/8 unbalanced
(sexually compatible)‘ ro'rAr: 3/15 unbalanced
N. sylvestnlr (24) Petunioides S.A. I 7 II 5 NO SELECTION:
alatae cybrid MS M/M 9/2/4 unbalanced
N. biglovii (48) Peamr'or'der NA. 8 6,9 III (syl) (90% level)
biglovianae

(sexual compatibility not available)

 

35
Table 3 (Cont’d.)

PARENTAL SPECIES SOMATIC HYBRIDIZA’TIONS

 

 

ISUBGENUS a ISPECIES 21m- 3

   
    

     

 

 

 

 

 

 

 

SPECIES (2a) m NUCIEAR VERTI- ‘TISSUE PLASITD STATISTICAL
Nico- ., -. _- 1 mm , .880 lso __ t
N. sylvmr's (24) Pcmru'or'der SA. I 7 II 5 NO SELECTION:
alatae cybrid MS M/M 10/0 unbalanced
N. undulara (24) Peauuorda SA. S 6,9 III (syl)
undulatae
(sexually compatible; unilaterally?)4
N. W (24) Pearm‘or'der SA. I 7 II 5 NO SELECTION:
alatae cybrid MS M/M 7/0 unbalanced
N. alara (l8) Paunr'oides SA. S 3,5 III (syl)
alatae
(unilaterally sexually compatible; ala x cyl)‘
N. zabaeunr (48) Tabacum SA. 7,9 II 20 NO SELECTION:
Genuianae cybrid MS M/M 11/15 N.S.
N. debucyr' (48) Pemnl'oider Aus. 1,3, A 2 (tab)
Saveolentes 5 ybrid int-R S/M 2/7 unbalanced
(Mb)
(sexually compatible)4 TOTAL: 13/22 unbalanced
(90% level)
N. tabacunr (48) Tabacum SA. 7,9 II 22 SELECTION: pigment deﬁciency
Genuianae cybrid cp-T M/M 0/ 12 unbalanced
N. waveolens (23) Peamior'da Aus. 2,5, A (tab)
Saveolentes 6,8 23 NO SEIECTION: .
4 cybrid a Micro- C/M 0/7/1 —
(sexually incompatible) 2‘llybrid Iso
hybrid Cl S/M 6/19 unbalanced
zscybﬁd Micro— S/M 9/1/73 unbalanced
7 (tab) lso
hybrid Cl S/M 5/ 15 unbalanced
mm 20/57 unbalanced
N. tabacunr (48) Tabacum SA. 7,9 II 9 NO SELECTION:
Genuianae hybrid NA. S/M 0/5 N.S.
N. nesoplu'la (48) Peamr'or‘des NA. 9 I
repandae
(sexually incompatible)4
N. zabaeum (48) Tabacum SA. 7,9 II 25 NO sawmon:
Genuianae cybrid Micro- S/M 41/2/32 N.S.
N. repanda (48) Petunior'der NA. 9 I (tab) lso
repandae
(unilaterally sexually compatible; rep x tab)4
N. tabacum (48) Tabacum SA. 7,9 II 23 NO SELECTION: ,
Genuianae cybrid a Micro- C/M 0/6/3 --—
N. glauca (24) Rum'ca SA. 9 l 26hybrid lso
panicuulatae hybrid Ci S/M 3/0 N.S.
(unilaterally sexually compatible; gla x tab)4 7hybrid car S/M 11/10 N.s.
(tab)
’IO'I’AL: 14/10 N.S.

 

Table 3 (cont’d)

PARENTAL SPECIES SOMATIC HYBRIDIZATION S

 

 

1SUIGENUSt lSPECIES 2TEN~ 3mm NUCLEAR VERII'I- TISSUE PIAS'TID STATISTICAL

SPECIES (2.)
Nic . SECTION ORIGIN TOXIN SSU LSU GENOME CATION SOURCE RA‘ITO INFERENCE

 

 

 

 

 

N. langsdodir' (36) Peanu'or’da SA. S 3,5 II 27 SEIECTION‘! electmhnlon
Alatae hybrid SSU M/M 7/0 unbalanced
N. glauca (48) [harried SA. S 9 I 28 .. (90% level)
Paniculatae hybrid SSU M/M All/0 —-
4 29 NO SELECTION: e e
(unilaterally sexually compatible; Ian x gla) hybrid SSU S/S O/AIl/O -—--
”hybrid ssu M/M 0/1 N.S.
”hybrid ssu M/M 9/1/7 N.S.
TOTAL- 9/8 N.S.
N. tabacum (48) Tabacum SA. I 7,9 II 32 SELECTION: antibiotic resistance
Genuianae cybrid cp-T M/M 117/0 unbalanced
N. syhmir (24) PM SA. I 3 II (syl)
Alatae 6 NO SELECTION:
4 cybrid NC M/M 8/18/9 N.S.
(sexually compatible) 9(tab)
hybrid NA. S/M 1/0 N.S.
‘I‘O‘I‘AL: 9/9 N.S.
N. tabacum (48) Tabacum SA. 1 7,9 II 33 SELECTION: antibiotic resistance
Genuianae cybrid cp-T M/M All/0 —-
N. phambagr’m’folia Pcamr'oida SA. 5 3 II 34(plu)
(20) Alatae cybrid CI, cp-T S/M 0/12 unbalanced
(tab)
(sexually compatible)4 35 SELECTION? iodoacetate
cybrid IZ M/C 31/5/3 unbalanced
(Pin)
23 NO SELECTION: .
hybrid C! C/ M 0/1/0 ~—
”hybrid IZ M/C 7/3/6 N.S.
N. tabacum (48) Tabacum SA. I 7,9 II 36 SELECTION: antibiotic resistance
Genuianae hybrid cp-T M / M 9/1 unbalanced
N. tabacum (48) Tabacum SA. I 7,3 II
Genuiane 37 NO SELECIION:
4 cybrid & NC M/M 76/87 N.S.
(sexually compatible) hybrid
N. debneyr' (48) Petwu'oides Aus. I 1,3, 38 NO SELECTION:
Saveolentes 5 hybrid IZ M/M 4/2 N.S.
N. debneyr' (48) Peauu'oides Aus I 1.3,
Saveolentes 5
(sexually compatible)4
N. biglovir' (24) Peamr'or'des NA. S 4.6 III 39 NO SELECIION:
bigelovianae cybrid mt-R M/M 10/ 12 NS.
N. undulata (20) Petunior'des SA. S 6.9 III (syl)
Undulatae

(sexual compatibility not available)

 

Table 3 (cont’d)

PARENTAL SPECIES

37

 

SOMATIC HYBRIDIZA'TIONS

 

 

 

 

 

 

 

 

 

 

 

 

spacras (2n) «TAXONOMIC “SEXUAL NUCLEAR vaun- nssua HAS‘TID sums-next.
Sol REIATIONSHIP CErOSS GENOME CATION SOURCE RA'ITO INI-‘ERENCE
S. tuberosunr (48) Subgenus; Pctotoc incompatible NO SELECTION:
section; Tuberosa 42
S. nigrwn (72) Subgenus; Solanum hybrid Cl M/M 3/1/8 N.S.
section; Solarium
S. arbemsum (48) Subgenus; Paolo incompatible SELECTION: pipnent deficiency
section; Tuberosa 43
S. brevidars (24) Subgenus; Pctota hybrid Cl M/M 0/29 unbalanced
section; Eutuberosa
S. pinaa'sceaun (24) Subgenus; Paola compatible “ NO SEIECTION:
section; Pinnatisecta hybrid IZ M/M 0/6 NS.
5. phurcja Subgenus; Pctota (pin +
section; Tuberosa phu x tab)
S. melongala (24) Subgenus; leptostanonum incompatible NO SELECTION:
section; Melongena ‘5
S. sitybn’r'foliwn (24) Subgenus; Leproaemanum hybrid Cl, I2 S/S 0/8 unbalanced
section; Olignethes
S. melongaro (24) Subgenus; Leprosranonum not available NO SELECTION:
section; Melongena 46
S. roman (24) Subgenus; lzptosrcmonum hybrid CI, I2 M/M 8/2/0 unbalanced
section; Torva
S. melongcna (24) Subgenus; Leprosranomun not available SEIECIION: herbicide resistance
section; Melongena 47
S. m'grum (24) Subgenus; Solarium hybrid Cc, IZ M/M 0/3 N.S.
section; Solarium
Lgom'cw.
L. csculauum (24) 'csculenarm complex' unilateral by NO SELECHON:
ovule culture 48
L. peruvr'anum (24) 'pcruvr'anum complex“ (esc x per) hybrid CI NA. 0/5 N.S.
L. csculauum (24) 'csculenaun complex' unilateral NO SELECIION:
(we x pen) ,,
L. pcnncllr'r’ (24) 'esculenrum complex' hybrid CI, 12 C/M 1/1/2 N.S.
Petunia
P. parodir' (14) unilateral NO SELECTION:
(par x inf) 50
P. inﬂara (14) Subgenus; Eupeaau'a hybrid Cl, 12 M/S 10/0 unbalanced
Datum
D. innoxr'a (24) compatible by NO SELECTION:
ovule culture 51
D. mammalian (24) hybrid Cl, 12 M/M 6/1 N.S.
D. innaxr'a (24) compatible by NO SELECTION:
ovule culture 51
D. discolor (24) hybrid Cl, 12 M/M 2/1/1 N.S.

 

Table 3 (Cont’d)

PARENTAL SPECIES

 

SOMATIC HYBRIDIZATIONS

 

 

 

 

 

 

 

SPECIES (2n) 4o'I‘AXONOMIC “SEXUAL NUCLEAR VERTI- TISSUE PLASTID STATISTICAL
Br . RELATIONSHIP CROSS GENOME CATION SOURCE RATIO INFERENCE
B. naplas (38) compatible 52 NO SELECTION:
B. compacts (20) hybrid Micro- M/ES 15/4 unbalanced
(nap) lso
W + molar:
L escalator»: (2A) Subfamily; Solanoideae incompatible NO SELECTION:
Tribe; Solaneae 53
S. nigrunr (24) Subfamily; Solanoideae hybrid Cl, 12 MN 0/3 N.S.
Tribe: Solaneae
L csculcnaun (24) Subt’amily; Solanoideae incompatible 5‘ NO SELECTION:
Tribe; Solaneae hybrid Cl, SSU S/M 11/‘7 N.S.
S. mbaosum (24) Subfamily: Solanoideae 55
Tribe; Solaneae hybrid Cl, SSU M/M 0/4 N.S.
‘I‘O‘TAI: ll/ 11 NS.
L esculentum (2A) Subfarnily; Solanoideae incompatible NO SELECTION:
Tribe; Solaneae 56
S. rich'i (24) Subfamily; Solanoideae hybrid CI, 12 M/S 0/4 N.S.
Tribe; Solaneae
L. esculenamr (24) Subfamily; Solanoideae unilateral by NO SELECTION:
Tribe; Solaneae ovule culture 57 .
S. heopasr'coidec Subfamily; Solanoideae (esc x tab) hybrid Cl, IZ M/S 68/1/1 unbalanced
(24) Tribe; Solaneae
ELM + m
N. tabocum (48) Subfamily; Cestroideae incompatible $8 SELECTION: pigment deficiency
Tribe; Nicotineae hybrid a cp-T M/M 0/83 unbalanced
P. hybrida (28) Subfamily; Solanoideae cybrid
(tab)
59 SELECTION: pigment deﬁciency
hybrida cp-T M/M 1/0 N.S.
cybrid
, (pet)
Brassrca + Raphanus
B. napus (38) Family; Cruciferae compatible NO SELECTION:
R scrim: Family; Cruciferae ”cybrid Micro- M/HC 4/2 Ns.

(nap) lso

 

39
Table 3 (Cont’d)

References: 1(Goodspeed, 1954; Burbidge, 1960; Smith, 1979,) 2(Durbin and Uchytil, 1977a;
Durbin and Uchytil, 1977b; Burk and Durbin, 1978) 3(Chen and Wildman, 1981) 4(Goodspeed, 1954;
Burbidge, 1960; Durbin and Uchytil, 1977; Pandy, 1979) 5(Aviv er al., 1984a) 6(Fluhr et al., 1983)
7(Bonnett and Glimelius, 1983) 8(Gleba et al., 1985) 9(Flick and Evans, 1982) 10(Horn et al., 1983)
11(Pental et al., 1984) ”(Nakata and Oshkima, 1982) 13(Mai ct al., 1980) 1“(Hamill et al., 1984)
15 (Douglas et al., 1981) 16(Menczelet al., 1981) l7(Maliga et al., 1978; Maliga er al., 1980) 18(Aviv et
al., 1984b) 19(Gleddie et al., 1983) 20(Kumaaliiro and Kubo 1986) 21(Iielliarcl ct al., 1978; Belliard and
Pelletier, 1980)22(Fluhr et al., 1984) 23(Gleba er al., 1984) 2“(Glimelius et al., 1981) 25(Fliclt ct al.,
1985) 26(Evans et al., 1980) 27(Morikawa et al., 1987) 28(Chapel at al., 1986) 29(Akada and Hirai,
1983) 30(ng et al., 1975) 31(Chen et al., 1977), 32(Medgyesy et al., 1980) 33(Menczel et al., 1982)
34(Menczel et al., 1986) 3S(Sidorov et al., 1981) 36(Bourgin et al., 1986) 37(Glimelius and Bonnett,
1981) 38(Seowcrcft and Larkin, 1981) ”(Aviv and Galun, 1986) 40(Hawks er al., 1978; Sink, 1984)
41(Hogenboom, 1979; Hermsen and Sawicka, 1979; Rao, 1979; Hawkes, 1979; Rick, 1979) 42(Gressel
er al., 1984) 43(Barsby et al., 1984; Kemble et al., 1986) 44(Sidorov et al., 1987) 45(Gleddie at al., 1986)
46(sari and Sink, 1988a) “(Guri and Sink, 1988b) 48(Pivca et al., 1986) 49(O’Connel et al., 1987)
50(Schnabelrauch et al., 1985; Clark et al., 1986) 51(Schieder, 1978; Muller-Gensert and Schieder,
1985) 52(Y arrow er al., 1986) 53(Guri er al., 1988) “(Melchers er al., 1978; Schiller er al., 1982)
5 5 (Shepard et al., 1983) 56(O’Connel and Hanson, 1986) 57(Handley et al., 1986; Levi er al., 1988)
58(Glimelius and Bonnert, 1986) 59(Pental et al., 1986) 60(Morgan and Maliga, 1987)

*The numbers indicate the number of calli which regenerated shoots with plastids from only one
parent or from both parents. cg. shoots of 1st parent/shoots of both parents/shoots of 2nd parent.

"The plastid type of hybrid calli is reported.

40
CHAPTER 2

Non-random plastid transmission in somatic hybrids of Lycopersicon esculentum
and Solarium lycopersicoides

Introduction

Maternal inheritance of plastids during sexual reproduction predominates among
angiosperms, but a pr0portion of species have biparental inheritance (Sears 1980). Uniparental
inheritance precludes the independent assortment of organelles genomes in most crop species
examined so far. However, chemically or electrically mediated fusion of plant protoplasts can
produce heteroplasmic cells. Subsequent sorting-out of the organelles in these cells during the cell
divisions leading to regenerated plants often results in novel combinations of nuclear and cytoplasmic
genomes (reviewed by Kumar and Cocking 1987).

The plastids in the somatic hybrids examined to date have sorted-out rapidly, without plastid
DNA (chNA) recombination (Akada and Hirai, 1986; Chapter 1 of this thesis), excepting two
demonstrations of chNA recombination using a very effective method of in vitro selection
(Medgesey, et al., 1985; Thanh and Medgyesy, 1989). Rapid sorting-out is a consequence of the rarity
of plastid fusion, which precludes mixing of DNA between plastids and recombination of plastid
genomes (Possingham and Lawerence, 1983). Sorting-out is usually complete before shoot
regeneration, but occasionally chimeric shoots regenerate (Glimelius et al., 1981; Nakata and
Oshkima, 1982; Aviv er al., 1984; Guri and Sink, 1988b). Shoots with mixed plastids are more
common in fusions with balanced plastid transmission (Chapter 1) as predicted by a mathematical
model of stochastic plastid assortment (Michaelis, 1967a). However, somatic hybrids can inherit
plastids predominantly from one parent, contrary to the expectations of purely stochastic assortment
(Chapter 1; Appendix I; Levi et al., 1988; Clark et al., 1986).

In comparison, mitochondrial genomes almost always undergo recombination or
rearrangement in somatic hybridization experiments (Belliard et al., 1979; Galun et al., 1982;

Boeshore er al., 1983; Rothenberg er al., 1985; Robertson et al., 1987; reviewed by Hanson 1984). The

41

mitochondria in somatic hybrids probably sort-out more slowly than the plastids, and this may stem
from the prevalence of mitochondrial fusion (Bendich and Gauriloff, 1984) and mitochondrial DNA
(mtDNA) recombination (Aviv and Galun, 1986; Aviv and Galun, 1987). For example, mtDNA-
encoded cytoplasmic male sterility in Petunia somatic hybrids continued to segregate for more than
three meiotic cycles (Izhar et al., 1983). A few investigations imply that non-random transmission of
mitochondria also can occur in somatic hybrids, but interpretation of the data is ambiguous due to the
complexity of the mitochondrial genome and the prevalence of mtDNA recombination and
rearrangement (O’Connell and Hanson, 1985; O’Connell and Hanson, 1986; Levi et al., 1988).

The possible causes of unequal transmission of organelles in somatic hybrids are; (1) an
initially larger contribution of organelles from one fusion partner, (2) more efficient replication the
organelles from one partner, (3) genetic incompatibility of the nuclear genome with the organelles
from one partner, or (4) artiﬁcial selection of the organelles from one partner in vitro (Kumar and
Cocking, 1987). Understanding the factors determining organelle transmission in somatic hybrids will
lead to increased understanding of the interactions among the three different genomes within plant
cells.

This chapter describes biased transmission of plastids in a large population of somatic hybrid
plants in which the mitochondrial transmission probably also was biased (Appendix I; Levi er al.,
1988). Fusion of leaf mesophyll-derived protoplasts from Lycopersicon esculentum with suspension-
derived protoplasts of Solomon beapersicoides produced intergeneric somatic hybrid plants (Handley
er al., 1986). Stringent selective conditions were not used to recover the hybrid calli or plants (Sink et
al., 1986). The S. lycopersicoides suspension-derived protoplasts did not divide and calluses of the
tomato cultivar rarely regenerated shoots. Putative hybrid calli were selected by their vigorous
growth, and their distinct morphology and pigmentation. All of the plants studied herein have been

veriﬁed as nuclear hybrids using Got-2 and Got-3 isozyme markers (Moore and Sink, 1987a).

42

Materials and Methods

All DNA analyzed was isolated from fully expanded leaves of plants grown in the greenhouse.
Lycoperxicon esculentum Mill. ‘Sub Arctic-Maxi’, Solarium lyeopem'coides Dun. (LA 1990), their
sexual hybrid, and 70 somatic hybrids were asexually propagated for the analysis. Whole cell DNA
was isolated from bulk leaf samples collected from several areas of each somatic hybrid. Hybrids 63
and 240 were selected for more detailed study. Three clones from shoot culture of regeneratant
number 63 and one shoot from regeneratant number 240 were rooted in vitro and transferred to the
greenhouse. Several cuttings of hybrid 240 were propagated after it was transferred to the
greenhouse. Whole cell DNA was isolated from several small samples (app. 0.5 g) collected from the
individual branches of each of the cloned plants.

Chloroplasts were isolated from freshly harvested tissue using 5-step 20-60% sucrose gradients
as described by Palmer (1986) with some modiﬁcations. The extraction and wash buffers both
contained 0.4 M sucrose, 50 mM Tris pH 8, 6 mM Na EDTA; but 0.1% BSA (w/v), 0.15 PVP—40
(w/v), and 30 mM 2-mercaptoethanol were also added to the extraction buffer. Chloroplast DNA
(chNA) was prepared by suspending the organelle fraction in CTAB buffer (10 mM Tris pH 8,
1.4 M NaCl, 20 mM Na EDTA, 2% cetyltrimethylammonium bromide (w/v), 120 mM
2-mercaptoethanol), extracting with 24:1 chloroform/isoamyl alcohol, then precipitating the nucleic
acids by adding 2—3 volumes of ethanol (Saghai-Maroof er al., 1984). The chNA pellets were
dissolved and further puriﬁed on CsCl/ethidium Bromide (EtBr) gradients.

Plant total cellular DNA was isolated from fresh tissue or from frozen tissue that had been
stored at -70 0C. The DNA was isolated by grinding 0.5 g of leaves in a mortar with 10 ml of the
same buffer used for chloroplast extraction and centrifuging the slurry at 4,500 x g; 15 min. The pellet
was resuspended in CI‘ AB buffer and extracted with 24:1 chloroform/isoamyl alcohol. The nucleic

acids were precipitated and further puriﬁed on CsCl/EtBr gradients. Electrophoresis, Southern

43
blotting, nick translation, hybridization, autoradiography, and photography were performed using
standard procedures (Maniatis et al., 1982).
A 15 kilobase (kb) tomato chloroplast Psi 1 clone (P—6) was used for the chNA analysis
(Phillips 1985). Puriﬁed nuclear and mitochondrial DNA from both L esculentum and S.
lyeopersicoides were the kind gifts of Dr. Patrick Moore (Moore and Sink, 1988b) and Mr. Amnom

Levi (Levi er al., 1988) respectively.

Results

Whole cell DNA was isolated from bulk leaf samples collected from several branches of each
somatic hybrid in order to screen more regions for rare plastid sectors. S. chopersicoides has an
additional PstI site which splits the tomato 15 kb chNA fragment corresponding to clone P-6 into
9 kb and 6 kb fragments (J. Palmer personal communication). When Pst I digests from 70 different
somatic hybrids were blotted onto nitrocellulose and probed with P-6, all but two had the tomato
chNA restriction pattern (Figure 1). Hybrid 240 had the S. lyeopersicoides chNA pattern and
hybrid 63 had a pattern corresponding to a mixture of parental chNAs (Figure 1). Hybrid 240 had
S. Mopersicoides mitochondria; the mtDNA of hybrid 63 was not analyzed (Levi et al., 1988). The
sexual hybrid had the L. esculentum plastid pattern as expected (data not shown). No nuclear or
mitochondrial homologies were detected when puriﬁed nuclear, mitochondrial and chloroplast DNAs
from these species were probed with P-6 (Figure 1; data not shown).

The mixed chNA pattern of hybrid 63 could have been due to the maintenance mixed cells, a
stable periclinal chimera, or sorting-out between branches. The orig'nal analysis using bulk samples
could not distinguish these possibilities. In addition, bulking leaves from many branches reduces the
sensitivity of the analysis by diluting the chNA from rare sectors. For these reasons hybrids 64 and

240 were analyzed using separate samples from several branches of each plant.

 

0 PO 0910 cc
mdvmhmgwmzoaagﬁ
JCDNLDVVQ'U’VVVVCOCOCO

15
9 .O'
6

 

 

Figure 1. Southern analysis of plastid DNA from the parental lines and thirteen somatic
hybrids. Pst I digests of whole cell DNA from the somatic hybrids or puriﬁed chN A from L.
esculentum (LE) and S. lywpersicoides (SL) were electrophoresed on a 0.8 96 agarose gel, blotted to
nitrocellulose, and probed with the 32? labeled chNA clone P-6. The sizes of the fragments are given
in kilobase pairs.

45

SN- ohq'mqnn...
nnMNN—;""

. 0000’
FFJQQQQQQQ§§§§
7.3”, o
. , --
.’bl""00

J , 7 .
i.i
2+ .00 0.1 0 al.
t' i
'4’ . a ‘
4 7‘

T+L(50:l) _

 

l‘tv

 

 

Figure 2. Southern analysis of the plastid DNA from the parental lines S. lycopersicoides (SL),
and L. esculentum (LE), and eleven samples of somatic hybrids 240 and 363. Eco RV-Pst I double
digests of whole cell DNA were electrophoresed on an 0.8% agarose gel, blotted to nitrocellulose, and
probed with the 32F labeled chNA clone P-6. The DNA samples are labeled ﬁrst with the hybrid
number, then with the plant clone number, and ﬁnally with the DNA sample number separated by
colons. The lane marked LE + SL contains a 50:1 ratio of tomato to S. lycopersicoides whole cell
DNA.

46

Seven samples from hybrid 240 and 13 samples from hybrid 63 were double-digested with Sal I
and Pr: I, or with Eco RV and Pr: 1. Southern blots of these double-digests were probed with the
cloned chNA fragment P-6. In Sal I- Per digests S. chopersicoides has an additional PstI site
which cleaves the tomato 8.6 kb fragment to yield 5.9 and 2.7 kb fragments (data not shown). The
same Pstl site results in a missing 7.3 kb fragment and additional 5.7 and 1.4 kb fragments in
Eco RV- Psrl digests of S. lyeopersicoides (Figure 2). A faint 7.3 kh L esculenarm band could be
detected in Eco RV- PM I digests when a 50:1 mixture of L esculentum and S. lycopersicoides DNAs
was analyzed to test the sensitivity of the procedure (Figure 2). All seven samples from hybrid 240
had S. lycopersicoides plastids (Figure 2). Seven hybrid 63 samples from various clones had S.
lycopersicoides plastids and ﬁve had L esculentum plastids (Figure 2). The remaining hybrid 63
sample (63:3:14) from in vitm clone 63:3, had a faint 7.3 kb band which may have been due to a small
amount of L esculentum chNA or partial digestion (Figure 2). Similarly, a faint 8.6 kb band was
observed in Sal I- PstI digests of sample 63:3:14 (data not shown). All other DNA samples from
clone 63:3 had only S. lycopersicoides fragments. Similarly, all samples from hybrid 63 in viva clone
63:1 had the L esculentum pattern, and all samples from hybrid 63 in virm clone 63:2 had the S.

lycopersicoides pattern.

Discussion

The two solanaceous species utilized in these experiments are unilaterally sexually compatible
when tomato is the female parent, but the hybrids are highly sterile (Rick, 1979). Hence, these
somatic hybrid plants afford the opportunity to study unique combinations of organelle genomes not
normally possible following sexual mating. It is striking that the majority of these somatic hybrids
seem to have chloroplasts and mitochondria from opposite fusion parents. They have predominantly
(97%) tomato plastids and probably have exclusively S. chopcrsicoides mitochondria (Appendix I;
Levi et al., 1988).

The plastid transmission in these somatic hybrids could have been determined by the relative

number of plastids donated to the heterokaryon by the parental protoplasts. In these experiments,

47

the tomato protoplasts were isolated from leaf mesophyll cells and fused with suspension-derived S.
lycopersicoides protoplasts. Mesophyll cells have about 10-fold more plastids than meristematic cells
(Possingham and Lawrence, 1983), more than any other tissue that has been examined (Steele-Scott
er al., 1984). To my knowledge, the number of plastids found in long-term suspension cells has not
been determined. However, these rapidly dividing cells are similar to meristematic cells of whole
plants and probably have relatively few prOpIastids. For this reason tomato chloroplasts probably
predominated in the heterokaryons. Following fusion, the partitioning of plastids during cell division
would have ﬁxed the predominant type (Akada and Hiria, 1986; Michaelis, 1967a). This process
would have been quite rapid if the number of plastids in the hybrid cells decreased from around 100
to about 10 within 3—4 divisions as they did in cultured mesophyll protoplasts of N. tobacum (Thomas
and Rose, 1983).

There are eight reports of mesophyll-suspension cell fusions of various species where
mesophyll-derived plastids clearly predominated, none where the plastids from the suspension partner
predominated and two reports of relatively large populations having balanced transmission (Chapter
1). The cumulative ratio from all the mesophyll-suspension fusions without plastid selection which
are tabulated in Chapter 1, Table 3 is 185 with plastids from mesophyll partners to 97 with plastids
from suspension partners. Clearly, our ratio of 68 hybrids with plastids from the mesophyll partner to
2 with plastids from the suspension partner much more skewed, suggesting other factors may be
involved. However, the cumulative ratios are subject to uncertainty because the number of
comparisons is small and many uncontrolled factors among the various experiments could have
affected the result. Differential transmission of plastids due to unequal input from the gametes is a
well documented aspect of sexual reproduction in higher plants (Whatley, 1982).

Another possibility is that more rapid proliferation of the plastids from L. esculean resulted
in biased transmission (Chapter 1). Plastids can divide continuously during the cell cycle but the
maximum number per cell seems to be limited in a developmentally coordinated fashion (Possingham
and Lawrence, 1983). Assuming that plastid multiplication is a stochastic process, plastids that can

divide more rapidly when the number per cell is below the limit will predominate in subsequent cell

48

generations. Oenothem, with biparental plastid inheritance, has yielded genetic evidence supporting
this concept. Schotz (1954) discovered differences in competition between ﬁve different plastomes
(plastid chromosomes) of Euoenothem when they were brought together by sexual matings. Each of
the ﬁve plastome types was assayed using reciprocal crosses in which one parent carried a plastome
encoded albino mutant, and the other carried the wild-type plastome to be tested. The germinating
seeds from these crosses were scored by measuring the relative size of the green sectors on their
cotyledons, which gives a quantitative estimate of the relative competitiveness of each plastome. This
”variegation rating“ ranks the ﬁve plastomes in nearly the same order regardless of the direction of
the cross in a constant nuclear background (Chiu er al., 1988). Schbtz (1968) attributed this to
differences in the rate of plastid multiplication.

Perhaps the under-represented S. choperricoides plastids were less competitive in the hybrid
cells due to incompatibility with the hybrid nuclear genome. Biased sorting out due to plastome-
genome incompatibility may have occurred at any time between cell fusion and the time of analysis.
Genetic evidence for the incompatibility of plastomes with certain nuclear genomes has been
obtained for Oenothem. Stubbe (1960) studied the reaction of the ﬁve different plastomes of
subsection Euoenothera and found profound diﬂ'erences in their ability to green when combined with
different nuclear complements. The manifestation of this “plastome-genome“ incompatibility ranged
from small changes in chloroplast pigmentation to complete lethality. Each plastome type is more
compatible when associated with a genome more closely related to the one with which it co- evolved
(Kutzelnigg and Stubbe, 1974).

Incompatibility during plant regeneration, which is a complex process involving coordinate
expression of many genes, may be particularly important in determining organelle transmission.
Menczel er al., (1981) reported two somatic fusion experiments using Nicotiana tobacum and N.
knightiana with completely different results. In the ﬁrst experiment they observed only N. hlightiana
chloroplasts in the hybrid plants, and in the second they observed balanced transmission of both types.
They suggested the culture conditions used in the ﬁrst experiment precluded regeneration from cells

with N. (abacum plastids. Schnabelrauch and Sears (personal communication) studied regeneration

49
of isonuclear lines of Oerlorhem with ﬁve different plastomes, and observed an inﬂuence of plastome
type on the efficacy of regeneration. Perhaps in our experiments, cells with different plastomes also
responded to the culture medium differently.

The bulk DNA sample from hybrid 63 had chNA restriction fragments corresponding to
both parents. Originally, the hybrid 63 shoot which regenerated had plastids from both species, but
the plastids sectored as the plant was propagated via shoot culture. Segregation must have been
nearly complete by the time the original shoot could be cloned in shoot culture since only one of the
hybrid 63 in vitro clones may have had plastids from both species. This is in accord with observations
of plastid segregation in other somatic hybrids. Mixed cells usually sort prior to shoot regeneration
making hybrid plants with mixed plastids uncommon (Chapter 1; Kumar and Cocking, 1987). Stable
plastid chimeras have not been reported after somatic hybridization, but are fairly common after
sexual crosses. Furthermore, there is only one report of the stable maintenance of different chNAs

within plant cells and it did not involve somatic hybridization (Moon et al., 1987).

CHAPTER 3

Conclusion

The prevalence of uniparental inheritance of plastids and the scarcity of intergenomic
recombination of plastomes can impede breeding efforts to improve crop plants. Some agronomically
important traits encoded by plastomes are atrazine resistance (Robertson, 1985), resistance to certain
diseases (Vaughn and Duke, 1984), and control of photorespiration (Ogren, 1984). The primary
function of plastids is photosynthesis, but they also have other important metabolic functions. Plastids
participate in chlorophyll, amino acid, and fatty acid biosynthesis; nitrate and sulfate reduction; and
starch metabolism (Kirk and Tilney-Bassett, 1978). Therefore, it is implicit that plastomes encode
other traits which could be manipulated for agronomic beneﬁt. Somatic hybridization of plants by in
vitro fusion of protoplasts produces heteroplasmic cells. Unique genetic complements of both
cyt0plasmic and nuclear genomes can result from sorting and recombination of genetic elements
during the subsequent cell generations.

The evolutionary origin of plastids and the manner in which they transmit their genetic
information has inﬂuenced the structure of modern plastomes. Examination of plastomes with this in
mind provides information about the mechanisms governing plastome inheritance. Hence,
consideration of certain aspects of plastid evolution and genetics is relevant in understanding plastid
transmission in cell fusion hybrids.

Plastids are thought to have arisen through endosymbiosis of an organism related to blue
green algae within a primitive eucaryotic cell. Plastomes from evolutionarily diverse groups such as
algae and angiosperms encode nearly the same complement of genes. The plastomes of all the land
plants examined to date encode about 120 genes, and those with known functions encode components
of the plastid transcription and translation systems, photosynthetic complexes, and chlororespiration
system. Plastid genes are transcribed and translated in the plastid by enzymes which are almost

entirely distinct from those found in the nucleus and cytosol.

50

51

However, most proteins found in plastids (about eighty to ninety percent) are encoded by nuclear
genes, translated on cytoplasmic ribosomes and transported across the plastid envelope (Kirk and
Tilney-Bassett, 1978). Most of these genes were probably transferred from the plastome to the
nucleus during the course of evolution. The fact that plastomes from diverse species encode nearly
the same set of genes implies that most genes were transferred to the nucleus early in the evolution of
the symbiosis, and that the plastome has been relatively stable ever since. In support of this, a
bryophyte (Marehantia polymorpha) and an angiosperm (Nicotiana tabocum) have their plastid genes
arranged in nearly the same order with the exception of a 30 kb inversion.

Plastids may be inherited maternally, paternally or biparentally in higher plants. Genetic
analysis using appropriate plastid markers is needed to deﬁnitively establish the means of plastid
inheritance, since cytological observation of organelles during reproduction can be unreliable (Sears,
1980b). Markers such as restriction fragment length polymorphisms, pigment mutations and
antibiotic resistance have been used to follow organelle inheritance in plants. Some criteria that may
be used to demonstrate that a trait is cytoplasmically encoded are: 1) non-Mendelian inheritance such
as reciprocal differences after reciprocal crosses, 2) the sorting out of genetic entities during
development, and 3) molecular genetic evidence that a trait is cytoplasmically encoded (Kirk and
Tilney-Bassett, 1978; Medgyesy er al., 1986). Cyt0plasmic traits also must be assigned to either
plastids of mitochondria by morphological, cytological or biochemical characterization (Michaelis,
1966; Kutzelnigg and Stubbe, 1974; Fluhr ct al., 1985; Bérner and Sears, 1986).

Plastome encoded pigment-deﬁciency mutants in plants with biparental inheritance provide a
convenient means to study the processes involved in sorting-out of plastids during development. In
plants developing from biparental embryos the plastids from each parent segregate during vegetative
growth until pure or chimeric shoots are formed (Kirk and Tilney-Bassett, 1978). Plastid chimeras
may sort further, but can be relatively stable during continued plant growth (Tilney-Bassett, 1986).
When a pigment-deﬁciency marker is used to follow the sorting process there are four visibly
discernible stages: 1) the initial phase with only mixed cells and no visible manifestation, 2) a ﬁne

mosaic phase during which the mixed cells sort-out as they divide producing pure white and pure

52
green cells, 3) a coarse mosaic phase with few remaining mixed cells during which pure tissues sort-
out from a mosaic of pure cells and 4) a ﬁnal phase during which pure white, pure green, and
chimeric shoots segregate from pure tissues (Kirk and Tilney-Bassett, 1978; Tilney-Bassett, 1986).

When the different plastids replicate at an equal rate, partition randomly into daughter cells,
and do not differentially effect cell division or plant development, plastid segregation is a random
process (Michaelis, 1967a). Michaelis (1967a) developed a mathematical model to predict the
characteristics of plastid segregation when it is a stochastic process. The model is based on the
additional assumptions that the number of plastids per cell is a constant (2N) prior to division, and
each daughter cell receives half (1N) during cell division. Also, each plastid must replicate only once
each cell cycle. It is clear from many investigations of plastid behavior that each of these assumptions
is normally violated at different phases of the plant’s life cycle (Sears, 1980b; Whatley, 1982;
Possingham and Lawrence, 1983; Thomas and Rose, 1983; Bendich, 1987). However, Michaelis’
model provides an excellent basis for understanding plastid behavior and plastids often sort-out as
predicted, although exceptions are fairly common (Michaelis, 1967b; Kirk and Tilney-Bassett; 1978;
Birky, 1978; Kumar and Cocking, 1987).

Several general features of plastid somatic segregation can be inferred from this model (Kirk
and Tilney-Bassett, 1978). The summation of all the ratios of different plastid types in the progeny
cells is always the same as the ratio the initial cell originally had. Hence, after sorting-out is complete
the proportion of cells with each plastid type reﬂects the ratio in the initial cell. Furthermore, a cell
must undergo about 10 x N cell divisions to attain nearly complete (>99%) sorting-out, where N is
the number of plastids per cell immediately after division.

It is useful to at this point to digress and consider whether individual plastids or plastomes are
appropriate as the units of segregation. Angiospcrm cells each have about SOD-50,000 copies of the
plastome (Possingham and Lawrence, 1983; Bendich, 1987). Since meristematic tissues have the
lowest chNA ploidy and are most important in the sorting-out process, at least 2500 (10 x N) cell
divisions would be required for nearly complete sorting of chNA molecules. Most annual plants

would require several reproductive cycles for this number of cell divisions. In comparison,

53

meristematic cells of angiosperms have about 10-30 plastids, corresponding to 50-150 divisions for
nearly complete sorting-out of plastids (Possingham and Lawrence 1983; Steele-Scott er al., 1983).
Annual plants usually sort-out in one or two reproductive cycles and two cycles could usually be
completed within 50-150 cell divisions (Michaelis, 1967b). It should be noted that the eﬂ'ective
number of segregation units is always reduced when there is plastid selection, unequal eﬁciency of
plastid replication, unequal partitioning into daughter celk or incomplete mixing of plastids between
divisions (Michaelis, 1967a). Hence, calculations using the number of plastids per cell give the
maximum number of divisions needed for sorting-out.

The high ploidy of the plastome and the large number of plastid per cell probably play a key
role in the strong evolutionary conservation of chNAs. In the few instances where intermolecular
chNA recombination has been demonstrated the recombinant molecules have many closely spaced
crossover points (Sears, 1980a; Mets and Geist, 1983; Lemieux er 01,1984; Medgyesy er al., 1986). This
suggests that recombination occurred through gene conversion. There is also evidence that
intermolecular copy correction through gene conversion occurs between the large inverted repeats
(Chapter 1; Dron er al., 1983; Erickson er al., 1985). This implies that copy correction also occurs
between multiple copies of chNA within each plastid. It follows that a mutant chNA molecule
could either be eliminated or ﬁxed through consecutive rounds of gene conversion (Birky, 1978).
Since a mutant molecule must arise as one of a population of 20-200 wild type molecules in a plastid,
it is much more likely to be eliminated than ﬁxed (Possingham and Lawrence, 1983; Steel-Scott er al.,
1984; Bendich, 1987). If a mutation is to be passed on to subsequent generations it must be ﬁxed in
one plastid, compete successfully in a population of about 10-200 plastids per cell and be maintained
in a cell lineage that gives rise to germ cells (Kirk and Tilney-Bassett, 1978; Possingham and
Lawrence, 1983). Since this process is largely stochastic the chances for successful establishment of a
mutation are low.

Cell fusion has been used to study the somatic genetics of plastids in plant species lacking
biparental inheritance (for reviews of plastid transmission following somatic hybridization see: Fluhr,

1983; Harms, 1983; Gleba and Syntnik, 1984; Pelletier and Chupeau, 1984; Pelletier, 1986; Maliga and

54

Menczel, 1986; Grun, 1986; Kumar and Cooper-Bland, 1986; Kumar and Cocking, 1987). As
discussed in Chapters 1 and 2 of this thesis, the behavior of plastids in somatic tissues is similar after
sexual and somatic hybridizations. However, caution must be used since the in vitro culture
conditions used during somatic hybridimtion could inﬂuence plastid transmission in ways that are not
always readily apparent (Chapter 1). In additiml, somatic hybridization procedures are both diﬁcult
and laborious so the number of experimental observations is restricted. Perhaps as a consequence,
somatic hybridization experiments designed speciﬁcally to study plastid genetics have been rare
(Akada and Hirai, 1983; Akada et al., 1983; Akada and Hirai, 1986). Therefore, most conclusions
about plastid inheritance in somatic hybrids are based in comparisons of several experiments from
different laboratories (Chapter 1). Such conclusions are subject to error due to the inﬂuence of many
uncontrolled factors, such as differences in the method of fusion, selection of hybrids, or plastid
analysiS-

The chNA coding complement and arrangement is stable during somatic hybridization, as
expected given the striking evolutionary conservation of plastomes. In contrast, mitochondrial
genomes regularly recombine or rearrange after somatic hybridization, as expected given their
evolutionary ﬂuidity (Palmer, 1985a; Hanson, 1984). Therefore in is not surprising that we observed
chNA stability and mtDNA rearrangement in our studies of the L esculentwrl + S. beapersicoider
somatic hybrids (Chapter 1; Appendix 1).

The contribution of plastids from each fusion partner can inﬂuence plastid transmission after
hybridization. When sorting-out of plastids after somatic hybridization is completely random, the
proportion of pure cell lines when sorting-out is complete should reﬂect the original plastid ratio in
the heterokarion (Chapter 1; Michaelis, 1967a; Birky, 1978; Akada and Hiria, 1986). There are more
plastids in mesophyll cells than any other tissue examined, and mesophyll derived plastids often
predominate when mesophyll and suspension derived protoplasts are fused (Kumar and Cocking,
1987). I am aware of no reports of mesophyll- suspension fusions where the plastids from the
suspension derived partner clearly predominated (Chapter 1). The cumulative ratio of mesophyll to

suspension derived plastids from all the mesophyll- suspension fusions tabulated in Chapter 1 is 185

55
to 97. In our experiments the ratio of plastids from the mesophyll (L escrdeutum) and suspension (S.
chopersicoides) partners is 68 to 2 (Chapter 2; Appendix 1). Diﬂ'erences in the plastid input probably
contributed signiﬁcantly to our results. However, ours is the most unbalanced ratio reported for a
mesophyll- suspension fusion suggesting that other factors may have also been involved.

Therelative rephcaﬁwcompeﬁﬁvenessofdiffemﬂplasﬁdsisimMamindetaminingphsﬁd
transmissioninsenlalcrossesinEuoarodlem (Chapter 1;Schétz, 1954;KirkandTilney-Bassett,
1978), and may have had a role in the evolution of this subsection (Stubbe, 1964). More rapid division
oftheplastidsfrom oneparentcanincreasetheirfrequencyinsuhsequentcellgenerations. Contrary
to an assumption the Michaelis model, each plastid probably does not replicate exactly once each cell
cycle. Plastid division can occur continuously during the cell cycle but the maximum number per cell
is under stringent developmental regulation (Possingham and Lawrence, 1983); Thomas and Rose,
1983). When the number of plastids per cell is belowthe maximum, those that divide more rapidly
can reproduce more frequently and predominate in subsequent cell generations. The relative
replicative competitiveness of diﬂ'erent plastids is very likely to inﬂuence plastid transmission in
somatic hybrids as well (Appendix 1), and there is evidence for such an inﬂuence in hybrids from the
genus Nicotiano (see Chapter 1). It is possible that the highly unbalance plastid transmission we
observed in our L esculenmm + S. choperricoider somatic involved diﬁerential replicative
competitiveness.

Plastid transmission in somatic hybrids is almost certainly inﬂuenced by compatibility with the
nuclear genome. Eighty to ninety percent of proteins needed for normal plastid function are encoded
in the nucleus (Parthier, 1982; Palmer, 1987). There is ample evidence of nucleo—cytoplasmic
incompatibility from sexual hybridization in Euoenothem and Pelargonium (Chapter 1; Stubbe, 1960;
Metzlaff er al., 1982; Polheim, 1986). However, evidence for a nuclear inﬂuence of plastid
transmission in somatic hybrids is scarce, primarily because of the lack of relevant nuclear-encoded
genetic markers (Chapter 1; Pelletier er al., 1983; Menczel et al.,1987; Kumar and Cocking, 1987).
Our two somatic hybrids with S. beapersicoides plastids grew vigorously and were visibly

indistinguishable from the 68 with L esculentum plastids (Appendix 1). Clearly, both plastid types

56
were compatible with the hybrid nuclear genome. However, the nuclear genome could have had a
quantitative inﬂuence on plastid segregation in these hybrids.

The rapid pace and stochastic attributes of plastid segregation in somatic hybrids are
congruent with observation of the segregation process after sexual crosses (Chapter 1; Michaelis,
1967a; Birky, 1978; Akada and Hirai, 1986). In summary, the possible causes of unequal transmission
oforganellesinsomatichybridsare:1)alargerorganellecontributionﬁomonefusionpartner,2)
more eﬁcient replication of the organelles from one partner, 3) incompatibility of the nuclear
genome with the organelles from one partner, or 4) in viva selection of the organelles form one
partner with antibiotics or culture conditions (Kumar and Cocking, 1987). My research determined
the origin of the plastids in our somatic hybrids, but gave little information about the cause(s) of the
unbalanced plastid transmission I observed.

Combinations of fusions between mesophyll and suspension derived protoplasts isolated from
L esculentum, S. lyeopersicoides, and a third ’tester' species such as L permellii could be used to
distinguish the inﬂuences of plastid replication efﬁciency and the initial input of plastids. Observation
of the number and morphology of the plastids in each type of protoplast could help determine the
role of plastid input. Combinations of hybridizations and cybridizations with {L. esculentum} and { S.
lycopersicoides} could be used to examine the inﬂuence of the nuclear genome on plastid
transmission. Understanding the role of these factors in determining organelle transmission in
somatic hybrids will lead to better understanding of the interactions among the three genomes within

plant cells.

APPENDIX

APPENDIX

Non-random organelle transmission in somatic hybrids of Lycopersicorr esculentwn
and Solution lyeopersicoides

A. Levi, B.L. Ridley and rcc. Sink

The following manuscript has been published in Current Genetics (14: 177-182). The author of this
thesis was responsible for the chloroplast analysis conveyed herein, and a large portion of the writing of
the mannuscript.

57

Summary.

Restriction fragment length polymorphisms (RFLPs) were used to determine the transmission
of organelle genomes in somatic hybrid plants of tomato and its wild relative Solarium lycopersicoides.
Biased frequencies of organelle combinations were observed in a population of 70 somatic hybrid
plants each derived from a separate callus. The plastids in 68 of70 hybrids examined were from L
esculenaun. One of the remaining hybrids, plant 240, had S. lycopersicoides plastids and the other,
plant 63, had a mixture of parental plastids. Forty-six of the same 70 plants were analyzed for
mtDNA and all had that of S. heapersicoides including plant 240. One of these hybrids had novel
mtDNA fragments which may have resulted from recombination or rearrangement. The non-random

transmission may have resulted from an initial unequal input of organelles, differential replication of

organelles, or nucleo-organelle incompatibility.

Key words: RFLP - tomato - plastid - mitochondria . protoplasts

59

Introduction

Maternal inheritance of organelles predominates among angiosperms during sexual
reproductionwith onlya fewspecies exhibitingbiparental inheritance(Sears 1980;8mithetal. 1986).
Suchmhedmnwachwapredudehdependemmwofmgandkgenomumalmoaaﬂaop
plants. Conversely,ceﬂfudmofphﬂprmopluuinducedbychemicalmelearialmcthods
produces heteroplasmic cells. Subsequent random sorting-out of the organelles during cell divisions
and organogenesis often results in novel combinations of organelle genomes in the regenerated
plants. The plastomes (plastid chromosomes) usually quickly sort-out in a random manner (Chen et
al. 1977; Belliard et al. 1978; Scowcroft and Laan 1981; review by Kumar and Cocking 1987) or
unilaterally when a chloroplast marker is selected (Medgyesy et al. 1980; Fluhr 1983; Menczel et al.
1986). Each regenerated shoot usually has plastids from only one parent, but some experiments have
produced chimeric shoots (Gleba et al. 1985; review by Kumar and Cocking 1987). These results
stem from the lack of plastid fusion which precludes chNA recombination (Possingham and
Lawrence 1983), and from rapid sorting-out of plastids during cell division (Akada and Hirai 1986).
However, Medgyesy et al. (1985) demonstrated plastid recombination when the heteroplasmic state
was prolonged. In contrast, mitochondrial genomes almost always undergo recombination or
rearrangement in somatic hybridization experiments (Belliard et al. 1979; Galun et al. 1982; Boeshore
et al. 1983; Rothenberg et aL 1985; Robertson ct al. 1987; reviewed by Hanson 1984). Although the
evidence is as yet meager, mitochondria may not sort-out as quickly as plastids (Aviv and Galun
1987). For example, cytoplasmic male sterility in Petunia somatic hybrids continued to segregate for
more than three meiotic cycles (Izhar ct al. 1983).

In this report we describe the biased transmission of both plastids and mitochondria in a large
population of somatic hybrid plants. Fusion of leaf protoplasts from tomato with suspension— derived

protoplasts of Solarium chopersicoides produced intergeneric somatic hybrid plants (Handley ct al.,

60

1986). Stringent selective conditions were not used to recover the hybrid calluses or plants (Sink et al.
1986). S. lycopersicoides suspension-derived protoplasts did not divide and calluses of the tomato
cultivar line used rarely regenerated shoots. Putative hybrid calluses were selected by their distinct
morphology, pigmentation and vigorous growth. All of the plants studied herein have been veriﬁed as
nuclear hybrids using Got-2 and Got-3 isozyme markers (Moore and Sink, 1987).

These two solanaceous species are unilaterally sexually compatible when tomato is the female
parent but the hybrids are highly sterile (Rick 1951). Hence, the somatic hybrid plants afford the

opportunity to study unique combinations of organelle genomes not normally possible following
sexual mating.

Materials and Methods

All DNA analyzed was isolated from fully expanded leaves of plants grown in the greenhouse.
Lycopersicon esculentum Mill. ‘Sub Arctic-Maxi’, Solarium lyc0persicoides Dun. (LA 1990), their
sexual hybrid, and 70 somatic hybrids were asexually propagated for analysis. Chloroplasts were
isolated using 5-step 20-60% sucrose gradients as described by Palmer (1986) with some
modiﬁcations. The extraction and wash buffers both contained 0.4 M sucrose, 50 mM Tris pH 8, 6
mM Na EDTA; whereas, 0.1% BSA (w/v), 0.15% PVP-40 (w/v), and 30 mM 2—mercaptoethanol
were added to the extraction buffer. Mitochondria were isolated according to Hanson et al. (1986)
using DNAse treatment and omitting the sucrose gradient. Chloroplast DNA (chNA) or
mitochondrial DNA (mtDNA) was prepared by suspending the organelle fraction in CI‘AB buffer (10
mM Tris pH 8, 1.4 M NaCl, 20 mM Na EDTA, 2% cetyltrimethylammonium bromide (w/v), 120 mM
2-mcrcaptocthanol), extracting with 24:1 chloroform /isoamyl alcohol, then precipitating the nucleic
acids by adding 23 volumes of ethanol (Saghai-Maroof et al. 1984). The chNA pellets were
dissolved and further puriﬁed on CsCl/ethidium Bromide (EtBr) gradients. The mtDNA pellets were
used without further puriﬁcation, and the yields were 3 to 15 ug of mtDNA/50 g of leaves, depending
on genotype. Whole cell DNA was isolated by grinding 0.5 g of leaves in a mortar with 10 ml of the

same buffer used for chloroplast extraction and centrifuging the slurry at 4,500 g; 15 min. The pellet

61

was resuspended in CTAB buffer and extracted with 24:1 chloroform/isoamyl alcohol. The nucleic
acids were precipitated and further puriﬁed on CsCl/EtBr gradients. Electrophoresis, Southern
blotting, nick translation, hybridization, autoradiography, and photography were performed using
standard procedures (Maniatis et al. 1982).

A 15 kilobase (kb) tomato chloroplast Pst 1 clone (P-6) was used for the chNA analysis
(Phillips 1985). The mtDNA was probed using pZmEl, a 2.4 kb Eco RI clone encoding corn
cytochrome C-oxidase subunit II (Fox and Leaver 1981) and pmt SylSa8, a 22.1 kb clone containing

8.9, 8.4, and 4.8 kb Sal I mitochondrial fragments from Nicotiana rylvesarlr (Aviv et al. 1984).

Results

Total DNA was isolated from 70 hybrids, digested with Pst I, blotted onto nitrocellulose and
probed with P-6. S. lycopersicoides has an additional Pst I site which splits the tomato 15 kb chNA
fragment into 9 kb and 6 kb fragments (J. Palmer personal communication). Sixty-eight of the 70
hybrids examined had the L esculentum 15 kb fragment (Fig. 1). Hybrid 240 had the S. lycopersicoides
pattern and hybrid 63 had a pattern corresponding to a mixture of parental chNAs (Fig. 1). Hybrid
240 had S. lycopersicoides mitochondria; the mtDNA of hybrid 63 was not analyzed. ‘The sexual hybrid
had the L esculentum plastid pattern as expected (data not shown). No nuclear or mitochondrial
homologies were detected when puriﬁed nuclear, mitochondrial and chloroplast DNAs from these
species were probed with P-6 (data not shown).

To detect RFLPs between tomato and S. I‘ycopersicoides mtDNA, digestion patterns produced
by 7 restriction enzymes were screened using the 2 mtDNA speciﬁc probes. No differences in
restriction patterns of the parents were detected when the Pst I, Sst I and Xho I digests were probed
with pZmEl; or when the Sst I digest was probed with pmt SylSa8 (Table 1). However, differences in
the parental mtDNAs were detected using eight enzyme-probe combinations. To perform the
mitochondrial analysis, Hind III or Eco RI digests of puriﬁed mtDNA from hybrid plants were
probed with pZmEl and pmt SylSa8, and Bam hi or Xho 1 digests were probed only with pmt SylSa8.

The sexual hybrid had the maternal tomato mitochondrial genome as camected (data not shown).

 

O POT-OHOv-wv-c-oe—
l
mJemnmeuu oo
nmwovevvvgeeggg
15
9
6

 

Figure 1. Southern analysis of plastid DNA from the parental lines and thirteen somatic
hybrids. Pst I digests of whole cell DNA from the somatic hybrids or puriﬁed chNA from L.
esculentum (LE) and S. chopersicoides (SL) were electrophoresed on a 0.8% agarose gel, blotted to
nitrocellulose, and probed with the 32P labeled chNA clone P-6. The sizes of the fragments are given
in kilobase pairs.

63

Table 1. Restriction fragment lengths of L esculentum (LE) and S. lycopersicoides (SL)
mitochondrial DNA in kilo-base pairs.

 

 

 

 

 

Enzyme Probes
WW ernEl
SL LE 81. LE
Kpn I - - - None 23 24
Pst I - — — i 18, 15, 8.2 None None
Sst I 24, 22.5, 12, 10, 85, 2.8 None None 14, 8.2, 5.0, 0.8 None None
Hind III 14.2, 12.5, 8.4, 5.9, 5.2, 25 6.8 None 9.4, 7.2. 3.4, 1.8 4.3 None
EcoRI 6.6, 65, 6.4, 35, 3.2, 3, 2.3, 5.6, 5.1 6.3, 5.7 2.6 3.6, 1.2 4.6, 1.9
1.8, 1.1
Barn III 21, 205, 20, 13, 7.4, 5.4, 22, 215, 21.7 10, 1.9 7.5 73
4.7, 2.2 2.8
Xho I 17.5, 16, 13, 7.9, 4.6, 40, 21, 32, 30, 6.7, 2.8, 0.8 None None
2.8, 1.3 83, 6.7 22

 

To determine the mt-DNA composition of the somatic hybrids, a random subset of 46 plants was
analyzed. Forty-two hybrids were analyzed using both pmt SylSa8 and pZmEl, and 4 were analyzed
using only one probe. Forty-ﬁve hybrid plants analyzed by Southern blotting had the same pattern as
S. chopersicoides (See hybrids 8 and 57, 38 and 51, and 69 in Figs. 2a, 2b and 3, respectively).

In contrast, when a Hind III digest of hybrid 23 was probed with pint SylSa8 the pattern was
the same as that of S. lycopersiciodes except a 14.2 kb fragment was missing and a novel 10.5 kb
fragment appeared (Fig. 2a). In Bani HI digests of mtDNA from hybrid 23, the same probe
hybridized to a novel 9.3 kb fragment and a 4.7 kb fragment with altered stoichiometry (Fig. 2b). This
plant also had a more intense 8.0 kb band when a Hind III digest was probed with pZmEl (data not
shown). Hybrid 23 had no other changes from the S. chopersicoides mitochondrial pattern when
Southern blots of Eco RI digests were probed with pZmEl, or when the Hind III restriction pattern
was examined on EtBr stained gels (data not shown). Interestingly hybrid 73 has tomato plastids, a

highly abnormal leaf morphology, and is slow growing.

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Fig. 3. Southern analysis of puriﬁed mtDNA from L. csculemmn (LE), S. lycopersicoides (SL),
and somatic hybrid 69 digested with Eco RI, electrophorescd on a 0.8% agarose gel and hybridized to
pZmEl. Fragment sizes are given in kilobase pairs.

Discussion

It is striking that the majority of the somatic hybrids have chloroplasts and mitochondria from
opposite fusion parents. They have predominantly (97%) tomato plastids and exclusively S.
beapem’cofda mitochondria. Hybrid 63 had chNA restriction fragments corresponding to both
parents. Mswuldbeduetochlmophstwrﬁng-ombetweenbranchesapeﬁcﬁnﬂchimermor
maintenance of the m cell type.

Plastid transmission in these intergeneric somatic hybrid plants could have been determined by
the relative number of plastids donated to the heterokaryon by the parental prot0plasts. For example,
mesophyll cells have about 10—fold more plastids than meristematic cells (Possingham and Lawrence
1983). In these experiments, the tomato protoplasts were isolated from leaf mesophyll cells and fused
with suspension-derived S. chopersicoides protoplasts. Mesophyll cells have more chloroplasts than
those of any other tissue that has been examined (Steele-Scott et al. 1984). To our knowledge, the
number of plastids found in long-term suspension cells has not been determined. However, these
rapidly dividing cells are similar to meristematic cells of whole plants and probably have relatively few
proplastids. For this reason tomato chloroplasts probably predominated in the heterokaryons.
Following fusion, the partitioning of plastids during cell division would have ﬁxed the predominant
type (Akada and Hiria 1986). This process would have been quite rapid if the number of plastids in
the hybrid cells decreased from around 100 to about 10 within 3-4 divisions as they did in cultured
mesophyll protoplasts of N. tabacum (Thomas and Rose 1983). Differential transmission of
chloroplasts due to unequal input from the gametes is a well documented aspect of sexual
reproduction in higher plants (Whatley 1982).

Another possiblity is that more rapid division of the plastids from one fusion parent resulted in
biased transmission. Plastid division can occur continuously during cell development and division but

the maximum number of plastids seems to be limited in a developmentally coordinated fashion

 

67

(Possingham and Lawrence 1983). Assuming that plastid multiplication is a stochastic process after
cell fusion, when the number of plastids per cell is below the limit, those that divide rapidly will
predominate in subsequent generations. Oeuothem, with biparental plastid inheritance, has yielded
genetic evidence supporting this concept. Schotz (1954) discovered differences in competition
between the ﬁve different plastomes of Omothem when they were brought together by sexual
mating. Each of the ﬁve plastome types was assayed using reciprocal crosses in which one parent
carried a plastome encoded albino mutant, and the other carried the wild-type plastome to be tested.
Thegerminatingseedsfrom thesecrosseswerescoredbymeasuringtherelativesizeofthegreen
sectors on their cotyledons, which gives a quantitative estimate of the relative competitiveness of each
plastome. This “variegation rating“ ranks the ﬁve plastomes in nearly the same order regardless of the
direction of the cross or the nuclear background (Chiu et al. 1987). Schotz (1968) attributed this to
differences in the rate of plastid multiplication.

Our ﬁndings as well as the reports of others indicate that protoplast fusions involving species
that are partially or entirely semally incompatible are more likely to exhibit non-random plastid
transmission (Menczel et al. 1981; Fluhr et al. 1983; Muller-Gensert and Schieder 1985; Clark et al.
1986; Gleddie et al. 1986; O’Connell and Hanson 1986). Conversely, fusions between highly sexually
compatible species are likely to produce a random distribution of plastids (Glimelius and Bennett
1981; Scowcroft and Larkin 1981; Sidorov et al. 1981; Flick et al. 1985; Kumashiro and Kubo 1986).
This suggests some cytoplasmic genomes are less able to function physiologically with a foreign
nucleus and this is more likely in hybrids between distantly related sexually incompatible species.

Genetic evidence for the incompatibility of plastomes with certain nuclear genomes has been
obtained for Oenothem. Stubbe (1960) studied the reaction of the ﬁve different plastid types of the
subsection Euoenothem and found profound differences in their ability to green when combined with
different nuclear complements. The manifestation of this ”plastome-genome" incompatibility ranged
from small changes in chloroplast pigmentation to complete lethality. Each plastome type is more
compatible when associated with a genome more closely related to the one with which it co—evolved

(Kutzelnigg and Stubbe 1974). Perhaps the under-represented S. chopem'coides plastids were less

68
competitivein thehybrid cellsdue toincompatibilitywiththe hybridnuelear genome. Biasedsorting
out due to plastome-genome incompatibility may have occurred at any time between cell fusion and
the time of analysis.

Incompatibility during plant regeneration, a complex process involving coordinate expression of
many genes, may be particularly important in determining organelle transmission. Menczel et al.
(1981) reported two somatic fusion experiments using N‘rcotiana taboaam and N. kniym'ana with
completely different results. In the ﬁrst experiment they observed only N. larigita'ata chloroplasts in the
hybrid plants, and in the second they observed balanced transmission of both types. They suggested the
culture conditions used in the ﬁrst experiment precluded regeneration from cells with N. tobacum
plastids. Schnabelrauch and Sears (personal communication) studied regeneration of isonuclear lines
of Oenothem with ﬁve different plastomes, and observed an influence of plastome type on the efﬁcacy
of regeneration. Perhaps in our experiments, cells with different plastome contents also responded
differentially to the culture medium.

Our mitochondrial results parallel the ﬁnding of two other somatic hybridimtion experiments
involving tomato with a related wild species. O’Connell and Hanson (1985) examined nine L.
esculentum + L. pennellii somatic hybrid cell lines using one mitochondrial probe and found eight
with the L. pennellii pattern and one which was a mixture. When the same workers used three probes
to examine four L. esculenaam + S. ricla'i somatic hybrid plants, all derived from one callus, they
found only mitochondrial bands speciﬁc to S. n’cla'i (O’Connell and Hanson 1986). They also noted
changes in the relative intensities of some of the hands when these hybrids were probed with a
mitochondrial ribosomal RNA clone, which suggests rearrangement or changes in the stoichiometry
of sub—genomic circular molecules (Grayburn and Bendich 1987).

Of the forty-six hybrid plants examined in this study, only hybrid 23 had restriction pattern
changes relative to S. lycopersicoides mtDNA, and no tomato speciﬁc fragments were detected in any
hybrids. This suggests the tomato mitochondrial genome was rapidly eliminated and the changes in
hybrid 23 represent rearrangement of the S. lycopersicoider genome rather than intergenomic

recombination. It is, however, possible that tomato speciﬁc fragments in the hybrids were not

 

69

detected. When mtDNA digests of the hybrids were visualized on ethidium bromide stained agarose
gels only S. lycopasicoider speciﬁc fragments were observed. Unfortunately, many regions of the gel
were difﬁcult to interpret due to dense spacing of the bands. Consequently, Southern analysis was
usedtoobtainmoredcﬁnitivedata. Sincethisprocedureismoreinvolvcd,weuscdtwoprobes
representing less than one tenth of the tomato mitochondrial genome (Palmer 1985) for the analysis.

TheS. Monasicoides mtDNA inthese hybridswasderived from one year-old suspension
cultures, and there are reports of mtDNA rearrangement in such cultures of tobacco (cg. Dale et al.
1981). Recent evidence indicates that the sequence arrangement of the master mtDNA chromosome
is stable, but the stoichiometry of subgenomic circular molecules can change in suspension culture
(Bailey-Scrres ct al. 1987; Grayburn and Bendich 1987). These changes in stoichiometry may have
caused many of the changes in the mtDNA restriction patterns that have been reported (Negruk et al.
1986). Since no differences were observed between the restriction patterns of mtDNAs isolated from
leaves of S. lyeopersicoides and the somatic hybrids, except for hybrid 23, the mtDNA in the S.
lycopersicoides cell suspension must have been fairly stable. The mtDNA changes in hybrid 23 cannot
easily be explained by altered mtDNA stoichiometry since they involve novel fragments as well as
changes in intensity of parental fragments. However, there is at least one report of a novel mtDNA
fragment which was probably induced by tissue culture (Dewey ct al. 1986).

The number of mitochondria may vary with the protoplast donor tissue (Bendich and Gauriloff
1984) in a manner analogous to that of chloroplasts. However, the mtDNA ploidy (the number of
cepies of mtDNA per cell) as well as the number of mitochondria per cell may influence mtDNA
transmission. This is because mitochondria probably fuse frequently allowing the resident mtDNAs to
interact (Bendich and Gauriloff 1984), whereas, plastids rarely fuse (Medgyesy ct al. 1985).
Mitochonrial fusion is probably a prerequisite of recombination and such recombination after somatic
hybridization is common (Robertson et al. 1987; Rothenberg and Hanson 1987; Vedcl et al. 1986;
Rothenberg et al. 1985). Thus, depending on the rate of mitochondrial fusion, the mitochondrial
genome from donors with higher mtDNA ploidy would be more likely to predominate in hybrid plants.

However, the amount of mtDNA per cell remains relatively constant between developmentally distinct

70

tissues in pea, the only plant examined thus far (Lamppa and Bendich 1984). The role of these factors
in mitochondrial transmission remains unknown since neither the mtDNA ploidy nor the number of
mitochondria has been determined in plant suspension cells or compared between these two species.

Itseemslikclythatthe parcntalmitochondriawere notmaintainedasamixturesinccwedid
not observe extensive mtDNA recombination. This may suggest that nuclear-mitochondrial
incompatibility was a contributing factor. However, we are not aware of any direct evidence for
nuclear-mitochondrial incompatibility in plant somatic hybrids. Mitochondria and plastids have

similar interactions with the nucleus (Douce 1985), suggesting mitochondria may be similarly capable

 

of nuclco- cytoplasmic incompatibility relationships.

WSW“ -'-—|; T: ' .

 

The authors gratefully acknowledge Drs. BB. Sears and JD. Palmer for technical assistance and advice
during this research, Dr. A.L. Phillips for the tomato chNA clone bank, Dr. D. Aviv for mtDNA
clones and Dr. CM. Rick for seed of LA 1990.

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71

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