|Table of Contents|
Table of Contents
Chapter 1. Introduction
Chapter 2. Literature review
Chapter 3. Material and methods
Chapter 4. Results
Chapter 5. Discussion
Chapter 6. Conclusions
INHERITANCE OF ORGANELLE GENOMES IN CITRUS
MARIA CRISTINA DRUMMOND MOREIRA
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1997
I do not think there is a way to thank Dr. Christine Chase enough for infinite discussions, suggestions, support and expertise. I also want to thank all Chase's lab group, especially my mentor, Mr. Victor Ortega, for his constant presence and incentive.
I want to thank Dr. Jude W. Grosser for his advice, practical approaches, encouragement and friendship. Without his and Francisco Mourao' s incentive, the possibility of a Ph.D. in the USA would be no more than a dream.
Dr. Fred Gmitter and his lab supported this research since the very beginning and are responsible for part of the
results. Dr. Gloria Moore, Dr. Ken Kline, Dr. Darryl Pring and Dr. Mickey Parish also played an important role in this research's history.
My eternal gratitude goes to my friends Cecilia Ritzinger, Viviana Faundes and the Quaresma family for their constant support.
Thanks are also expressed to CNPq for the financial support.
This research is dedicated to Dr. Celio Moreira, who is present in each of my steps, to my husband Eduardo, for constant love, support and incentive and to my children, Thais and Fabio, that are the reasons for everything.
TABLE OF CONTENTS
ACKNOWLEDGMENTS ........................................ ii
ABSTRACT ............................................... vii
1 INTRODUCTION ....................................... 1
Citrus ............................................... 1
Somatic Hybridization ................................ 2
Organelle Genetics ................................... 3
Research Objectives .................................. 4
2 LITERATURE REVIEW .................................. 7
Organelles ........................................... 7
Organelle Genome Organization ........................ 7
Organelle Division, Distribution and Inheritance ..... 11 Types of Organelle Inheritance ....................... 13
Heteroplasmy ......................................... 16
Uniparental x Biparental ............................. 21
Mechanisms involved in Organelle Inheritance ......... 25 Why Uniparental Inheritance? ......................... 32
The Nuclear Influence ................................ 37
Citrus ............................................... 40
History of Somatic Hybridization ..................... 41
Methods Used for Somatic Hybridization ............... 42
Organelle Analysis in Somatic Hybrids ................. 47
Methods of Organelle Analysis ..................... 47
Organelle Genome Stability ........................ 48
Factors Influencing Organelle Inheritance ......... 52
Summary ............................................... 55
3 MATERIAL AND METHODS ............................... 57
Plant Material ....................................... 57
Sexual Crosses .................................... 57
Somatic Hybrid and Cybrid Combinations ............ 58
RFLP Analysis of Nuclear, Chloroplast and MtDNA ...... 60
Total DNA Extraction ............................... 60
Restriction Enzyme Digestion and Electrophoresis... 61 Southern Blot and Hybridization ................... 61
Hybridization Probes .............................. 62
PCR Reaction ...................................... 65
Primer Sequences .................................. 66
Transformation Techniques ......................... 67
Plasmid Prep ...................................... 68
Insert Recovery ................................... 70
RAPDs ................................................ 71
PCR Reaction ...................................... 71
DNA Copy Number Analysis ............................. 72
4 RESULTS ............................................ 73
Sexual Cross ........................................ 73
DNA Polymorphisms Distinguishing the Parents ...... 73 Mitochondrial DNA Inheritance ..................... 75
RAPDs ............................................. 78
Chloroplast DNA Inheritance ....................... 81
Summary ........................................... 83
Somatic Fusions ..................................... 83
DNA Polymorphisms Distinguishing the Parents ..... 83 Nuclear and Organelle Inheritance ................... 87
'Succari' + Citropsis gilletiana ..................... 87
'Succari' + Atalantia ceylanica ...................... 89
'Willowleaf' + 'Duncan' ............................ 91
Swinglea glutinosa + Sour Orange .................... 91
'Rohde Red Valencia' + 'Dancy' ..................... 94
'Willowleaf' + 'Valencia' .......................... 96
"Hamlin' + 'Ponkan' ................................ 96
Cleopatra + Sour Orange .......................... 98
Summary .......................................... 98
DNA Copy Number Test ............................. 103
5 DISCUSSION ......................................... 107
Sexual Cross ........................................ 107
Mitochondria Inheritance ......................... 107
Chloroplast Inheritance .......................... 108
Nuclear Influence ................................ 109
Biparental Inheritance : A Hybrid Phenomenon? ..... 109
Models . . . . . . . . . .. 110
Discussion of the First Model....................... 111
Discussion of the Second and Third Models........... 113
Segregation Of the Paternal Configurations..........116
Somatic Hybrids......................................... 118
MtDNA from the Embryogenic Parent.................. 118
Random Chloroplast Inheritance..................... 121
DNA Copy Number Test................................ 122
Cybrids as unexpected outcome...................... 124
Cybrids Definition................................... 125
6 CONCLUSIONS........................................... 127
Sexual Cross............................................ 127
Somatic Fusions......................................... 128
BIOGRAPHICAL SKETCH....................................... 140
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
INHERITANCE OF ORGANELLE GENOMES IN CITRUS By
Maria Cristina Drummond Moreira December, 1997
Chair: Dr. Jude W. Grosser
Cochair: Dr. Christine D. Chase Major Department: Horticultural Science
The citrus nuclear genome has been investigated for a number of years. In contrast, little is known about the citrus organelle genome. Restriction fragment length polymorphisms (RFLPs) were the method used in this research for the characterization of citrus organelle inheritance. Six mitochondrial and two chloroplast heterologous probes were used to study the organelle inheritance in two different sources of materials--the Fl progeny of a citrus intergeneric sexual cross and the products of eight different citrus protoplast fusions.
The most common mode of organelle inheritance in
angiosperms is uniparental-maternal; however the sexual
cross produced unexpected results in terms of apparent biparental inheritance of mitochondria for three vii
mitochondrial genes: atpA, coxIlI and coxIII. The same F1 hybrids presented a strict maternal inheritance of the mitochondrial coxl and 26SrRNA. This raised the possibility that there was a nuclear influence over the mitochondrial genome organization, which has been ignored in previous studies where the biparental inheritance of organelles has been reported. A maternal inheritance of the chloroplast cytf and petD was observed in the same cross.
All the cybrids and somatic hybrids in this work inherited their mitochondrial genome from the embryogenic fusion partner (callus or cell suspension). In some of the combinations, non-parental bands were observed among the mitochondrial configurations. In contrast, the cybrids and somatic hybrids inherited plastid DNA from either the embryogenic or the nonembryogenic (leaf) fusion partner. The relative abundance of organelle DNAs in the embryogenic and leaf cells may be the basis of these inheritance patterns. Both sexual crosses and somatic cell fusions can therefore produce novel combinations of organelle genes in citrus.
"Citrus fruits have been cultivated and enjoyed for over 4,000 years" (Davies and Albrigo, 1994, p. vii). Citrus includes many commercially important fruit species such as sweet orange (C. sinensis), mandarin (C.reticulata), lemon (C.limon), grapefruit (C. paradisi), and it is grown in over 100 countries in tropical and subtropical areas, between the latitudes 40'N to 400S.
The world produces around 70 million tons of citrus fruits/year. Brazil is now the largest producer of citrus worldwide having an industry oriented towards production of oranges for processing. This country is followed closely by the United States of America, the second largest producer of citrus and the largest producer of grapefruit (Davies and Albrigo, 1994).
Most of the citrus grown today originated by chance during seedling selections or bud mutations of existing cultivars and not from breeding programs (Grosser and Gmitter, 1990). The lack of success in breeding programs is
due to several aspects of the reproductive biology of citrus like heterozygosity and inbreeding depression, pollen and ovule sterility, apomixis, nucellar polyembryony and juvenility (Chapter 2). According to Grosser and Gmitter (1990): "future success will be achieved by combining conventional breeding methods with new biotechnologies" (p.344).
Somatic hybridization via protoplast fusion is a useful technique for the production of novel plants which cannot be obtained by conventional breeding methods. In 1975, Vardi et al. reported the isolation and culture of sweet orange protoplasts. Ohgawara et al. (1985) produced the first citrus somatic hybrid plants, followed by Grosser et al. (1988) producing the first somatic hybrid plants from sexually incompatible parents (reviewed by Grosser and Gmitter, 1990) In these more than 20 years, different methodologies were developed, and the protoplast fusion technique is an important tool for citrus breeding programs. Another possible application for somatic hybridization is the direct fusion-mediated transfer of cytoplasmic encoded traits (Chapter 2).
In the 1960s, the study of organelle genetics gained importance with the discovery that plastids and mitochondria contain their own genomes. In the 1970s, the study of
organelle heredity became a mature field. Much was learned about organelle DNA, the genes encoded by plastids and mitochondria, and the protein synthesizing apparatus of these organelles (Gillham, 1994).
Organelle inheritance is a very complex phenomenon and ever since the first demonstrations of maternal inheritance of plastid mutations, numerous exceptions have been
discovered. About one-third of all higher plant genera at least occasionally, inherit plastids biparentally (Chapter 2) Unless very low probabilities of paternal or biparental inheritance can be detected, researchers may incorrectly assume strict maternal inheritance of organelles.
When making wide crosses, plant breeders should be aware of the nature of organelle transmission, since
biparental transmission could have a lasting effect on the final product. Especially significant would be cytoplasmic male sterility genes, because any paternal transmission could
lead to undesirable results (Soliman et al., 1987) Indeed, even a very small amount of paternal leakage has to be taken into account in the studies of relatedness between
individuals that assume the maternal inheritance of organelles. Also, regular biparental inheritance of organelles could represent a method for achieving cytoplasmic hybridization.
The objective of this work was to analyze the organelle composition of plants recovered from eight different protoplast fusion experiments performed at the CREC (UF/Lake Alfred/FL), in Dr. Grosser lab. Fusion products were obtained when combining embryogenic culture-derived protoplasts with leaf-derived protoplasts. Two of these combinations produced tetraploid plants, with morphology intermediary between the two parents, characteristic of somatic hybrids. The other six analyzed combinations produced diploid plants with the leaf parent morphology and are probably cybrids (Chapter 2).
Restriction endonuclease technology, which allows the identification of restriction fragment length polymorphisms (RFLPs) has been found useful for studying organelle inheritance in hybrid plants. In this work, six mitochondrial and two chloroplast heterologous probes were used for organelle inheritance characterization.
Before proceeding to the organelle analysis of the somatic hybrids and cybrids, we decided to check the
organelle inheritance in a citrus sexual cross, to test the assumption of maternal inheritance. The citrus nuclear genome has been investigated for a number of years. In contrast, little is known about the citrus organelle genome. Dr. Gmitter supplied us with an intergeneric sexual cross between LB 1-18 (a hybrid of Citrus reticulata Blanco cv. 'Clementine' x C. paradisi Macf. cv. 'Duncan') and Poncirus trifoliata (L.)Raf.
Citrus and Poncirus belong to the family Rutaceae. Poncirus consists of two trifoliate species, Poncirus trifoliata and P. polyandra. P. trifoliata is a deciduous tree native to cold regions in Central China. It is an important rootstock in some parts of the world because it is cold hardy and it is resistant to the Tristeza virus, citrus nematode (Tylenchulus semipenetrans) and Phytophthora. For these reasons it has also been used as a parent in citrus rootstock breeding. Citrus and Poncirus have different
morphologies. However, both genera are diploid (2n=18) and they are sexually compatible (Davies and Albrigo, 1994).
The sexual cross produced unexpected results, in terms of a possible biparental inheritance of mitochondria for three mitochondrial genes: atpA, coxI and coxIII. We thought citrus would be one more exception to the uniparental inheritance of organelles, but further analyses revealed that the same Fl hybrids presented apparently a strict maternal
inheritance of the mitochondrial coxI and 26SrRNA. So, added to the original objective of analyzing the organelle inheritance of citrus somatic hybrids and cybrids, this intergeneric cross provided us with some extra exciting questions in terms of the mitochondria genome organization and the nuclear influence over the organelle inheritance in citrus.
According to Gillham (1994), one of the major features distinguishing eukaryotic and prokaryotic cells is that eukaryotic cells are populated with a variety of subcellular organelles. Of the many types of organelles found in the cell, chloroplast and mitochondria are described by this author as very distinctive, in possessing their own genomic system. Mitochondria are the principal source of energy in the cell. They contain the enzymes for the Krebs cycle, carry out oxidative phosphorylation, and are involved in fatty acid biosynthesis (Russell, 1992). Chloroplasts are defined by the same author as cellular organelles found in green plants, in charge of the photosynthetic process.
Organelle Genome Organization
According to Warren and Wickner (1996), "every membrane-bordered organelle in a particular cell type has a characteristic copy number, size, and position, which reflects its cellular function" (p.395). Mitochondria, for example, are often located near organelles that consume
energy, and their number is determined by the energy needs. For example, in mammals, they are numerous in muscle cells and are tightly wrapped around the flagellum. The cytoplasmic DNA forms DNA-protein complexes, called nucleoids (organelle nuclei), that can be seen clearly as bright dots under epifluorescence microscopy by staining with DAPI (4'6-diamidino-2-phenylindole) (Sodmergen et al., 1994).
Mitochondrial genomes of higher plants are unusually large, when compared with the animal mitochondrial genome (usually around 16 kb). Values in the range of 200 to 2400 kb were indicated by Ecke et al. (1989). This complex organization was first described for Brassica campestris, by Palmer and Shields (1984), mentioned by Ecke et al. (1989), as a 218 kb master chromosome, with one pair of a 2 kb direct repeat, that can be resolved into two subgenomic circles of 135 and 83 kb by recombination within this repeat.
Other plant species have more than one set of repeats. The multicircular organization of the normal maize mitochondrial genome presents a master circle of 570 kb with six pairs of repeats. Many possible subgenomic molecules can arise, due to recombination between direct repeats. The cytoplasmic male sterile (cms) maize mitochondrial genome is
even more complex, since six permutations of the master circle alone are possible (Gillham, 1994).
According to Ecke et al. (1989), the reason for this multipartite organization and high frequency of recombination remains unknown. Also, they mentioned that this kind of mitochondrial organization is not always the rule, as the Brassica hirta mitochondrial genome consists of a 208 kb master chromosome only, with no subgenomic circles.
However, despite their large size, plant mitochondrial genomes do not contain many more genes encoding proteins. The majority of the mitochondrial proteins are encoded in the nuclear genome (Gillham, 1994).
Birky (1994) indicated that only a minority of plant mitochondrial DNA (mtDNA) molecules are really circular in vivo. According to Bendich and Smith (1990), cited by Birky (1994), the maxicircle (or "genomic chromosome") could be a long linear concatamer of minicircles (or "subgenomic circles") that replicates as a rolling circle. However, since the minicircles do not contain the complete mitochondrial genome, every daughter cell must receive at least one mastercircle.
According to Stoneking (1996), one of the properties that makes mtDNA very useful for evolutionary studies is that it is present in multiple copies per cell. There are
several mtDNA genomes per mitochondrion, several hundred mitochondria per cell, and several thousand billion cells per individual. This author suggested that this makes us a population of mtDNA genomes.
According to Gillham (1994), the chloroplast genomes of a few land plants (mostly angiosperms) are very well characterized. The plant chloroplast genome is defined as one circular chromosome, with a very conserved DNA sequence. In striking contrast between the mitochondrial genomes of liverwort (Marchantia polymorpha) and angiosperms, their chloroplast genomes are very similar in organization and gene content. According to this author, the chloroplast genomes possesses two unique sequence regions and two large inverted repeats (typically 10-30 kb), always positioned asymmetrically, splitting the chloroplast genome into unique small and a large sequence regions.
Nuclear genes are organized in chromosomes, which are replicated once and each daughter cell receives one copy at cell division. In contrast, organelle chromosomes are chosen for replication and partitioned at random to daughter cells during cell division. According to Birky (1994), there is no loss of genes, because, in general, each organelle chromosome contains a complete genome (a complete set of mitochondrial or chloroplast genes) Organelle genes obey non-Mendelian laws: vegetative segregation (alleles of
organelle genes segregate during meiosis and mitosis), uniparental inheritance and intracellular selection (some organelle chromosomes may replicate faster than others, in the same cell, depending on their genotype). This author called the organelle genome the "relaxed genome." Birky (1994) also indicated that mutant alleles can be fixed by intracellular selection.
Organelle Division, Distribution and Inheritance
Kuroiwa and Uchida (1996) suggested that organelles in higher organisms might divide with the use of prokaryotelike "division rings" that we cannot yet visualize. They believe that "microbodies" are in control of organelle division, at least in certain Eukaryotes. Microbodies are common and consist of at least two types: glyoxysomes and peroxisomes, both of which contain the enzyme catalase. When studying the Chlamydomonas merolae organelle division, these authors observed the initial signs of a mitochondrial division(MD)-ring formation, with the movement of a microbody to the region between the mitochondrion and the cell nucleus. An MD-ring that is 40 nm wide and 40 nm thick forms in the cytoplasm at the point where the microbody has attached to the mitochondrion. These authors also pointed out that the separation of organelles following their
divisions to daughter cells is important for cytoplasmic inheritance.
Berger and Yaffe (1996) indicated that mitochondria will arise only from pre-existing mitochondria, in a coordinated expression of nuclear and mitochondrial genes. There is a role for the cytoskeleton in the positioning and movement of the mitochondria. This was observed when
chemical agents that perturbed the cytoskeletal network also altered the mitochondrial distribution. According to these authors, the microtubules largely co-align with the mitochondria during Saccharomyces pombe cell division and have a central role in the mitochondrial distribution. However, the authors pointed out that there are differences in the mitochondrial behavior between the fission and
budding yeast (Saccharomyces cerevisae) These authors also mentioned that a mitochondrion may not necessarily behave as a single, unitary structure, and that different components (e.g. matrix and the inner membrane) may possess different motility properties. These authors described two classes of proteins that facilitate the movement and the inheritance of mitochondria in yeast: cytoskeletal proteins and mitochondrial proteins. Two general mechanisms are probably involved: one has to do with "molecular motors" that bind the mitochondrial surface and pull the mitochondria along the cytoskeletal tracks. In the other mechanism, the
mitochondria would move via internal changes in morphology and the sequential binding and release of surface structures along a cytoskeletal scaffold.
According to Mogensen (1996), the polarization of chloroplasts during cell division may be mediated by microtubules, actin filaments and/or a biochemical gradient. However, this author indicated that more studies are necessary in this area.
Types of Organelle Inheritance
The study of organelle genetics began over 80 years, when Correns and Baur discovered in 1900 that mutations affecting the chloroplast phenotypes of higher plants frequently exhibit non-Mendelian inheritance (Kumar and Cocking, 1987) Interest in organelle inheritance was aroused only 10 years later, after the rediscovery of Mendel's laws. The study of organelle inheritance began with the use of phenotypic markers (mainly chlorophyll deficiency) and has more recently been extended by cytological and molecular approaches (Reboud and Zeyl, 1994).
Kuroiwa and Uchida (1996) mentioned that, in primitive cytoplasmic inheritance associated with sexual reproduction,
male and female derived organelles are mixed in the zygote, and in some cases fused, permitting recombination of their
plastid DNA. They also mentioned that recombination of mtDNA in the slime mold Physarum polycephalum depends on mitochondrial plasmids and thus is analogous to bacterial sex (conjugation). According to these authors, in more advanced forms of cytoplasmic inheritance, uniparental transmission of organelles is generally seen.
The inheritance of organelles has been categorized into three types: maternal, paternal and biparental. The most common mode of organelle inheritance in angiosperms is uniparental-maternal. About one third of all higher plant genera at least occasionally inherit plastids biparentally (Mogensen, 1996). Most of these results have been obtained from studies with chlorophyll-deficient mutants, as chlorophyll markers (Smith,1989) Biparental, or even predominantly paternal transmission of chloroplasts has been documented in a number of angiosperms such as Nicotiana tabacum (Medgyesy et al., 1986; Yu et al., 1994), Medicago sativa (Lee et al., 1988), Oenothera (Chiu and Sears, 1993), Coreopsis grandiflora (Mason et al., 1994), Turnera ulmifolia (Shore et al., 1994), Lens culinaris (Rajora and Malhon, 1995), Pelargonium (Guo, 1995) and Actinidia (Cipriani et al., 1995).
Examples of paternal inheritance of mitochondria in gymnosperms are common and have been reviewed by Reboud and Zeyl (1994): in Sequoia sempervirens (Neale et al., 1989),
in Pinus banksiana x P.contorta (Wagner et al, 1991) and Calocedrus decurrens (Neale et al., 1991).
In contrast, there are few reports of deviations from maternal inheritance of mitochondria in angiosperms. This may be the result of either inherent differences between these organelles or the lack of mitochondrial phenotypic markers (Rebould and Zeyl, 1994). Erickson and Kemble (1990) reported that the paternal transmission of the mitochondrially associated plasmid can occur in Brassica napus. Fairbanks et al. (1988) observed that F1 progenies of alfalfa (Medicago sativa) inherited several large RNA molecules from both maternal and paternal parents. More recently, a predominant paternal transmission of the mitochondrial genome in cucumber (Cucumis sativus), was documented by Havey (1997).
Biparental inheritance of chloroplasts and mitochondria has been observed in intergeneric crosses involving Hordeum and Secale (Soliman et al. 1987) and Festuca pratensis and Lolium perenne (Kiang et al. 1994). Maternal inheritance of chloroplasts and paternal inheritance of mitochondria were observed in bananas (Musa acuminata) by Faure et al.
Examples of biparental inheritance of mitochondria in animals have been described for the mussel Mytilus edulis
(Hoeh et al., 1991) and the bark weevil (Pissodes sp.). An interesting point was raised with the work of Kaneda et al. (1995), where paternal mtDNA was detected in crosses between two different species of mice, but not in intraspecific crosses. The authors presented a model where the interaction between sperm mitochondria and components in the egg cytoplasm involves a receptor-ligand system. According to the model, receptor and ligand are mismatched in interspecific hybrids because of the evolutionary divergence, thus preventing efficient recognition and elimination of the sperm mitochondria. Hoeh et al. (1991) also agreed that "biparental inheritance of mtDNA may be a hybrid phenomenon" (p. 1489).
Heteroplasmy can be defined as the presence of different types of mtDNAs coexisting within a cell or individual. Transplanting germ plasma into an egg carrying a different type of mtDNA should generate germ cells possessing two types of mtDNA in the cytoplasm, provided mitochondria in the donor germ plasma survive and are successfully transmitted to the next generation. Matsuura et al. (1989) induced mtDNA heteroplasmy by intra- and interspecific transplantation of germ plasma in Drosophila. They demonstrated that the heteroplasmic state persists in
female germ cells at least from heteroplasmic Go fly to the G2 progeny. The authors concluded that since no significant decrease or elimination of donor mtDNA was observed and the heteroplasmic state was obviously retained in germ cells, it is clear that rapid purification of mitochondria with
respect to mtDNA type does not occur, at least in early generations.
Organelle heterosis is defined by Smith (1989) as the occurrence of a heterotic response when organelles from different genotypes are mixed, either in vitro or in vivo, in a cytoplasmic hybrid. The author mentioned the work of Sarkissian and Srivastava (1967) who observed enhanced oxidative phosphorylation efficiency in artificially mixed mitochondria of particular maize inbreds when compared with the mitochondria of inbreds alone. Organelle
heterosis/complementation requires that regular biparental inheritance of organelles occurs, although this was not always stated. According to Smith (1989), regular biparental inheritance of organelles could represent a method for achieving cytoplasmic hybridization.
Laipis et al. (1988) and Koehler et al. (1991) reported that nucleotide sequence differences can arise within a few generations in the mtDNA of Holstein cows. The authors implied that heteroplasmy can be considered a source of evolution of the mitochondrial genome. Koehler et al. (1991)
observed the replacement of Holstein cows leukocyte mtDNA by a sequence variant in just one generation. This implies, according to the authors, the existence of heteroplasmic cells in the cell lineage between the germ line of the mother and leukocytes of the progeny, and that the heteroplasmic state is transient relative to the total number of cells in this cell lineage.
Lee and Taylor (1993) detected a complete mitochondrial replacement during mating in the filamentous fungus Neurospora tetrasperma. In only three days following hyphal fusion, the nuclear acceptor strain mtDNA replaced donor mtDNA throughout the entire colony. They raise the point that perhaps both mitochondrial types have existed for short periods of time or in unequal stoichiometries and have gone undetected by their methods. These authors indicated that replacement can be due to the migration of one mitochondrion throughout the recipient mycelium coupled with destruction of the other type, to an organelle replication advantage of one of the types, or even to recombination between mitochondria, with the identifying features of one genome always being incorporated in the recipient. The possibility that variant mitochondrial genomes can be fixed in one generation should be considered in analyses of evolutionary relationships based on divergence of mitochondrial sequences (Koehler et al., 1991).
Kondo et al. (1990) found heteroplasmy due to an incomplete maternal transmission of mtDNA in Drosophila. This group used radioactively labeled probes that were specific to paternal mtDNAs. This method could detect as little as 0.03% paternal mtDNA when present in a sample. MtDNA heteroplasmy was also observed in Drosophila mauritiana by Solignac et al. (1983). Petri et al. (1996), found an extraordinary level of mtDNA sequence heteroplasmy within a species of European bat, and there was as much variation within an individual bat as there was within the entire population. These authors provided evidence to suggest that such heteroplasmy also exists in humans,
although in reduced levels, compared to the bats.
Jazin et al. (1996) estimated the spectrum of mtDNA sequence heteroplasmy in the brain of normal individuals in order to determine the importance of heteroplasmy to aging and neurodegenerative disease. The level of heteroplasmy was assessed in five regions of the mtDNA in human brain by denaturing gradient gel eletrophoresis (DGGE). Analyses
revealed high levels of heteroplasmy in the D-loop (a
triple-stranded displacement loop, with no coding function), while no variability was detected in the coding regions. These authors observed a higher frequency of mitochondrial heteroplasmy in the older individuals (about 90 years old) in comparison with a 28 year old individual. Small
insertions and deletions increased 7.7-fold, but point
mutations did not show any significant increase with the aging process. This group indicated that further experiments measuring the accumulated damage in the coding region will be needed to clarify the functional importance of heteroplasmic mutations in aging human brain.
Turner et al. (1995) studied the inheritance of kinetoplast DNA in Tripanosoma brucei (a protist). The kinetoplast is a unitary organelle that replicates and segregates into each daughter cell at mitosis. It is found in the matrix of the cell's single, giant mitochondrion (Gillham, 1994). It contains maxi and minicircular DNA molecules and the maxicircles are equivalent to mtDNA, except they appear not to contain tRNA genes. Minicircles encode the vast majority of guide RNAs involved in the mitochondrial RNA editing. In the progeny, maxicircles are inherited uniparentally, and minicircles are inherited biparentally. However, Turner et al.(1995) demonstrated that the inheritance of maxicircles is biparental in some clones during the early growth stages of hybrid progeny resulting from a genetic cross. Subcloning and further vegetative expansion showed that the mixtures of maxicircle genotype were unstable and quickly became fixed. They pointed out that 140 generations (35 days) would be necessary to achieve
stability. In contrast, there is no requirement for the minicircles to segregate with strict fidelity.
In contrast to mitochondria, Smith (1989) and Chiu and Sears (1988) in studies using species that inherited the plastids biparentally, did not observe evidence of chloroplast recombination in a population of sexual hybrids.
Uniparental x Biparental
The inheritance strategy adopted by a particular organelle can vary among cell types and organisms (Warren and Wickner, 1996). Even with a particular cell type, different organelles could use different strategies. The inheritance of cellular organelles, therefore, differs fundamentally from the inheritance of chromosomes, where a single, universal strategy is used, based on the mitotic spindle. Reboud and Zeyl (1994) also suggested that uniparental and biparental inheritance are not fixed alternatives. According to these authors, they are reversible conditions, whose frequencies in the population can respond to selection. These authors also raised two hypotheses that can give rise to opposite expectations:
(i)If only the nuclear genome is responsible for organelle inheritance, selection for such control should be relaxed in selfing angiosperms, where a potential conflict between organelles of different origins is much less frequent.
Biparental inheritance should therefore be more common in selfing than in outcrossing species; (ii)But, if organelles can drive their own inheritance, a biparentally inherited plastid will invade an outcrossing population, while in selfing species such plastids would only replace other copies of themselves. Thus, according to this second hypothesis, biparental inheritance should be more common in outcrossing than in selfing species. After reviewing different studies analyzing organelle inheritance in different plants, these authors concluded that these data support the hypothesis that plastids can influence their inheritance. For example, paternal and biparental inheritance of plastids is found in all gymnosperms, which are known to be outcrossers.
The probability of failing to recognize occasional paternal transmission is usually high. The number of hybrid plants, according to Smith (1989), typically subjected to organelle inheritance analysis has usually been quite small. Also, the organelle extraction, DNA isolation and visualization procedures used in most of the organelle studies generally are not sensitive enough to detect small amounts of paternal DNA (stoichiometrically
underrepresented in hybrid plants) should it be present. Milligan (1992) and Avise (1991) also mentioned that unless very low probabilities of paternal or biparental
inheritance can be detected, biologists may incorrectly
assume strict maternal inheritance. Milligan (1992) used a binomial approach to show that a progeny may contain paternally inherited chloroplasts with a probability of P=0.0001-0.025. Extremely large sample sizes would be
required to distinguish between uniparental and biparental organelle inheritance. This distinction is very important, according to Milligan (1992), when considering the population genetics of organelles. The possibility of a paternal transmission opens the possibility of recombination among distinct organelle genomes, and this
can alter the patterns of diversity observed in organelle genomes.
Erickson and Kemble (1993) also suspected that there may be varying degrees of paternal or biparental inheritance of mitochondria in plant species but that studies to detect it have been too few and inadequate.
These authors used cytoplasmic male sterility as a marker to prove that paternal mitochondria can be transmitted to the progeny of a sexual cross in rapeseed (Brassica napus).
They were able to transfer paternal mitochondria to the progeny in only one of the four crosses. An average of 1 in 15 progeny of this cross contained the paternal
mitochondria. Also, Avise (1991) suggested that a paternal mtDNA might rarely, but quickly, colonize and dominate a
maternal line. Under a neutral model, according to this author, the expected frequency of such takeovers is simply the ratio of sperm:egg mtDNA numbers in a zygote, which is probably less than 1:2000 in most animal species.
Erickson and Kemble (1993) suggested that the female genotype has an influence on whether paternal mitochondria will survive and replicate in the progeny of a sexual
cross. But, the authors also indicated that they cannot determine if the genetic factors responsible for pollen transmission of mitochondria on the female side are
cytoplasmic, nuclear or both. The authors concluded that genes active during pollen development and following
fertilization are responsible for the maternal inheritance of mitochondria in higher plants.
Erickson and Kemble (1993) reported the effect of ploidy level on the transmission rate of paternal
chloroplasts through the pollen. The authors mentioned the work of Massoud at al. (1990) where tetraploid alfalfa
plants transmitted plastids to their progeny at a higher rate than diploid genotypes, regardless of whether they were males or females in a cross.
Environmental conditions also play a role in the male cytoplasmic inheritance, according to Yu and Russell (1994) They studied the sperm-cell organization of Nicotiana tabacum under different environmental conditions.
Sperm cells were serially reconstructed to evaluate their quantitative cytological organization and that of their
organelles. They observed that the size of the nucleus and the number of mitochondria were larger in flowers grown in growth chambers under warmed controlled conditions, whereas the number of plastids was the same or maybe slightly
higher in flowers under cooler greenhouse conditions. The authors pointed out that further research would be necessary to verify if this kind of environmental influence occurs in other flowering plants.
Mechanisms Involved in Organelle Inheritance
Vaughn (1981) described two contrasting theories to explain organelle inheritance in higher plants: 1) physical exclusion of the organelles from the generative cell and 2) organelle alteration leaving debilitated organelles that would be incapable of genetic transmission, even if
included in the generative cell. This author investigated the ultrastructure of pollen grains of a severely debilitated plastome mutant of Pelargonium that is not transmitted via pollen. Usually, this is one of the angiosperms presenting paternal inheritance of
chloroplasts. Because the plastids are not physically
excluded from the generative cell, although they are not transmitted via pollen, the theory of physical exclusion is
inadequate to explain the chloroplast inheritance in this mutant. Debilitated organelle genomes are not transmitted to the progeny with success despite the inclusion of these organelles in the generative cell. According to Vaughn
(1981), this conclusion is consistent with the organelle alteration hypothesis for the control of organelle inheritance. The same author described myelin-like figures, that are no longer recognizable as mitochondria, as
possible degenerate mitochondria. Similar "unknown bodies" were found by other authors (Clauhs and Grun, 1977, and Hagemann, 1979, mentioned by Vaughn, 1981) and were assumed
to be degenerate mitochondria or plastids. Hause (1986) also observed myelin-like structures, besides intact
plastids and mitochondria. The author pointed out that by the first pollen mitosis during the pollen development of Pisum sativum, the number of mitochondria appears to be 510 times higher than that of plastids. By this time,
mitochondria and plastids are randomly distributed within the generative and vegetative cells. The author mentioned that the mitochondria of the vegetative cell contain a
higher number of cristae and they are bigger in diameter than the generative cell mitochondria.
The organelle exclusion hypothesis, first put forward by Sager (1972), is supported by Ikehara et al. (1996). When studying the maternal chloroplast inheritance in
Chlamydomonas reinhardtii, these authors noticed that in the zygotes, the chloroplasts from the male parent (mating type minus, mt-) disappear within 40-50 minutes after mating, while those from the female parent (mating type plus, mt ) persist. Thus, the preferential digestion of chloroplast nucleoids was suggested as the main reason for the maternal inheritance of the plastid genes. According to these authors, soon after the zygote formation, specific mRNAs are synthesized in the cell nucleus of mt+ cells, which code for proteins that directly or indirectly activate a calcium dependent nuclease to digest mt- nuleoids in the zygotes. They mentioned the nuclear gene zyslB as tightly related to preferential digestion of male origin chloroplast nucleoids. The ZyslB protein was detected in early stages of the zygote formation, then decreased to an undetectable level with the completion of the preferential digestion. The turnover of the transcript is rapid and a putative amino acid sequence of this protein has cysteine and glutamine rich domain, indicating that it could be a transcription regulator.
Ikehara et al. (1996) also investigated whether higher number and higher DNA amount (fivefold) of chloroplast
nucleoids could disturb this process of preferential digestion and maternal inheritance. Their results showed that this preferential digestion occurred with the appearance of the ZyslB protein, although a slightly longer
period was needed to complete the digestion of chloroplast nucleoids in large number.
In Chlamydomonas, according to Nakamura et al. (1992), endo-exonucleolytic activity of Nuclease C is high enough to completely digest about 10 chloroplast nucleoids, dispersed in the male chloroplast, in just 10 min. In their research, when this enzyme was added externally to the cells whose cell wall, membrane and chloroplast envelope have been biochemically punctured, it specifically digested male chloroplast nucleoids, rather than those of female. According to these authors, plants like Rhododendron kaempfer, Zygocactus truncatus, Oenothera lacinata and O. speciosa, showing biparental chloroplast inheritance, probably have a low activity nuclease C, allowing the intact paternal chloroplast nucleoids, or plastid DNA to remain. Nucleases are also referred by Lee and Taylor (1993) and Kuroiwa and Uchida (1996), as possible destruction agents of one of the parent's organelle genome. They also mentioned that no mechanism of methylation seems to be involved in the process of organelle inheritance.
Lee and Taylor (1993) referred to the theory of exclusion as the heterokaryon incompatibility associated with the mating type alleles, which immobilizes the cytoplasm of the male. Migration of nuclei into one partner without concomitant mitochondrial migration has been
reported by these authors in a work with the filamentous fungus Neurospora tetrasperma. These authors mentioned a possible extrinsic feature, maybe associated with nuclear acceptance or female behavior, which confers a replication advantage or destructive ability on the maternal mtDNA.
Mogensen (1988) described several mechanisms that have been considered responsible for the uniparental inheritance of organelles, in even more detail: "(i)exclusion of the organelles from the generative cell or one sperm cell during the first or second pollen mitosis, respectively;
(ii)deletions in plastid DNA, degeneration of organelles, or elimination of organelle-containing cytoplasm during microspore, generative, or sperm cell maturation; (iii)organelle exclusion during gamete fusion; (iv) degeneration, lack of replication, or compartmentalization into suspensor cells after zygote formation" (p. 2594). This author indicated that structural evidence for the exclusion of male cytoplasm has been reported for spinach and cotton, in which two membrane-bound, enucleate cytoplasmic bodies were found within the degenerated synergid shortly after fertilization. Mogensen's (1988)
hypothesis has to do with the relocation of the sperm nucleus once an opening in the egg is created, which would result in the sperm cytoplasm being left behind within its original plasma membrane. Subsequent closing of the opening
at the point of fusion could occur by a process of membrane reformation, involving vesicles present within the egg and sperm cell or by a membrane constriction process. The author pointed out that this mechanism may be important during sexual reproduction for the nontransmission of genetically altered male organellar genomes, such as large deletions in the chloroplast DNA, known to occur in microspores of wheat and barley.
Hause (1986) indicated that Hagemann (1983) summarized three modes of plastid behavior that lead to uniparental inheritance: (i)Lycopersicum-type, where an extreme unequal distribution of plastids during the first pollen mitosis causes the lack of these organelles in the generative cell;
(ii)Solanum-type, when after the first pollen mitosis some plastids are present in the generative cell. They degenerate and are not present in the mature sperm cells. (iii)Triticum-type, when intact plastids are transmitted into the sperm cells, but during the fertilization only the sperm nucleus enters the egg cell. The cytoplasm of the sperm cell remains outside the egg.
Reboud and Zeyl (1994) affirmed that during gametogenesis, organelle loss results primarily from the formation of "cytoplasmic projections" that are subsequently discarded from the sperm cell body. These authors also pointed out that no exclusion mechanism is
completely effective on its own. Probably they operate in sequence, during both gametogenesis and embryogenesis. There must be a trend, where the more evolved is the organism, more numerous are the mechanisms employed.
Kuroiwa and Uchida (1996) noted a huge amplification of organelle DNA in egg cells during Pelargonium zonale oogenesis, which accompanies an increase in the egg cell volume. This phenomenon complements processes such as unequal division of first pollen mitosis and preferential digestion of organelle DNA in generative cells to yield a complete uniparental inheritance of organelle genomes.
Hause (1986) mentioned the theory of Russel and Cass (1983) concerning the biparental inheritance of plastids in Plumbago zeylanica. During the prophase of the second pollen mitosis, they observed a polarization of plastids in the generative cell. In consequence, one sperm cell contains all plastids and a small amount of mitochondria; in the other sperm cell, there was the majority of
mitochondria and no plastids. During the fertilization process, only the sperm cell containing the plastids fuses with the egg cell, and causes biparental inheritance of plastids in this species. The other sperm cell, according to Reboud and Zeyl (1994), will fuse with the central cell, which will become the endosperm. The number of paternal mitochondria to the endosperm is greater than that
transmitted into the egg as the result of preferential fertilization by the mitochondrion-rich dimorphic sperm cell. In an evolutionary paradox: mitochondria behave suicidally by entering a sperm cell which will never transmit them, although this probably increases the fitness of the embryo (Reboud and Zeyl, 1994) According to these authors, in such cases, mitochondrial segregation must be controlled by nuclear genes. Paternally-derived nuclear genes in the embryo probably benefit from the presence of mitochondria in the endosperm, perhaps increasing the allocation of material resources to the embryo.
Why Uniparental Inheritance?
Hastings (1992) postulated that isogamy (where all gametes are identical in size and function) is disadvantageous as it allows deleterious cytoplasmic organisms (such as intracellular parasites) to spread
through the population when cytoplasms are shared at fertilization. Conversely, according do this author, the uniparental inheritance restricts deleterious genomes and they cannot spread through the population. He even argued that once uniparental inheritance is established, the "male" gametes which contribute no cytoplasm may become small and anisogamy may evolve. Therefore, according to Hastings (1992), uniparental inheritance of cytoplasm arose
in response to the presence of deleterious cytoplasmic agents.
one example of a deleterious genome, mentioned by this author, would be the so-called "selfish mitochondria," which increases its own rate of replication, at the cost of reduced metabolic activity. The overall frequency of
selfish mitochondria is a function of two processes: their initial frequency rises due to their replicative advantage, but eventually falls, as cytoplasms without selfish mitochondria dominate the population due to metabolic advantage. The number of mitotic divisions would be
determinant in the ability of the selfish mitochondria to be transmitted. A cytotype initially containing a mixture of "selfish" and wild type mitochondria will eventually, after enough mitotic divisions, produce cytoplasms containing exclusively wild type or "selfish mitochondria." This phenomenon was described by Hastings (1992) as Mitotic Segregation. This author indicated that mutations producing
"selfish mitochondria" are likely to arise continuously, and their gradual accumulation in the population may result in a continuous decline in mean fitness.
Reboud and Zeyl (1994) suggested that monogametic
transmission and selective silencing may have evolved to avoid organelle recombination. According to these authors, uniparental inheritance may prevent the invasion by
transposons, which seem to be less abundant in chloroplast genomes than in the mitochondria and nuclei.
Hastings (1992) indicated that, in extant species, it appears that a small amount of paternal "leakage" may occur. This would be a transitory phenomenon, as natural selection favors nuclear alleles which reduce this leakage. The amount of leakage observed in extant species represents the balance between the benefit of decreased leakage which inhibits the spread of deleterious cytoplasmic agents and the advantages of increased leakage which aids the incorporation of advantageous mutations across the lineages.
Allen (1996) indicated that the respiratory electron transport and ATP synthesis in mitochondria are accompanied by the generation of mutagenic free radicals of oxygen, causing damage to the mitochondrial genetic system. The author indicated that mtDNA suffers oxidative damage at about ten times the rate of nuclear DNA. There is a positive feedback loop: free radical mediated mutagenesis of mtDNA may initiate and promote further mutagenesis through its effects on the structure and function of respiratory chain proteins, and on mitochondrial gene expression. The author pointed out that there is an inverse correlation between life span and metabolic rate, and that in plants, chloroplast genomes may be subject to a similar cycle of
redox damage from electron transport in photosynthesis.
Allen (1996) even indicated that the proximity of certain regions of plastid and mtDNA to the membrane-bound photosynthetic and respiratory electron transport chains
should be expected to lead to mutation "hot spots" (regions of DNA close to sites Of 02 generation) Allen (1996) also described that in animals, a similar positive feedback loop
("vicious circle" of energy loss) has been proposed as an explanation of aging, whereby mitochondrial division is impaired and becomes incapable of replenishment in postmitotic cells such as those of muscle.
However, the offspring do not inherit their parents' somatic degeneration. The author suggested that the positive feedback loop is broken in mitochondria of the
female germ line, from which all the mitochondria derive (escape of female germ line from mitochondrial ageing) In a very interesting hypothesis, Allen (1996) indicated that the mitochondria of the female germ line have a repressed bioenergetic function, avoiding mutagenesis from products of respiratory electron transport. In consequence, their genomes would survive and replicate with minimal damage.
Allen (1996) named "Promitochondria" as the mitochondria of the male germ line and of somatic cells of both sexes:
bioenergetically functional but genetically disabled. This would lead to the elimination of damaged, paternal mtDNA at
or before fertilization. The promitochondria persists in plants in meristematic cells, prior to differentiation of somatic and germ cells. The author suggested that the size, motility and number of gametes can be secondary characteristics to define male and female. According to his hypothesis, oxidative phosphorylation is the difference between sexes.
Specific predictions arise due to Allen's (1996) hypothesis: (i)female germ-line mitochondria have a repressed bioenergetic function; (ii)cells of the female germ line are sequestered from somatic cell lines at an early stage in development, prior to differentiation of
promitochondria (not true for plants); (iii)female gametes are relatively long-lived, having the lowest metabolic rate consistent with viability, and usually are few in number;
(iv)mitochondrial damage may eventually accumulate and natural selection would favor the elimination of individual females that are still capable of passing mutant mitochondria. This would have to do with the limit of the female reproductive phase; (v) female gametes depend on import of ATP from somatic cells for energy; (vi) the male germ-line mitochondria is a genetic dead end, committed to short-term energy production. So male gametes would be short lived, produced at any stage of a lifetime.
Hastings (1992) mentioned that the spread of deleterious elements, as "selfish mitochondria," may constitute another action pressure favoring the transfer of genes from mitochondrial to nuclear DNA. Allen (1996)
proposed that the major reason for retention of certain genes in the organelles is a requirement for redox control of plastid and mitochondrial gene expression. This minimizes free radical damage to the cell as a whole. "Eukaryotes and their nuclear genes owe a debt to the altruistic mitochondria and chloroplast, which occupy the eukaryotic cell's most hostile internal compartments" (p. 139). Avise (1991), referred to the endosymbiont origin of the organelles, when the author postulated that "the symbiont ensures its own survival by keeping its fingers on the jugular vein of cellular energy flow" (p. 55).
The Nuclear Influence
The majority of components necessary for mitochondrial and chloroplast functions are supplied by genes encoded in the nucleus. The nucleus is involved in mtDNA replication, recombination, and/or mitochondrial segregation (Mackenzie et al., 1994). One example of nuclear gene regulation of mitochondrial function is the existence of a single nuclear gene (Fr) able to restore the fertility of CMS (cytoplasmic
male-sterility) in common bean, by altering the mitochondrial genome (Mackenzie and Chase, 1990).
Another example of nuclear genome influencing the
mitochondrial genome organization was described by Sakamoto et al. (1996) with a (maternal inherited) distorted leaf mutant of Arabidopsis, induced by the recessive nuclear mutation: chloroplast mutator(chm). This mutation causes the preferential amplification of substochiometric mtDNA configurations (present in very low amounts). CHM induces mutations not only in mtDNA, but also changes in chloroplast morphology and function. However, the authors pointed out that it is difficult to correlate the morphological change with chloroplast mutations, because mitochondrial mutations can affect chloroplast structure and function. They propose that this nuclear gene in Arabidopsis has a function similar to Fr in common bean. The function of CHM and other nuclear products affecting mitochondrial genome organization is
still to be elucidated. According to Sakamoto et al (1996), it is possible that these proteins interact with a specific region of the mitochondrial genome and preferentially maintain master molecules, maybe securing their distribution along with the mitochondrial division.
The fact that Gymnosperm plants transmit mtDNA maternally, but chloroplast DNA paternally, suggests some active regulation of organelle transmission, since both
genomes must be subject to the same physical constraints imposed by the relative sizes of the egg and pollen
cytoplasms (Avise, 1991). Tilney-Bassett (1994) has shown that the genetic transmission of plastids is under nuclear control. The relationship between nuclear and chloroplast genomes was discussed by Smith (1989) when describing a special type of chlorophyll deficiency, termed "hybrid
variegation" that appears to result from an incompatibility of some sort between the nuclear and cytoplasmic genomes of the parents involved in a cross.
Gotoh et al. (1995) described nuclear-cytoplasmic interactions and sometimes lethal recombination of genes, causing reproductive incompatibility between two populations of the spider mite, Tetranichus quercivorus. They mentioned that the cytoplasmic factor responsible for the incompatibility in this species may be cytoplasmic heritable agents such as mitochondria. These authors indicated that examples of incompatibility between the cytoplasmic factors of one population and the nuclear genes of another population are known in insects, plants such as wheat, and in cybrid plants possessing the Atropa genome and the Nicotiana plastome.
Another example of nuclear influence over the mitochondrial genome has to do with RNA editing. This process results in mitochondrial RNA alterations, which may
change the meaning of the genetic information. Most are C-U transitions that occur in open reading frames, but a few are
observed in intron sequences (Pring et al. 1993 and Wilson and Hanson, 1996). According to these authors, the specificity of RNA editing in plant mitochondria is probably determined by nuclear gene(s), according to site affinity and accessibility.
Small et al. (1987) observed in maize changes in stoichiometry of different molecules in response to different nuclear backgrounds. They suggested that an explanation for sudden genomic reorganization of the mitochondrial genome is selective amplification of preexisting sub-stoichiometric (possibly undetectable)
molecules (perhaps together with a reduction of previously abundant molecules).
According to Smith (1989), in the zygote of at least some species, a signal is produced possibly by nuclear gene(s), which determines whether maternal or paternal plastids or both will be propagated and in what proportions as the embryo develops.
Most of the citrus scion and rootstock cultivars grown today are not a product of breeding programs, but originated by chance during seedling selections or bud mutations
(Grosser and Gmitter, 1990). Despite the immense genetic variability in the genus Citrus and related genera, the conventional breeding has had a limited role in terms of developing new varieties. According to these authors, this is due to several aspects of the citrus reproductive
biology: heterozygosity and inbreeding depression, pollen and ovule sterility, sexual incompatibility, apomixis and nucellar polyembryony, and juvenility.
History of Somatic Hybridization
Regeneration from Citrus protoplasts began after Kochba et al. (1972), cited by Grosser and Gmitter (1990), reported the production of Citrus sinensis embryogenic callus from the nucellar tissue of cultured ovules. According to these authors, the first example of a successful somatic hybridization in Citrus was an intergeneric allotetraploid hybrid produced by Ohgawara et al. in 1985 between embryogenic protoplasts of Citrus sinensis 'Trovita' and Poncirus trifoliata leaf protoplasts. The production of a somatic hybrid plant between the sexually incompatible C. sinensis and Severinia disticha by Grosser et al. in 1988, demonstrated that protoplast fusion is a viable means of bypassing barriers to sexual hybridization. Other authors also reported their achievements in terms of somatic hybridization/cybridization including Ohgawara et al.
(1994), Vardi et al. (1987a, 1987b and 1990), Grosser et al. (1996) and Saito et al. (1994).
Methods Used for Somatic Hybridization
Protoplasts can be fused by chemical or electronic methods. Grosser and Gmitter have been using the polyethylene glycol (PEG) method since 1984. It is recommended because it is simple, efficient, inexpensive and does not seem to interfere with protoplast viability.
Protoplasts can be isolated from various sources including leaves, embryogenic callus, embryogenic suspension cultures, nonembryogenic callus, and flower bud tetrads (for haploid protoplasts) (Grosser and Gmitter, 1990). According to these authors, the requirement for plant regeneration following fusion has been that protoplasts from one of the parents must have embryogenic capacity. For general somatic hybridization, protoplasts from embryogenic callus or suspension cultures should be fused to a non-totipotent source of protoplasts such as those derived from leaves.
According to these authors, the somatic hybrid selection (the identification and separation of somatic hybrid plants from unfused parental material) is a key part in the whole process. Protoplasts of one parent are obtained from non-embryogenic tissue, reducing the possibility of a whole plant recovery from this parent. Grosser and Gmitter
(1990) cited the work of Ohgawara et al. (1985), where a high concentration of sucrose in the protoplast culture medium can inhibit the regeneration of the embryogenic parent. Together these processes prevent unfused protoplasts from undergoing somatic embryogenesis.
True somatic hybrids, according to Grosser and Gmitter (1990), have a vegetative morphology generally intermediate to the donor parents, tetraploid chromosome numbers, and what the authors called a composite expression of DNA or gene products markers. Among the techniques available for hybrid verification they described: mitotic chromosomes counting, visual evaluation, molecular characterization by gel eletrophoresis of DNA or isozymes, or even chromatographic separation of leaf oils. These authors
pointed out that none of these methods is sufficient by itself; each is subject to limitations. The hybrid verification should include morphology evaluation, cytogenetic, and molecular characterization.
In contrast to somatic hybrids, "cybrids" contain the nucleus and exhibit the general morphology of the recipient parent, but contain the organelle genomes of the donor parent. Vardi et al. (1989) used mitochondrial probes for cybrid verification. These authors have been using the "donor-recipient" protoplast fusion method. The nuclear division of donor protoplasts is arrested by either X or
gamma irradiation, and the cytoplasm of recipient protoplasts is inactivated by an antimetabolite such as iodoacetate. Success in the "donor-recipient" method of cybridization depends on the ability of organelles to withstand the radiation that will disrupt the nuclear DNA. The differential effect of radiation on the nuclear and organellar DNA is probably due to the high number of organelles and organelle genomes present. Also, the mitochondrial structure may serve as a shield for the DNA, against the irradiation effects.
A different definition for cybrids was presented by Bonnema et al. (1992) and Kumar and Cocking (1987), where cybrids or cytoplasmic hybrids are defined as special cases of asymmetric somatic hybrids since they contain the nuclear genome of only one of the protoplast fusion partners and cytoplasmic organellar genomes of both parents.
According to Earle (1995), as the fusion product divides and produces callus or plants, the organelle populations segregate to give different, more or less stable combinations (Birky, 1978, reviewed by Earle, 1995). This process can be affected by the specific experimental conditions used during the creation of the fusion product.
Saito et al. (1993) observed that through the cell fusion between nucellar callus cells and mesophyll cells in two different citrus combinations, they obtained regenerated
plants resembling the mesophyll parent, in addition to the expected somatic hybrids. These authors investigated the mitochondrial genome composition of these plants. All the clones that resemble the mesophyll parents (and contain
their diploid nuclear genome) have the nucellar callus parent mitochondrial genome. These plants are therefore cybrids. Considering the fact that the mesophyll parent does not have the regeneration ability, possibly the mitochondria of nucellar-derived cells play a significant role in Citrus embryogenesis. Grosser et al (1996) discussed the cybridization requirement for plant regeneration from citrus leaf protoplasts following somatic fusion. These authors mentioned that the origin of these cybrid cells could be from successful protoplast fusion accompanied by unsuccessful nuclear fusion and loss of the embryogenic parent nuclei, or by incomplete fusions where the protoplast of the embryogenic parent ruptured during the fusion process and resulted in only partial transference of its contents.
According to Smith (1989), one of the goals of organelle research is the transfer of a cytoplasmic trait from a given genotype into the nuclear background of a second genotype, often in cases where the two genotypes could not be hybridized sexually. Direct fusion-mediated transfer of mitochondrially encoded traits (like cms) to other lines selected as parents for hybrid production is an
attractive application for the somatic hybridization (Earle, 1995 and Akagi et al., 1995). Yamamoto and Kobayashi (1995) reported the production of a cybrid having the nuclear genome of C. sinensis and the cytoplasmic genome of C. unshiu, usually male sterile, a very desirable characteristic in terms of obtaining seedless fruits.
In a plant breeding program it would be quite valuable to achieve unilateral cytoplasm transfer relatively quickly leading to the exchange of cytoplasm without backcrossing. This is particularly important when we consider that methods for gene transfer into higher plant mitochondria are not yet available. Stable transformation of higher plant chloroplasts has been accomplished via particle bombardment (reviewed by Earle, 1995) However, this author observed that unlike backcrossing, only some of the plants recovered are likely to contain the introgressed cytoplasm, and these will probably differ in their mtDNA arrangements. Also, somaclonal variation and/or transfer of some unwanted nuclear DNA from the donor to the recipient in spite of irradiation pretreatment, may occur during the use of methods like the "donor-recipient" cybridization process.
Organielle Analysis in Somatic Hybrids Methods of Organelle Analysis
Two general approaches for the analysis of mtDNA in putative somatic hybrids, are described by Earle (1995). First is the use of ethidium bromide stained gels of restriction enzyme-digested mtDNA. Efficient isolation of mtDNA may be a limitation in this approach. The second strategy is the hybridization analysis of total DNA isolated from the fusion products and parents. This permits the examination of small amounts of fusion-derived material
(such as calli weighting as little as 50 mg) and multiple loci (by hybridizations with nuclear and organelle probes on a single DNA gel blot) The first step is to find combinations of restriction enzymes and probes that reveal clear differences between fusion parents. Usually the probes are cloned genes. However, gene-containing fragments, mtDNA regions from homologous and heterologous sources, cosmid clones carrying mtDNA, or simply total mtDNA from one fusion partner, also can be used.
Most studies of mitochondrial genomes after fusion, include only the types of analysis described above. However, a few studies (reviewed by Earle, 1995) provided more
detailed information, such as mapping of the DNA regions contributed by each fusion parent (Morgan and Maliga, 1987; Honda and Hirai, 1992), sequencing of a recombinant
mitochondrial gene (Rothenberg and Hanson, 1988) and Northern analysis of transcription (Rothenberg and Hanson, 1988).
Organelle Genome Stability
Samolylov et al. (1996) indicated that in contrast to the array of recombinations that occur between the two nuclear genomes, the heteroplasmic state of somatic hybrid cells almost always sorts out quickly leading to uniparental transmission of chloroplast DNAs. By contrast, according to these authors, the mitochondrial genomes often undergo
rearrangement and novel types of mtDNA in somatic hybrids are frequently observed. Motomura et al. (1996) observed mitochondrial recombination between parents in the product of an electrofusion between 'Seminole' tangelo and Severinia buxifolia. In contrast, no recombination was observed in the chloroplast genome of the products, and the sorting out of plastids was a rapid process.
Indeed, according to Earle (1995), mtDNA
"rearrangement" or "non-parental mitochondrial genome" are really more appropriate terms to describe the "novel" mtDNA configurations, considering that differential replication of subgenomic molecules from the fusion partners, rather than intergenomic recombination, might be responsible for these observed unique fragments. This author indicated that,
because regeneration from fused protoplasts always involves a callus stage, mtDNA changes seen in somatic hybrids or cybrids might result from spontaneous culture-related changes, rather than to the fusion event per se.
Honda et al. (1991) mentioned by Earle (1995), worked with tobacco fusion products. They observed many different types of mtDNA rearrangements in small fusion-derived calli. However, when checking leaves from regenerated plants, a more uniform DNA was found.
Earle (1995) indicated that in the work of Morgan and Maliga (1987) Brassica napus cybrid calli and calli from unfused protoplasts both contained the same apparent modification in the mtDNA. Its appearance may be related to an intragenomic recombination in vitro or maybe is just an amplification of a band present at very low levels in the original plant (a "sublimon"). However, Nagy et al. (1983), reviewed by Earle (1995), observed mtDNA changes only in tobacco plants derived from fusions, not in plants regenerated directly from protoplast or callus culture of similar protoplasts.
Gleba et al. (1985), cited by Kumar and Cocking (1987), showed that heterozygosity for parental types is relatively stable in hybrid/cybrid plants and can be maintained even in their sexual progeny. According to the authors, this would
provide an unique opportunity for chloroplast DNA recombination between the two types of parental chloroplasts. They raise the point that usually the protocols fail to achieve this goal due to a lack of a stringent selection system, and also possibly due to a very low frequency of chloroplast genome recombination. According to Earle (1995), both inter- and intramolecular recombination appears to be quite common among mtDNAs in plants. In contrast, this author and Malone et al. (1992), mentioned that recombination of chloroplast DNA after fusion is very rare and there is only a single case of a putative chloroplast recombination in higher plants (Medgyesy et al., 1985).
Changes in mtDNA from regenerated plant to progeny, were also observed by Sakai and Imamura (reviewed by Earle, 1995) in Brassica napus cybrids obtained after fusion with irradiated cms radish line. Indeed, Earle (1995) pointed out that the mtDNA alterations that occur after fusion are not completely random. Rather, some regions look like "hot spots" for rearrangements. Rothenberg and Hanson (1988), reviewed by this author, identified some of these regions, and through sequencing and computer searchers, even provided some information about how the novel fragments were produced. They did extensive studies of a novel recombined atp9 gene in progeny of a Petunia somatic hybrid obtained by
fusion of fertile and cms lines. By sequencing this gene, they showed that it contained the 5'end of one parental atp9 gene (fertile P. hybrida) and the 3'end of an atp9 gene from the other fusion partner (cms P. parodii) The coding region of the gene was identical in both parents and was not altered in the somatic hybrid. Portions of the 5' and 3' flanking regions were also the same in both parents. In the
somatic hybrids, recombination apparently took place within a 414 bp region of homology between the two genes. Homologous recombination within the coding region of a mitochondrial gene was also detected in somatic hybrid calli
of tobacco (Honda and Hirai, 1992) Rearrangements of mtDNA after fusion might facilitate wide hybridizations by achieving more favorable nuclear-cytoplasmic interactions through selection against mtDNA regions that did not
interact well with the nucleus of the other fusion partner (Earle, 1995).
Morgan and Maliga (1987), cited by Earle (1995),
examined small fusion-derived Brassica calli with one probe and concluded that mitochondrial segregation was complete in some (but not all) calli within 19-22 generations after fusion. More than half of these calli contained all regions
from one partner plus some regions from the other, as well as some novel fragments. This result suggests a gradual loss of some regions independent of others, perhaps by
"independent segregation or differential replication of subgenomic mtDNA molecules" (p. 568) Kumar and Cocking (1987) pointed out that although the fusion products initially contained a mixed population of parental chloroplasts, somatic hybrid/cybrid plants subsequently recovered usually possess only one or the other parental chloroplast type. Furthermore, the authors mentioned that chloroplast sorting out appears not to be influenced by the degree of sexual compatibility of the fusion partners.
Earle (1995) mentioned that chloroplasts and mitochondria segregate independently after the somatic fusion and suggested that there is no strong tendency for the two types of organelles to move together except insofar as compatibility with the nuclear genome is limiting or strong selection for chloroplast traits is applied. The contrast between the behavior of plastids and mitochondria may be related, according to Kumar and Cocking (1987), to the structural organization at the membrane level of these cytoplasmic organelles, and also to differences in their genomic organization. DNA recombination is the rule governing plant mitochondrial genome organization.
Factors Influencing Organelle Inheritance
Samolylov et al. (1996) found no correlation between the nuclear genome composition and the transmission of
chloroplast and mtDNA during the production of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant. A different result was obtained by Bonnema et al. (1992) They decided to test if the inheritance of the organelle genomes is influenced by the nuclear background of the protoplast fusion product. Working with a collection of tomato symmetric and asymmetric hybrids, and cybrids between tomato (Lycopersicum esculentum) and L. penelli, the authors observed that the inheritance of the organelle DNA was probably influenced by the nuclear background of the regenerant. An increase in the percentage of tomato alleles in the nucleus was accompanied by an increased probability of an individual inheriting the tomato chloroplast genome and having more tomato-specific mitochondrial sequences.
Kumar and Cocking (1987) pointed out that the presence of a single parental type mitochondrion in animal somatic hybrid cells (mouse+human) belongs to the parent whose nuclear chromosomes are more stable. They related the
propagation of mtDNA from one parent in the somatic hybrids to a particular set of chromosomes.
Earle (1995) mentioned that protoplasts from different tissue types may differ in the number and in the replication rate of the mitochondria they contain. Bonnema et al. (1992) suggested that when suspension cells (growing heterotrophically) are the source of protoplasts, this would
provide a more competitive form of mitochondria. Earle (1995) mentioned a work with Brassica, by Landgreen and Glimelius (1990), where there was a preferential transmission of mtDNA from hypocotyl protoplasts. These authors suggested that hypocotyl protoplasts may contain more mitochondria than mesophyll protoplasts because they come from young dividing cells.
Another factor raised by these authors is that leaf mesophyll cells are relatively enriched in plastids when compared to suspension-cultured cells. Kumar and Cocking (1987) mentioned that mesophyll protoplasts have approximately 200 chloroplasts/cell and cell suspension
protoplasts possess approximately 20 chloroplasts/cell. The prediction would be that the somatic hybrids would preferentially inherit the mesophyll parent chloroplast DNA. However, the authors did observe a random pattern of
inheritance of chloroplast DNA in the symmetric somatic hybrids.
Bonnema et al. (1992) mentioned that non-random organellar inheritance is more frequent in combinations of species that are sexually incompatible. Explanations for non-random inheritance include the tissue source of the protoplasts used in the fusion, intergeneric nucleocytoplasmic incompatibility, and differential replication rates for the organelles (Bonnema et al., 1992). Other
factors for plastid inheritance were mentioned by Malone et
al. (1992) :adequate time for vegetative segregation (even if there is selection for a certain organelle type) ; the unlimited and disoriented cell divisions in culture; and stringent conditions favoring multiplication of desired organelle population, such as light stimulation and preferential protein biosynthesis.
It is important to remember that techniques of somatic hybridization require that cells and organelles be placed in
conditions that are far different from those experienced in vivo. Howe (1986), mentioned by Smith (1989), suggested that
organelle recombination in vitro does not necessarily mean that this phenomenon occurs in nature.
Further research is necessary to elucidate the
mechanisms governing organelle inheritance. Sometimes rules are assumed without questioning the methodologies or all the factors that potentially influence the chloroplast and mtDNA inheritance.
It is interesting to note how chloroplast and mitochondrial genome inheritance can be completely
independent events. Chloroplast DNA apparently is inherited in an "organized" and consistent way, with few surprises in
terms of appearance of new configurations in the progeny of
a sexual cross or in somatic fusion products. Perhaps this is because recombination does not play a major role in the organization of the chloroplast genome.
Little is known about the plant mitochondrial genome organization. However, considering the array of possibilities (rearrangements, recombination and
differential replication) that may occur before, during or after a sexual cross or somatic fusion, there are probably no general rules. Each fusion or cross presents and entirely different situation. In each of these situations, a completely different nuclear genome is formed. This probably plays an important role in determining which mechanisms will be acting during the mitochondrial genome reorganization.
MATERIAL AND METHODS
The intergeneric sexual hybrid family analyzed in this work was developed by Dr. Fred Gmitter at the CREC (Citrus Research and Education Center/University of Florida), Lake Alfred. The seed parent used was LB 1-18, a hybrid of Citrus reticulata Blanco cv. 'Clementine' x C. paradisi Macf. 'Duncan' grapefruit. Seeds of LB 1-18 are monoembryonic, containing sexually derived zygotic embryos. The sexually compatible pollen parent was a seedling of Poncirus trifoliata (L.)Raf., which is no longer extant. Therefore, DNA from P. trifoliata cultivar Rubidoux was used for this analysis.
Twenty-six 7 year old progeny from this cross were screened for the inheritance of mitochondria and chloroplast DNA polymorphisms: Fls # 3, 4, 6, 7, 9, 10, 12, 13, 14, 15, 16, 18, 19, 21, 22, 23, 24, 27, 28, 30, 33, 35, 36, 44, 48, 55. Fls # 33 and 44 were open pollinated, and 6 progeny of each one were subjected to the same kind of analysis. Leaf 57
material from each one of these trees was collected in Lake Alfred (CREC) at different times of the year, but always at about 50% leaf expansion. The leaves were brought to the lab in an iced cooler, and frozen at -80'C.
Somatic Hybrid and Cybrid Combinations
All the somatic hybrid and cybrid combinations listed below were supplied by Dr. Jude Grosser, from CREC (Lake Alfred/FL). These fusions were performed using the PEG method
(Grosser and Gmitter, 1990), with the objective of obtaining tetraploid somatic hybrids for breeding purposes. After the fusion, regenerated plants were checked in terms of chromosome number and leaf isozyme banding patterns on starch
gels (Chapter 2). As mentioned in chapter 1, although the expected outcome is a symmetric hybrid containing both
parental nuclear genomes, some diploid plants resembling the leaf parent were obtained (putative cybrids). Description of the somatic hybrid and cybrid combinations in terms of number
of analyzed plants and source of materials is presented in Tables 3.1 and 3.2.
Table 3.1. Description and number (#) of the analyzed
somatic hybrid combinations.
EMBRYOGENIC PARENT LEAF PARENT #
'Succari' sweet Citropsis 14
(C.sinensis L.Osbeck) Swingle & M.Kell.
'Succari' sweet Atalantia 14
Table 3.2. Description and number (#) of the analyzed cybrid combinations.
EMBRYOGENIC PARENT LEAF PARENT #
'Willowleaf' mandarin a 'Duncan' 4
(C.reticulata Blanco) grapefruit
Swinglea glutinosa a Sour Orange 3
Swingle (C.aurantium L.)
'Rohde Red Valencia' b 'Dancy' mandarin 5
(C.sinensis L. Osbeck) (C.reticulata Blanco)
'Willowleaf' mandarin a 'Valencia' sweet 9
'Hamlin' sweet orange b 'Ponkan' 2
(C.sinensis L. Osbeck) mandarin
Cleopatra mandarin a Sour Orange 3
(C.reticulata Blanco) (C.aurantium)
b suspension cultures
The leaves were collected from regenerated somatic
hybrid or cybrid plants growing in the CREC' s greenhouse (Lake Alfred) and brought to Gainesville in an iced cooler. They were kept at -800C.
The erbryogenic suspension cultures and callus were
obtained in Lake Alfred, from Dr. Grosser, and were at the same growth stage used at the somatic fusion. The callus was friable, about 2 to 3 weeks old which is "log phase" on a 6
week subculture cycle. The suspensions were about 12 days old in a 14 day subculture cycle.
RFLP Analysis of Nuclear, Chloroplast and Mitochondrial DNA Total DNA extraction
Total cellular DNA was isolated from lg of frozen leaf tissue, 1g of embryogenic suspension culture, or 3 g of
callus. The same phenol /chloroform extraction method (Durham et al., 1992) was used to obtain DNA from leaves of sexual hybrids and from leaves, callus and suspensions of the parents somatic hybrids and cybrids. The main difference
between these different materials was that leaves had to be frozen with liquid nitrogen to be ground. Callus and suspension cells were easily ground without liquid nitrogen.
Restriction Enzyme Digestion and Electrophoresis
The first objective was to find polymorphism between the parents, and then to characterize the F1 hybrids, somatic hybrids, and possible cybrids. With this in mind, total DNA from the sexual and somatic hybrid parents was digested using different restriction enzymes having a six base recognition sequence (EcoRI, HindIII, PstI, EcoRV, BamHl, SmaI, Dral and XbaI) according to the manufacturer's instructions (Life
Technologies Inc.). Restriction fragments from 5 ug (for
organelle analysis) and 20 ug (for nuclear analysis) of total DNA were separated by electrophoresis through a 0.8% agarose
gel in TPE buffer (300mM NaH2PO4, 360mM Trizma base and 10mM Na2EDTA.2H20) at 40v for 20 hours. The size of the gel was 380 cm2
Southern Blotting and Hybridization
These DNA fragments were blotted to Hybond-N supports (Amersham Corporation) by capillary transfer (Sambrook et al., 1989) and hybridized with radiolabeled mitochondrial, chloroplast and nuclear probes. The blots were pre-hybridized for at least 1 hour in 10xSSPE (1x SSPE = 1.8M NaCl, 0.1M NaH2PO4, 0.01M Na2EDTA), 50x Denhardts (50x = 1% w/v Bovine Serum Albumin, 1% w/v Ficoll, 1% w/v PVP 360) and 10% w/v SDS. To avoid background in the autoradiographs, herring sperm DNA (500 ug of DNA) was denatured by boiling for 10
min., chilled on ice and added to the prehybridization solution.
The probes described in table 3.4 were radiolabeled with alpha 32PdCTP through random priming (Feinberg and
Vogelstein, 1984), using a "Gibco BRL Random Primers DNA Labeling System." Following denaturation, the probes were added directly to the prehybridization solution, and the blots were hybridized for a minimum of 16 hours, at 600C.
The blots were washed after hybridization in a 2x SSPE, 0.1% SDS solution at room temperature for 10 minutes.
Following a second 2x SSPE, 0.1% SDS wash for 10 min. at room temperature, the blots were washed in lx SSPE, 0.1% SDS at 600C for 15 min. A final wash in 0.1x SSPE, 0.1%SDS was done at 600C for 10 min. Membranes were exposed to Kodak (X-Omat RP XRP-5) film for 7-14 days.
Tables 3.3 and 3.4 describe the heterologous probes used for both the sexual cross and somatic hybrid analyses. The 26SrRNA cDNA was constructed by Mr. Byoung Kim, at Dr. Chase's lab, using reverse transcriptase PCR (RT-PCR) techniques. The original cytf clone was supplied by Dr. Ken Kline (Horticultural Sciences Dept./Gainesville). Plasmid culture from a citrus lycopene cyclase clone was provided by Mr. John Melton, in Dr. Gloria Moore's lab.
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The same PCR protocol was used in the production of the lyc.cyc., atpA, cob, coxl, coxIl, coxIII and cp3 probes and is described in Tables 3.5 and 3.6.
Table 3.5. PCR amplification reaction.
Target DNA 2ul
Primer l(10ng/ul) 10ul Primer 2(10ng/ul) 10ul HPLC water 65ul
10 x Taq bufferx 10ul
2.5 mM Mg beads 1
dNTPsY (2.5mM) 2ul
Taq polymerasex lul
Total Volume 100ul
x supplied by Promega Corp. Y dNTPs = deoxyribonucleoside triphosphates
Table 3.6. PCR Program.
1 94oC/1 min.
2 55oC/2 min
3 720C/3 min
4 Go to step 1-3,
29 more times
The primers used in PCR reactions are described in Table 3.7.
Table 3.7. Primer sequences.
PROBES PRIMER SEQUENCE
lyc.cyc., NEB1211x 5'dGTA.AAA.CGA.CGG.CCA.GT 3'
atpA, and cp3 CC33Y 5'CAG.GAA.ACA.GCT.ATG.ACC 3'
cob CC52Y 5'CTA.TTC.CGT.GTA.ATA.TTT.TGG 3'
CC53Y 5'ATG.ACT.ATA.AGG.AAC.CAA.CG 3'
coxl CC7 9 Y5'GGT.CCG.ATG.GCT.CTT.CTC 3'
CC80 Y 5'GAT.AGT.TGG.AAG.TTC.TCC 3'
cox11 CC81 Y5'GCG.GAA.CCA.TGG.CAA.TTA 3'
CC82 Y 5'GGC.ATG.ATT.AGT.TCC.ACT 3'
coxill CC66 Y5'GTA.GAT.CCA.AGT.CCA.TGG 3'
CC67 Y' 5'GCA.TGA.TGG.GCC.CAA.GTT 3'
X supplied by New England Biolabs Y'supplied by DNA Synthesis Core ICBR/University of Florida
NEB121l and CC33 hybridize to plasmid DNA sequences. In
contrast, CC52, CC53, CC79, CC8O, CC81, CC82, CC66 and CC67 primers were designed based on the mitochondrial cob (Dawson et al., 1984), coxI (Isaac et al., 1985), cox 11 (Moon et al., 1985) and cox1II (Malek et al., 1996) published
The following transformation protocol was used in Dr. Chase's lab to maintain the cytf and 26SrRNA cloned DNAs obtained from other investigators:
1. Thaw 100 ml of DH5alpha competent cells (Life
Technologies, Inc.) and mix gently.
2. Dispense to pre-chilled 4 ml snap-cap tubes.
3. Add 2ul of DNA and swirl gently to mix.
4. Incubate on ice for 30 min.
5. Heat pulse at 420C for 55 sec.
6. Add 400 ul room temperature LB (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride and 1.2% Difco agar), swirl gently to mix.
7. Spread over LB plates (with 0.1M IPTG, 20mg/ml
ampicillin and 2%w/v X-gal) for control over transformers.
8. Allow media to soak into plates. Incubate upside down at 37 0C overnight.
9. In theory, only the white colonies are transformed, but our experience showed that sometimes blue colonies might harbor the insert.
10. Select single white colonies (and occasionally
blue) and proceed to a plasmid prep, for DNA recovery.
1. Inoculate each clone into 3ml of LB plus ampicillin (20 mg/ml), at 37 cC, with continuous shaking, for 12-18 hours.
2. Prepare fresh triton lysis (lM Tris pH8, 0.2M EDTA pH8, 0.1% Triton X100 and 94% distilled water) and 10 mg of lysozyme/ml of ST (50mM Tris pH8, 25% sucrose in 100 ml of distilled water). Keep lysozyme on ice and triton lysis at room temperature.
3. Transfer 1.5ml of each culture to Eppendorf tubes.
4. Spin 45 sec. at 12,000 rpm in microfuge (Eppendorf 6415C).
5. Discard supernatant and drain tubes upside down.
6. Place tubes on ice and add 38 ul of ice-cold ST to each tube.
7. Vortex until the cells are completely resuspended.
8. Add 13 ul of fresh lysozyme solution to each tube.
9. Mix gently and incubate on ice for 5 min.
10. Add 32 ul of 0.2M EDTA, pH 8.
11. Mix gently and incubate on ice for 5 min.
12. Add 80ul of triton lysis buffer to each tube; tilt tube gently one time to mix.
13. Incubate on ice for 15 min.
14. Spin in microfuge top speed for 20 min.
15. Discard pellets and pour off supernatant into clean microfuge tube.
16. Add 160 ul of water-saturated phenol, close tubes and vortex off and on for 5 min.
17. Remove top phase to clean tube.
18. Add 16ul of 8M ammonium acetate and 320u1 of
absolute ethanol. Mix.
19. Incubate at -200C, overnight.
20. Spin 10 min in microfuge to pellet DNA.
21. Pour off supernatants and fill tubes with 70% ethanol; incubate on ice for 15 min.
22. Spin 5 min. in microfuge to repellet (cold room).
23. Pour off supernatant carefully.
24. Briefly air dry pellets, before resuspending in 60ul of sterile 0.1X NTE (0.01M Tris, 0.001M Na2EDTA, 0.01M NaCl, at pH 8) by pipetting.
25. Assay 20 ul of each prep by agarose gel electrophoresis.
26. Proceed to Insert Recovery.
All probes were recovered using the following "Insert Recovery" protocol:
1. Run digested clones or PCR products on a 0.8% agarose gel in TAE buffer (1X TAE = 0.04M Tris, 0.001M EDTA,
0.11 % glacial acetic acid, pH 7.8).
2. Stain gel for 10 minutes in ethidium bromide.
3. Transfer gel to light box, quickly excise bands and transfer to Eppendorf tubes (already perforated 5 times, in the bottom, with a 16 gauge needle).
4. Introduce each Eppendorf in a 5cc syringe, with large pieces of silanized glass wool packed in the bottom.
5. Transfer the whole apparatus (Eppendorf inside syringe)to a llcc dispo receiving tube.
6. Spin the dispo tubes in a SA600 rotor (Centrifuge Superspeed Sorvall RC2-B), 4,000 rpm, 10min.
7. Transfer liquid from bottom of receiving tube to an eppendorf tube.
8. Estimate volume. Add 1/10 volume of 8M ammonium acetate and 2 volumes of absolute ethanol. Mix well.
9. Incubate -200C overnight.
10. Pellet DNA in microfuge at top speed for 20 minutes in cold room.
11. Pour off supernatant, fill tube with 70% ethanol and incubate on ice for 10 minutes.
12. Repellet in microfuge at top speed for 5 min. in cold room.
13. Pull off the supernatant with a pipette. Repellet in microfuge for 30 seconds and pull remaining supernatant with a fine pipet tip.
14. Resuspend pellet in 20 ul of sterile distilled water.
15. Read the OD of a 1/50 dilution at 260nm and 280nm.
The progeny from Fls 33 and 44 from the sexual cross were analyzed with random amplified polymorphic DNA markers (RAPDs), in order to obtain confirmation of their zygotic origin. These analysis were performed by Mr. Huang Shu in Dr. Gmitter's lab at the CREC, Lake Alfred.
The components for the reactions and the PCR amplification program used for the RAPDs analysis were
described by Gmitter et al. (1996) The primer that best characterized the outcross progeny of Fls 33 and 44 was "H01" supplied by Operon Technologies; its sequence is 5'GGTCGGAGAA 3'
DNA Copy Number Analysis
Total DNA from callus, suspension, or leaves was used to estimate the organelle input from each kind of plant material used as a parental source in somatic hybridization experiments. RFLP analysis of these materials proceeded for a cybrid (Sour Orange + Cleopatra) and a somatic hybrid ('Succari'+ Atalantia ceylanica) combination. DNA samples of 15 ug were digested with restriction enzymes, fractioned by agarose gel electrophoresis, and transferred to nylon supports. The same blot was hybridized with radiolabeled lyc.cyc., cob and cytf probes. The citrus nuclear lyc.cyc. was used as control for the amount of total DNA loaded for each of the plant materials. The cob and cyt f clones were used to examine the mitochondria and chloroplast input, respectively. Densitometry analysis of each RFLP band, based on the "Integrated Density Value", was performed with an IS1000 Digital Imaging System (Alpha Innotech Corporation).
DNA Polymorphisms Among the Parents
To assess DNA polymorphisms between parents, total cellular DNA from each parent was digested and hybridized with two chloroplast probes, cytf and petD and six mitochondrial probes atpA, coxl, coxIlI, coxIII, 26SrRNA and atp9. Due to the fact that the male parent is no longer extant, two different varieties of P. trifoliata (Gainesville and Rubidoux) were examined in order to compare their banding patterns (Fig 4.1). No difference was observed between these two varieties with all the probe/restriction enzyme combinations, and Rubidoux was chosen as a DNA substitute for the male parent.
Most hybridization profiles revealed polymorphisms between the paternal (P. trifoliata) and maternal (LB 1-18) mtDNA (Table 4.1). In some cases, the P. trifoliata configurations were composed of abundant unique bands plus low abundance configurations matching those of LB 1-18.
Table 4.1. Probe/enzyme Combinations Demonstrating Polymorphisms Between Parents.
Hybridizations of the atp9 mitochondrial clone to
EcoRV, HindIII, BamHI, EcoRI and PstI digested DNAs did not reveal any useful polymorphism between the parents. An interesting pattern was observed when EcoRV digested DNAs were hybridized with this mitochondrial clone. There was a distinct 11 kb fragment characteristic of the male parent and a 14 kb fragment characteristic of the female parent. However, the 14 kb maternal fragment can be also visualized at low abundance in the male parent (Fig. 4.1).
Seven polymorhic probe-enzyme combinations were selected for use in this inheritance analysis. Table 4.2 presents the size of the paternal and maternal fragments, revealed by hybridization with each of the heterologous probes.
1 2 3
Fig 4.1. Autoradiograph of Southern blot showing mtDNA of LB 1-18 (maternal parent lane 3) and P. trifoliata (paternal parent Rubidoux lane 1 and 'Gainesville' lane 2). Total DNA was digested with EcoRV and hybridized to an atp9 coding region clone.
Table 4.2. Fragments Characteristic of the Female and Male Parents.
PROBE ENZYME P.trifoliata LB 1-18
atpA HindIII 4.3 kb 3.4 kb
coxl PstI 6.5 kb 10 kb
coxII EcoRI 16 and 2.6 kb 9 and 2.6 kb
coxIII HindIII 6 kb 4.5 kb
26SrRNA SmaI 10 and 4.5 kb 18 and 10 kb
cytf BamHI 10 kb 13 kb
petD HindIII 5 kb 11 and 9.5 kb
Mitochondrial DNA Inheritance
Some mitochondrial loci appeared to be biparentally inherited in the sexual cross. Hybridizations of the atpA mtDNA probe to HindIII digests of the 26 F1 plants showed the intense 3.4 kb maternal fragment in all the progeny plants. However, 17 F1 plants (Fls # 6, 7, 9, 10, 12, 13, 14, 15, 16, 18, 23, 27, 28, 30, 33, 36 and 44) showed a
faint 4.3 kb fragment characteristic of the paternal
P.trifoliata parent. Fig 4.2-A shows 14 of the 26 progeny, where 8 Fis (lanes 4,5,6,7,8,11,12,14) exhibited the paternal configuration. The cox-ll probe identified the 9.0 and 2.6 kb maternal fragments in EcoRI digests from all Fl plants. Again, the same 17 Fl hybrids carried a faint 16 kb paternal fragment (Fig 4.2B) A similar result was observed when total DNA of the 26 Fl hybrids was hybridized to the coxIII clone. All hybrids presented the strong maternal 4.5 kb fragment and the same 17 Fl hybrids presented a faint 6 kb fragment, characteristic of P. trifoliata (Fig 4.2C) In summary, the same F1 hybrids carried the paternal configurations for all three loci. These three loci appeared
to be "linked" in the same seventeen progeny that carried the P. trifoliata configurations, whereas the other 9
progeny carried none of the paternal configurations. The intergeneric hybrids are therefore segregating for the presence or absence of the paternal mtDNA configurations.
However, this pattern of inheritance was not observed for all mitochondrial loci. Additional results were
obtained using the same 26 hybrids and the coxl and 26.rRNA clones. CoxI identified only the maternal 10 kb fragment in these sexual hybrids, with no presence of the paternal 6.5 kb in any of the 26 sexual hybrids. In the Fig. 4.3A, the same 14 Fl hybrids showed in the previous figures presented
1 2 3 4 5 6 7 8 910111213141516 1 2 34 5 67 8 910111213141516
1 2 3 4 5 67 8 9 10111213141516
Fig 4.2. Paternal mtDNA configurations in the progeny of an intergeneric Citrus x Poncirus Cross. Autoradiographs of Southern blots showing mtDNA of 14 of the Fl hybrids of LB 1-18 (maternal parent lane 2) x P. trifoliata (paternal parent lane 1) F1 hybrids are shown in lanes 3-16
(hybrids # 3, 6, 7, 10, 15, 16, 19, 21, 27, 33, 35, 44, 48 and 55) Enzyme-probe combinations are as follows: panel A, HindIII-atpA; panel B, EcoRI-coxII; panel C, HindIII-coxIII. The arrows mark bands possibly transmitted to the progeny through the paternal parent.
a straight maternal inheritance of the coxl locus. A similar result was observed when total DNA of the 26 Fl hybrids was digested with SmaI and hybridized to the 26SrRNA clone. Only the maternal bands (10 and 18 kb) were present in the F1 hybrids (Fig 4.3B) Therefore, the observation of paternal mtDNA configurations was not necessarily true for all loci tested. However, because we are substituting P. trifoliata Rubidoux for the original paternal parent, we cannot exclude the possibility that this parent lacked the polymorphic bands observed in Rubidoux.
In order to assess the consequences of paternal mtDNA configurations for subsequent plant generations, the outcross progeny of F1 #'s 33 and 44 (which carried the paternal configurations) were analyzed using the mitochondrial atpA coding region clone. Two of six outcross progeny from Fl # 33 carried the 4.3 kb P. trifoliata configuration (Fig 4.4A). Three of six outcross Fl # 44 progeny have the 4.3 kb band (Fig 4.4B). Therefore, paternal mtDNA configurations persisted into the next generation.
RAPDs were the chosen method of analysis to quickly prove the zygotic origin of the outcross progeny from F1#33 and #44. These analysis were performed by Mr. Huang Shu,
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Fig 4.3. Autoradiograph of Southern blot showing mtDNA of the intergeneric hybrids of LB 1-18 (maternal parent lane 2) x P. trifoliata (paternal parent lane 1) Fourteen F1 hybrids are shown in lanes 3-16 (hybrids # 3, 6, 7, 10, 15, 16, 19, 21, 27, 33, 35, 44, 48 and 55). Enzyme-probe combinations are as follows: panel A, PstI-coxI; panel B, SmaI-26SrRNA.
1 234 5678 9
kb 4.3 3.4
1 2 34567 8 9
Fig 4.4. Autoradiograph of Southern blot showing mtDNA of outcross progeny from intergeneric hybrid #33 (panel A, lane 3) and #44 (panel B, lane 3). Six progeny are shown in lanes 4-9. LB 1-18 (grand maternal parent lane 2), P. trifoliata (grand paternal parent lane 1) Total DNA was digested with HindIII and hybridized to the atpA clone.
working in Dr. Fred Gmitter's lab at the UF/IFAS CREC at Lake Alfred,FL. Among all the primers tested, H01 was the one that gave the more conclusive results indicating the outcross progenies of F1 # 33 and # 44 as segregating zygotic populations.
Chloroplast DNA Inheritance
In contrast to mtDNA inheritance, chloroplast DNA appeared to exhibit strict maternal inheritance. The cytf probe detected the maternal 13 kb BamH1 fragment in all the 26 F1 progeny. No paternal (10 kb) fragments were observed in any of the hybrids (Fig 4.5). Only LB 1-18 configurations (9.5 and 11 kb fragments) were observed in all the F1 hybrids analyzed, after total DNA was digested and hybridized to the chloroplast petD clone. Although the lack of polymorphisms limited the number of loci that could be tested, the results indicated a maternal inheritance of chloroplasts. Again, we cannot exclude the possibility that the original paternal parent lacked the polymorphic bands observed in P. trifoliata Rubidoux.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
kb 11 9.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516
Fig 4.5. Maternal inheritance of chloroplast genome in the intergeneric hybrids. Autoradiograph of Southern blot showing chloroplast DNA of the intergeneric hybrids of LB 118 (maternal parent lane 2) x P. trifoliata (paternal parent lane 1). Fourteen F1 hybrids ( hybrids # 3, 6, 7, 10, 15, 16, 19, 21, 27, 33, 35, 44, 48 and 55) are shown in lanes 3-16 Enzyme-probe combinations are as follows: panel A, BamHl-cytf; panel B, HindIII-petD.
There was an apparent transmission of the mitochondrial atpA, coxII and coxIII paternal configurations to 17 of the 26 sexual hybrids of LB 1-18 x P. trifoliata. These paternal configurations also segregated in the outcross progeny of two F1 hybrids. However, there was an apparent straight maternal inheritance of the mitochondrial coxl and 26SrRNA in the 26 sexual hybrids. Apparent maternal inheritance of chloroplast DNA was observed in this sexual cross for the cytf and petD loci.
DNA Polymorphisms Distinguishing the Parents Organelle Genomes
The first objective was to find polymorphisms that would distinguish the two somatic fusion parents. This was problematic, especially concerning the chloroplast genome. Chloroplast gene coding probes (such as cytf and petD) did not show any polymorphisms among the somatic fusion parents. Longer chloroplast probes that included both coding and noncoding sequences were the solution, revealing polymorphisms among these parents. The mitochondrial atpA probe was used to evaluate the mtDNA inheritance in these hybrids. For chloroplast inheritance cpl, cp2 and cp3 were
the clones that revealed the necessary polymorphisms. In order to discriminate between somatic hybrids and cybrids, two genomic probes were used: 18SrRNA and lycopene cyclase (lyc.cyc). No genomic polymorphism was observed during this research for the 'Succari' + Atalantia ceylanica and the 'Hamlin' + 'Ponkan' combinations. However, Grosser et al. (1996b) had previously used RAPD markers to characterize the nuclear genome of the 'Succari' + A. ceylanica and the 'Succari' + Citropsis gilletiana fusion products. Tables 4.3, 4.4 and 4.5 present the sizes of the parental (leaf and embryogenic) DNA restriction fragments from the nuclear, mitochondrial, and chloroplast genomes, respectively.
Table 4.3. DNA Restriction Fragments Revealed after Hybridization with Nuclear-Encoded Probes.
PROBE ENZYME EMBRYOGENIC LEAF PARENT
lyc. cyc. EcoRV 'Willowleaf' 'Duncan'
(4.5 kb) (3 and 4 kb)
lyc. cyc. HindIII Swinglea Sour Orange
glutinosa (1.8 kb)
lyc. cyc. EcoRV 'Rohde Red 'Dancy' mandarin
Valencia' (5 kb)
(4.5 and 5 kb)
18SrRNA EcoRI 'Willowleaf' 'Valencia' sweet
(6.5 and 8 kb) (6.5 and 7.5 kb) 18SrRNA EcoRI Cleopatra Sour Orange
mandarin (6.5 and 9.5 kb)
(6.5 and 9.5 kb)kb) (6.5 and 8 kb)
Table 4.4. DNA Restriction Fragments Revealed by Hybridization with the Mitochondrial atpA Clone.
ENZYME EMBRYOGENIC PARENT LEAF PARENT
PstI 'Succari' sweet Citropsis gilletiana
orange (2 and 2.8 kb)
(2.5, 3.5, 6 and
PstI 'Succari' sweet Atalantia ceylanica
orange (14 and 5 kb)
(2.5, 3.5, 6 and
EcoRV 'Willowleaf' mandarin 'Duncan' grapefruit (16 kb) (5, 8, 22 and 25 kb)
HindIII Swinglea glutinosa Sour Orange
(2.5, 6.5, 8 and 11 (1, 6.5 and 8 kb) kb)
EcoRV 'Rohde Red Valencia' 'Dancy' mandarin
(4.5, 7, 19 and 20 (16 kb)
HindIII 'Willowleaf' mandarin 'Valencia' sweet (3.2 kb) orange
(2.5, 5.5, 6 and 9
HindIII 'Hamlin' sweet orange 'Ponkan' mandarin (2.5, 5.5, 6 and 9 (3.2 kb)
EcoRV Cleopatra mandarin Sour Orange
(4, 6, 6.5 and 8 kb) (4.5 and 7.5 kb)
Table 4.5. DNA Restriction Fragments Revealed by Hybridization with Chloroplast Encoded Clones.
PROBE ENZYME EMBRYOGENIC LEAF PARENT
cp2 HindIII 'Succari' sweet Citropsis
(5, 6, 8.5, 11 (5, 6, 7.5, 11 and 14 kb) and 14 kb)
cp3 PstI 'Succari' sweet Atalantia
(7 kb) (4.5 kb)
cpl BamHI 'Willowleaf' 'Duncan'
(4 and 5 kb) (3.5 and 5 kb)
cpl BamHI Swinglea Sour Orange
glutinosa (4 and 6 kb)
(4.5 and 6 kb)
cp3 PstI 'Rohde Red 'Dancy' mandarin
Valencia (1.5 and 4.5 kb) (6.5 kb)
cpl BamHI 'Willowleaf' 'Valencia' sweet
(4 and 5 kb) (3.5 and 5 kb)
cp3 PstI 'Hamlin' sweet 'Ponkan'
(7 kb) (4.5 kb)
cpl BamHI Cleopatra Sour Orange
mandarin (3.5 and 5 kb)
(4(3.5 and 5 kb) (4 and 5 kb)
Nuclear and Organelle Inheritance
Plants from the eight somatic fusion experiments were analyzed to determine their nuclear, mitochondrial and chloroplast genome composition. Results are provided in the following pages. When analyzing the results with respect to the nuclear genome, it is important to take into account that somatic hybrids are tetraploid and that cybrids are diploid plants. Therefore, considering the definition stated in Chapter 2, somatic hybrids should have all the parental configurations and cybrids should match one of the parental configurations. In terms of organelle inheritance, as reviewed in Chapter 2, there is a consensus in the literature that citrus somatic hybrids and cybrids inherit their mitochondrial genome from the embryogenic parent. In terms of the chloroplast genome, the results demonstrate a random pattern of inheritance.
'Succari'+ Citropsis gilletiana
All the hybrids produced in this combination were verified to be somatic hybrids by Grosser et al. (1996b), using RAPD markers. In terms of the mitochondrial genome (Fig 4.6), all the 14 somatic hybrids inherited their mtDNA from 'Succari' (embryogenic parent), although some "novel" (non parental) bands can be observed in DNA from some of the
1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16
1 2 3 4 5 6 7 8 9 10 11 12 13141516
Fig 4.6. Autoradiograph of Southern blot showing mtDNA (A) and chloroplast DNA (B) of the somatic hybrids of 'Succari' (embryogenic parent lane 1) + Citropsis gilletiana (leaf parent lane 2) Fourteen somatic hybrids are shown in lanes 3-16. Enzyme-probe combinations are as follows: panel A, PstI-atpA; panel B, HindIII-cp2.
regenerants (Fig 4.6A). A very interesting result was obtained in terms of the chloroplast genome inheritance in this combination: all the somatic hybrids presented both parental chloroplast genomes, which is a very unusual result (Fig 4.6B). The origin of the non-parental mitochondrial bands and the chloroplast inheritance in this combination will be discussed further in Chapter 4.
'Succari' + Atalantia ceylanica
Analysis of the 'Succari' + A. ceylanica combination with the lyc.cyc. and 26SrRNA clones and more than 6 different restriction enzymes revealed no genomic polymorphism between the parents. However, the 14 hybrids analyzed in this research (Fig 4.7) were verified to be somatic hybrids by Grosser et al. (1996b) using RAPDs. Again the mtDNA was inherited from the embryogenic parent, with the presence of "novel" bands in some of the somatic hybrids. However, these non-parental bands may be due, in part, to incomplete DNA digestion. The chloroplast genome was also inherited from the embryogenic parent (although, in this case, only seven of the 14 somatic hybrids were analyzed).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 2 3 4 5 6 7 8 9
Fig 4.7. Autoradiograph of Southern blot showing mtDNA (A) and chloroplast DNA (B) of the somatic hybrids of 'Succari' (embryogenic parent lane 1) + Atalantia ceylanica (leaf parent lane 2). Somatic hybrids are shown in lanes 3-16 (panel A) and 3-9 (Panel B) Enzyme-probe combinations are as follows: panel A, PstI-atpA; panel B, PstI-cp3.
'Willowleaf' + 'Duncan'
The four products of the 'Willowleaf' + 'Duncan' fusion were confirmed cybrids when the lyc.cyc. genomic probe was used. The cybrids RFLP pattern matched the leaf parent's ('Duncan') configuration, not presenting the 4.5 kb band characteristic of the 'Willowleaf' (embryogenic parent) genome. The mitochondrial genome was inherited from 'Willowleaf', again with the presence of a "novel band" (9.5 kb). The chloroplast genome in this cybrid combination was inherited in a random fashion, with 1 cybrid presenting the 'Willowleaf' chloroplast pattern and 3 cybrids inheriting the 'Duncan' chloroplast genome (Fig. 4.8).
Swinglea glutinosa + Sour Orange
Three putative cybrids of S. glutinosa + Sour Orange were analyzed in terms of their nuclear, mitochondrial and chloroplast genomes (Fig 4.9). They presented the Sour Orange (leaf parent) genomic configuration, without the 3.5 kb band characteristic of S. glutinosa (embryogenic parent), as expected in cybrid plants. The mitochondrial genome was apparently inherited from the embryogenic parent. However, the presence in the cybrids of multiple non-parental bands resulted in difficulty of reaching an accurate interpretation. Apparently the cybrids inherited the 8, 6.5
kb kb kb
1 2 3 4 56 1 23 4 56
1 2 3 4 5 6
Fig 4.8. Autoradiograph of Southern blot showing genomic DNA
(A), mtDNA (B) and chloroplast DNA (C) of the cybrids of 'Willowleaf' (embryogenic parent lane 1) + 'Duncan' (leaf parent lane 2) Four cybrids are shown in lanes 3-6. Enzyme-probe combinations are as follows: panel A, EcoRVlyc.cyc.; panel B, EcoRV-atpA; panel C, BamHI- cpl.