Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 × 10⁸ protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2–5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 × 10⁸ protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2–5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.     

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.     

Plant mitochondrial DNA evolved rapidly in structure,but slowly in sequence

Summary We examined the tempo and mode of mitochondrial DNA (mtDNA) evolution in six species of crucifers from two genera,Brassica andRaphanus. The six mtDNAs have undergone numerous internal rearrangements and therefore differ dramatically with respect to the sizes of their subgenomic circular chromosomes. Between 3 and 14 inversions must be postulated to account for the structural differences found between any two species. In contrast, these mtDNAs are extremely similar in primary sequence, differing at only 1–8 out of every 1000 bp. The point mutation rate in these plant mtDNAs is roughly 4 times slower than in land plant chloroplast DNA (cpDNA) and 100 times slower than in animal mtDNA. Conversely, the rate of rearrangements is extraordinarily faster in plant mtDNA than in cpDNA and animal mtDNA.     

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

Summary The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using ³²P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.     

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.     

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Summary Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km⁺Sr⁺ with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts.     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and Solatium rickii

Summary A single somatic hybrid callus clone was identified following the fusion of Lycopersicon esculentum protoplasts and Solanum rickii suspension culture protoplasts. The hybrid nature of the callus and the plants regenerating from it was determined by assaying phosphoglucomutase-2 isozyme expression. The chloroplast genome present in four somatic hybrid plants was characterized by probing digests of total DNA with nick translated L. esculentum chloroplast DNA(cpDNA). All four somatic hybrid plants had inherited S. rickii cpDNA. Two clones of plant mitochondrial DNA (mtDNA), soybean 18S and 5S rDNA and maize cytochrome oxidase subunit II were used to characterize the mtDNA present in total DNA digests of four somatic hybrid plants. In both cases, the somatic hybrid plants had inherited most but not all of the S. rickii specific fragments, but none of the L. esculentum specific fragments.     

Natural hybridization of Yezo and Sakhalin spruce in central Hokkaido,revealed by DNA markers with contrasting modes of inheritance

Yezo spruce (Picea jezoensis var. jezoensis) and Sakhalin spruce (Picea glehnii) occur across Hokkaido and co‐occur in some forest habitats. This leads to the potential for natural hybridization between these two species, which has been shown to occur at low frequencies. The purpose of this study was to identify these hybrids and their possible mating patterns, using various Pinaceae DNA markers with different modes of inheritance. The markers used were maternally inherited mitochondrial DNA (mtDNA), paternally inherited chloroplast DNA (cpDNA) and biparentally inherited nuclear microsatellites (nSSRs). Seven putative natural hybrids, four artificially‐crossed F₁ hybrids, four parent plants from each species, and two artificially‐backcrossed hybrids of putative natural hybrids and their parents were analyzed using the diagnostic DNA markers developed in this study. We found Yezo spruce and Sakhalin spruce to be distinct (J and G types, respectively), and the modes of inheritance held true for the two species, as was previously reported to be the case in Pinaceae. Four of the seven putative natural hybrids harbored J‐type cpDNA, G‐type mtDNA and J/G‐type nSSRs, indicating that natural F₁ hybrids are likely to arise from a G (female) × J (male) crossing. One natural hybrid harbored G‐type cpDNA, J‐type mtDNA and J/G‐type nSSRs, which implies that hybrids produced by J (female) × G (male) crossings occur at low frequencies. The two remaining hybrids harbored J‐type cpDNA and mtDNA with either J/G or J/J‐type nSSRs, suggesting that they may be F₂ hybrids resulting from backcrossing between an F₁ hybrid and a Yezo spruce.     

Organelle DNA variation in parental<Emphasis Type="Italic"> Solanum</Emphasis> spp. genotypes and nuclear-cytoplasmic interactions in<Emphasis Type="Italic"> Solanum tuberosum</Emphasis> (+)<Emphasis Type="Italic"> S. commersonii</Emphasis> somatic hybrid-backcross progeny

Nuclear-cytoplasmic interactions can influence fertility and agronomic performance of interspecific hybrids in potato as well as other species. With the aim of assessing the potential value of a novel recombinant cytoplasm derived by interspecific somatic hybridization, backcross progeny were produced by crossing a somatic hybrid between Solanum tuberosum (tbr) and the wild incongruous species S. commersonii (cmm) with various potato clones. BC₁ clones were evaluated for male fertility and other agronomic traits. Male fertility clearly depended on the cross direction and the cytoplasm source. Genotypes with cytoplasms sensitive to nuclear genes derived from Solanum commersonii and inducing male sterility showed identical mtDNA composition, as based on mtDNA analyses with various PCR-based and RFLP markers. On the other hand, genotypes with cytoplasms not inducing male sterility in the presence of the cmm nuclear genes showed a different mtDNA organisation. Analysis of cpDNA confirmed similarity of cytoplasmic composition in CMS-inducing genotypes and clear differences with the others. Genotypes with recombinant cytoplasm induced by somatic hybridization generally showed similar agronomic performances in reciprocal hybrids with tbr cytoplasm, suggesting that the novel cytoplasm can be used in potato breeding.Contribution no. 24 from the Institute of Plant Genetics, Research Division of Portici     

Comparison of the organization of the mitochondrial genome in tomato somatic hybrids and cybrids

Summary The organization of the mitochondrial genome in somatic hybrids and cybrids regenerated following fusion of protoplasts from cultivated tomato, Lycopersicon esculentum, and the wild species, L. Pennellii, was compared to assess the role of the nuclear genotype on the inheritance of organellar genomes. No organellar-encoded traits were required for the recorvery of either somatic hybrids or cybrids. The organization of the mitochondrial genome was characterized using Southern hybridization of restriction digestions of total DNA isolated from ten cybrids and ten somatic hybrids. A bank of cosmid clones carrying tomato mitochondrial DNA was used as probes, as well as a putative repeated sequence from L. pennellii mitchondrial DNA. The seven cosmids used to characterize the mitochondrial genomes are predicted to encompass at least 60% of the genome. The frequency of nonparental organizations of the mitochondrial genome was highest with a probe derived from a putative repeat element from the L. pennellii mitochondrial DNA. There was no difference in the average frequency of rearranged mitochondrial sequences in somatic hybrids (12%) versus cybrids (10%), although there were individual cybrids with a very high frequency of novel fragments (30%). The frequency of tomato-specific mtDNA sequences was higher in cybrids (25%) versus somatic hybrids (12%), suggesting a nuclear-cytoplasmic interaction on the inheritance of tomato mitochondrial sequences.     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

Differential organellar inheritance in Passiflora’s (Passifloraceae) subgenera

Four chloroplast (cp), one mitochondrial (mt), and one ribosomal nuclear (ITS) DNA regions were studied in four artificial and one natural interspecific Passiflora hybrids. The ITS results confirmed their hybrid origin and all mtDNAs were maternally inherited. The same, however, was not true for cpDNA. The four hybrids (three artificial and one natural) derived from species of the Passiflora subgenus showed a cpDNA paternal inheritance, while the one involving taxa of the Decaloba subgenus gave evidence of maternal transmission. These results are of significance for the ongoing studies which are being performed on the molecular evolution of this genus and furnish important background for investigations aimed at clarifying the factors which determine cpDNA inheritance.     

Transmission of organelle genomes in citrus somatic hybrids

Restriction fragment length polymorphisms (RFLPs), were used to analyze the organelle composition of two-year-old trees, recovered from two different experiments: protoplasts from embryogenic cell suspensions of `Succari' sweet orange (C. sinensis L. Osbeck) were fused with leaf protoplasts of Citropsis gilletiana Swingle & M. Kell or to leaf protoplasts of Atalantia ceylanica(Arn.) Oliv. The somatic hybrids of both fusion combinations had the mitochondrial genome from the embryogenic partner. In some somatic hybrids, non-parental fragments were observed among the mitochondrial patterns. Somatic hybrids between `Succari' + Atalantia had plastid DNA from the embryogenic parent, while the somatic hybrids of `Succari' + Citropsis all had both parental chloroplast genomes. The relative abundance of organelle DNAs in the donor embryogenic and leaf cells may explain the consistent transmission of the embryogenic parent mitochondrial DNA and the inheritance of the chloroplast genome from either parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

RFLP analysis and genomic in situ hybridization (GISH) in somatic hybrids and their progeny between Lycopersicon esculentum and Solanum lycopersicoides

 RFLP (restriction fragment length polymorphism) and GISH (genomic in situ hybridization) analyses were employed to identify the chloroplast and nuclear genomes of the somatic hybrids and progeny between tomato ‘Ohgata zuiko’ and Solanum lycopersicoides (‘LA 2386’). A random distribution of the chloroplast genotype was determined using a cloned 19.6-kb BamHI fragment (Ba1) of tobacco chloroplast DNA. Eight selected hybrids were analyzed for their chromosomal compositions; 4 were tetraploids (2n=48) with an equal number of chromosomes derived from each parent as accurately determined by GISH, and the other 4 were hexaploids, containing an average of two sets of tomato chromosomes and one set from the wild parent. RFLP analysis with six tomato nuclear probes of known chromosomal locations revealed no major variation among the 44 hybrid plants surveyed. However, it also showed the presence of both parent-specific alleles and the loss of some and the presence of a few non-parental alleles, indicating rearrangement and/or recombination of the nuclear DNA. The relevance of the molecular and cytological methods and the potential use of somatic hybrids for plant breeding are demonstrated. Received: 20 July 1997 / Accepted: 6 October 1997     

Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

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.