Mitochondrial DNA rearrangements in somatic hybrids of Solanum tuberosum and Solanum brevidens

Summary Thirty somatic hybrids between Solanum tuberosum and Solanum brevidens were analysed for mitochondrial and chloroplast genome rearrangements. In all cases, the chloroplast genomes were inherited from one of the parental protoplast populations. No chloroplast DNA alterations were evident but a range of mitochondrial DNA alterations, from zero to extensive intra- and inter-molecular recombinations, were found. Such recombinations involved specific recombination hot spots in the mitochondrial genome. Not all hybrids regenerated from a common callus possessed identical mitochondrial genomes, suggesting that sorting out of mitochondrial populations in the callus may have been incomplete at the plant regeneration stage. Sorting out of organelles in planta was not observed.     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Analysis of chloroplast and mitochondrial DNAs in asymmetric somatic hybrids between tobacco and carrot

Summary Chloroplast and mitochondrial DNAs have been examined by comparison of restriction enzyme patterns in asymmetric hybrid plants, resulting from the fusion between leaf mesophyll protoplasts of Nicotiana tabacum (Solanaceae), and irradiated cell culture protoplasts of Daucus carota (Umbellifereae). These somatic hybrids with normal tobacco morphology were selected as a consequence of the transfer of methotrexate and 5-methyltryptophan resistance from carrot to tobacco. The restriction patterns of chloroplast DNAs in somatic hybrids were indistinguishable from the tobacco parent. However, we found somatic hybrids with mitochondrial DNA significantly different from either parent, as judged by analysis of fragment distribution after restriction enzyme digestion. The possible formation of altered mitochondrial DNA molecules as the result of parasexual hybrid production between two phylogenetically highly divergent plant species will be discussed.     

Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum

The chloroplast (cp) DNA type and mitochondrial (mt) DNA composition of 17 somatic hybrids between a cytoplasmic albino tomato and monoploid potato (A7-hybrids) and 18 somatic hybrids between a nitrate reductase-deficient tomato and monoploid potato (C7-hybrids) were analyzed. Thirteen A7-hybrids and 9 C7-hybrids were triploids (with one potato genome); the other hybrids were tetraploid. As expected, all A7-hybrids contained potato cpDNA. Of the C7-hybrids 7 had tomato cpDNA, 10 had potato cpDNA and 1 hybrid contained both tomato and potato cpDNA. The mtDNA composition of the hybrids was analyzed by hybridization of Southern blots with four mtDNA-specific probes. The mtDNAs in the hybrids had segregated independently from the cpDNAs. Nuclear DNA composition (i.e. one or two potato genomes) did not influence the chloroplast type in the C7-hybrids, nor the mtDNA composition of A7- or C7-hybrids. From the cosegregation of specific mtDNA fragments we inferred that both tomato and potato mtDNAs probably have a coxII gene closely linked to 18S+5S rRNA genes. In tomato, atpA, and in potato, atp6 seems to be linked to these mtDNA genes.     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

Isolation and characterization of intergeneric somatic hybrids in the Apocynaceae family

Summary Protoplasts were isolated from callus cultures of Rauwolfia serpentina Benth., Rhazya stricta Decaisne, and Catharanthus roseus (L.) G. Don, or from leaves of Vinca minor L. Protoplast isolation, culture, and fusion techniques as well as hybrid screening systems were developed for these species, and hybrids were obtained. Hybrid combinations were Rauwolfia + Vinca, Rauwolfia + Catharanthus, Rauwolfia + Rhazya, and Catharanthus + Vinca. For hybrid isolation, the physiological complementation method was utilized. Analyses of the material obtained included a cytogenetic study of the chromosomes, a study of multiple molecular forms of transferases and esterases, and the blot hybridization of restricted nuclear DNA using ribosomal DNA as a probe. Hybrids were identified in all species' combinations tried. A ten-fold increase in the accumulation of raucaffricine (relative to the parental Rauwolfia strain) was observed in one cell line of the Rauwolfia + Vinca hybrid. Our studies indicated the genetic stability of the great majority of the hybrid cell lines over a period of more than 20 months of in vitro growth. No shoot morphogenesis has so far been observed in this material.     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Interspecific somatic hybrids between wild potato Solanum acaule Bitt. and anther-derived dihaploid potato (Solanum tuberosum L.)

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition. Received: 19 January 1998 / Revision received: 27 March 1998 / Accepted: 20 April 1998     

Absence of mitochondrial and chloroplast DNA recombinations in Brassica napus plants regenerated from protoplasts,protoplast fusions and anther culture

Summary Over 400 Brassica napus plants regenerated from individual protoplasts, from protoplast fusions and from anther culture were analysed for chloroplast and mitochondrial genome rearrangements by restriction fragment length polymorphisms. None were detected, attesting to the fidelity of the tissue culture procedures employed. In the majority of protoplast fusion products, the cytoplasmic organelles had completely sorted out at the callus stage but three regenerated plants possessed mixed parental populations of mitochondrial genomes and one regenerant contained mixed chloroplast genomes. In all four examples, the cytoplasmic genome sorted out in planta in favor of one parental type which was faithfully maternally transmitted to progeny.     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

A variant mitochondrial DNA arrangement specific toPetunia stable sterile somatic hybrids

We have characterized two related regions of twoPetunia mitochondrial genomes in order to understand how plant mt genomes from a cytoplasmic male sterile (cms) line and a fertile line diverge from one another. Restriction maps of these regions indicate that a sequence arrangement shared by the two genomes adjoins sequences which are not shared at the corresponding locations in the two genomes. A point where the mt genomes from the cms line and the fertile lines diverge from each other was identified and mapped. Previously we had observed that somatic hybrids constructed from the cms and the fertile line contained mt genomes carrying new combinations of parental mtDNA restriction fragments (3). Using the restriction maps of the two related mtDNA regions, a mtDNA arrangement unique to the cms parent could be shown to be present in all 17 stable sterile somatic hybrids tested and none of the 24 stable fertile somatic hybrids tested. This data does not exclude the possibility that additional, as yet unidentified, mtDNA arrangements unique to the cms parent might also be found exclusively in sterile somatic hybrids. Whether or not the sterile parental mtDNA arrangement reported here is functionally related to cms, it apparently segregates with cms in somatic hybrids.     

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia

Summary Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.     

Selection of a universal hybridizer in Sinapis turgida Del. and regeneration of plantlets from somatic hybrids with Brassica species

A double-mutant cell line, which was unable to grow in a medium with NO ₃ ^- as the sole nitrogen source and was resistant to 5-methyl-tryptophan (5MT), was selected from cell suspensions of Sinapis turgida Del. (Brassicaceae) by culturing the cells in AA medium (Toriyama and Hinata, 1985, Plant Sci. 41, 179–183) supplemented with 50 mM chlorate and 229 M 5MT. Protoplasts of this cell line were fused with mesophyll protoplasts of Brassica oleracea L. with dextran, and six somatic hybrids were selected initially by culture in the NO ₃ ^- medium and then by transfer to the NO ₃ ^- medium supplemented with 229 M 5MT. The somatic hybrids produced embryoids, leaves and plantlets on a regeneration medium. The hybrid characters were confirmed by investigations of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) isoenzymes, chromosome number, growth on NO ₃ ^- medium, 5MT resistance, and capacity to regenerate plants. Somatic hybrids between S. turgida Del. and B. nigra (L.) Koch were also obtained using this method. These results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.Abbreviations B5 medium of Gamborg et al. (1968) - MS medium of Murashage and Skoog (1962) - 5MT 5-thethyltryptophan     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

Chloroplast DNA assorts randomly in intraspecific somatic hybrids of Nicotiana debneyi

Summary Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi. The two genotypes differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase (Pgm) isozymes. There was no conscious selection for hybrid genotypes during protoplast culture or plant regeneration. Among 225 plants screened for Pgm, six were identified as nuclear hybrids. Restriction endonuclease and filter hybridisation analysis revealed that the cytoplasms of the hybrids contained one or other but never both parental chloroplast DNAs. The sorting out of chloroplasts was random and complete; the limit of detecting a rare chloroplast-DNA type in a mixture was 0.1%.     

Non-random inheritance of organellar genomes in symmetric and asymmetric somatic hybrids between Lycopersicon esculentum and L. pennellii

Summary The organization of the mitochondrial genome and the genotype of the chloroplast genome was characterized using restriction fragment length polymorphisms in a population (82 individuals) of symmetric and asymmetric somatic hybrids of tomato. The protoplast fusion products were regenerated following the fusion of leaf mesophyll protoplasts of Lycopersicon esculentum (tomato cv UC82) with suspension cell protoplasts of L. pennellii that had been irradiated with 5, 10, 15, 25, 50, or 100 kRads from a gamma source. The chloroplast genome in the somatic hybrids showed a random pattern of inheritance, i.e., either parental genome was present in equal numbers of regenerants, while in asymmetric somatic hybrids, the chloroplast genotype reflected the predominant nuclear genotype, i.e., tomato. The mitochondrial genome in the symmetric somatic hybrids showed a non-random pattern of inheritance, i.e., predominantly from the L. pennellii parent; asymmetric somatic hybrids had more tomato-specific mitochondrial sequences than symmetric somatic hybrids. The non-random inheritance of the chloroplast and mitochondrial DNA in these tomato protoplast fusion products appears to be influenced by the nuclear background of the regenerant.     

Non-random chloroplast segregation inNicotiana tabacum (+)N. rustica somatic hybrids selected by dual nuclear-encoded resistance

Nicotiana tabacum (+)N. rustica interspecific somatic hybrids were produced by fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistantNicotiana tabacum L. with leaf mesophyll protoplasts of transgenic kanamycin-resistantN. rustica L. Somatic hybrids were selected on the basis of resistance to both methotrexate and kanamycin. Evidence for nuclear hybridization was obtained for 21 hybrids by restriction-fragment-length-polymorphism (RFLP) analysis using a heterologous wheat nuclear ribosomal-DNA (rDNA) probe and by analysis of glutamate-oxaloacetate transaminase (GOT) isoenzymes. Chloroplasts segregated non-randomly as 20 of the somatic hybrids possessedN. rustica chloroplasts and only one hadN. tabacum chloroplasts. Patterns of mitochondrial inheritance were examined by hybridization of a heterologous wheat cytochrome oxidase subunit II (coxII) gene with genomic DNA of the somatic hybrids. Four somatic hybrids with hybridization patterns similar toN. rustica and 17 with hybridization patterns consistent with mitochondrial DNA (mtDNA) rearrangement or recombination were obtained. None of the somatic hybrids had patterns ofcoxll hybridization identical withN. tabacum. Male-fertility levels in the hybrids ranged from undetectable to 87% and only nine hybrids produced a limited amount of viable seed. There was no apparent correlation between the patterns of organelle inheritance in the somatic hybrids and the relative degree of fertility.Contribution No. 1439 Plant Research CentreCurrent address: Plant Biotechnology Institute, National Research Council, 110 Gymmasium Road, Saskatoon, Saskatchewan S7N OW9, Canada