Correction of nitrate reductase defect in auxotrophic plant cells through protoplast-mediated intergeneric gene transfers

Summary Nitrate reductase-deficient cells of Nicotiana tabacum cv Gatersleben (coded cnx-68) lacking active molybdenum-cofactor were corrected by introducing the genes from Physalis minima and Datura innoxia into NR^- genomes. In these itergeneric reconstruction experiments, X-irradiated inactive mesophyll protoplasts of Physalis and Datura were fused separately with the cultured cell protoplasts of cnx-68 Nicotiana. A total of 45 cell colonies, 37 transformed by Physalis and 8 by Datura, were selected from about 1.7×10³ heteroplasmic fusion products. The selection of transformants was made by their ability to grow on a medium containing nitrate as the sole nitrogen source. Some of these transformants were further characterized with respect to nitrate reductase, xanthine dehydrogenase and glutamate dehydrogenase activities, chlorate sensitivity, and chlorophyll synthesis. The restoration of nitrate reductase and xanthine dehydrogenase activities confirm the presence of an active form of the molybdenum-cofactor by the expression of introduced genes of Physalis and Datura into the genome of cnx-68 Nicotiana. Such stable transformations via fusion of normal and highly irradiated protoplasts may have a considerable application in higher plants for introducing desirable characters from diverse genomes.     

Metabolic complementation for a single gene function associated with partial and total loss of donor DNA in interspecific somatic hybrids

Summary We report here on the obtainment of interspecific somatic, asymmetric, and highly asymmetric nuclear hybrids via protoplast fusion. Asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from a nitrate reductase-deficient cofactor mutant of N. plumbaginifolia with irradiated (100 krad) kanamycin resistant leaf protoplasts of a haploid N. tabacum. Selection for nitrate reductase (NR) and/or kanamycin (Km) resistance resulted in the production of three groups of plants (NR⁺, NR⁺, Km^R, and NR^-Km^R). Cytological analysis of some hybrid regenerants showed the presence of numerous tobacco chromosomes and chromosome fragments, besides a polyploid N. plumbaginifolia genome (tetra or hexaploid). All the regenerants tested were male sterile but some of them could be backcrossed to the recipient partner. In a second experiment, somatic and highly asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from the universal hybridizer of N. plumbaginifolia with suspension protoplasts of a tumor line of N. tabacum. Selection resulted in two types of colonies: nonregenerating hybrid calli turned out to be true somatic hybrids, while cytological analysis of regenerants obtained on morphogenic calli did not show any presence of donor-specific chromosomes. Forty percent of the hybrid regenerants were completely fertile, while the others could only be backcrossed to the recipient N. plumbaginifolia. Since the gene we selected for is not yet cloned, we were not able to demonstrate the transfer of genetic material at the molecular level. However, since no reversion frequency for the nitrate reductase mutant is known, and due to a detailed cytological knowledge of both fusion partners, we feel confident in speculating that intergenomic recombination between N. plumbaginifolia and N. tabacum has occurred.     

Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion

Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, ‘Telstar’, were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.     

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.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Intergeneric somatic hybridization and its application to crop genetic improvement

Related or distant species of cultivated cs are a large pool of many desirable genes. Gene transfer from these species through conventional breeding is difficult owing to post- and pre-zygotic sexual incompatibilities. Somatic hybridization via protoplast fusion is a possible alternative for gene transfer from these species to cultivated crops. Since the early days of somatic hybridization many intergeneric somatic hybrids have been developed through symmetric fusion, asymmetric fusion and microfusion. Somatic hybrids are mainly selected by using markers such as specific media or fusion parents with special features, biochemical mutants, antibiotic resistance and complementation strategy. The hybridity of the regenerants is determined based on morphological, cytological and molecular analysis. The inheritance patterns of nuclear and cytoplasmic genomes in the somatic hybrids are diverse. Nuclear DNA from both fusion parents co-exists congruously in some hybrids with translocation and rearrangement of chromosomes, but spontaneous elimination of chromosomes from either or both fusion parents has been observed very often. In asymmetric fusion, chromosome elimination is an important issue that is a complicated process influenced by many factors, such as irradiation dose, phylogenetic relatedness, ploidy level of fusion parent and regenerants. As for chloroplast genome, uniparental segregation is mainly detected, though co-existence is also reported in some cases. The mitochondrial genome, in contrast to chloroplast, undergoes recombination and very frequent rearrangements. Somatic cell fusion has potential applications for crop genetic improvement by overcoming sexual incompatibility or reproductive barriers, and by realizing novel combinations of nuclear and/or cytoplasmic genomes.     

Effect of radiation dose on the production of and the extent of asymmetry in tomato asymmetric somatic hybrids

Summary Asymmetric somatic hybrids were recovered following fusion of tomato leaf mesophyll protoplasts with irradiated protoplasts isolated from Lycopersicon pennellii suspension cells. The asymmetry was determined by scoring the regenerants at between 20 and 24 loci using isozymes and restriction fragment length polymorphisms. In addition, three quantitative traits, fruit size, leaf shape, and stigma exsertion, were measured in the regenerants. The recovery of asymmetric somatic hybrids was as high as 50% of the regenerants, and there was no requirement for the transfer of a selectable marker gene from the irradiated partner. The amount of nuclear DNA transferred from the irradiated protoplast fusion partner was found to be inversely proportional to the radiation dose. It was possible to recover tomato asymmetric somatic hybrids which were self-fertile and contained limited amounts of genetic information from L. pennelli.     

Production of intertribal somatic hybrids between Brassica napus L. and Lesquerella fendleri (Gray) Wats

Intertribal Brassica napus (+) Lesquerella fendleri hybrids have been produced by polyethylene glycol-induced fusions of B. napus hypocotyl and L. fendleri mesophyll protoplasts. Two series of experiments were performed. In the first, symmetric fusion experiments, protoplasts from the two materials were fused without any pretreatments. In the second, asymmetric fusion experiments, X-ray irradiation at doses of 180 and 200 Gy were used to limit the transfer of the L. fendleri genome to the hybrids. X-ray irradiation of L. fendleri mesophyll protoplasts did not suppress the proliferation rate and callus formation of the fusion products but did significantly decrease growth and differentiation of non-fused L. fendleri protoplasts. In total, 128 regenerated plants were identified as intertribal somatic hybrids on the basis of morphological criteria. Nuclear DNA analysis performed on 80 plants, using species specific sequences, demonstrated that 33 plants from the symmetric fusions and 43 plants from the asymmetric fusions were hybrids. Chloroplast and mitochondrial DNA analysis revealed a biased segregation that favoured B. napus organelles in the hybrids from the symmetric fusion experiments. The bias was even stronger in the hybrids from the asymmetric fusion experiments where no hybrids with L. fendleri organelles were found. X-ray irradiation of L. fendleri protoplasts increased the possibility of obtaining mature somatic hybrid plants with improved fertility. Five plants from the symmetric and 24 plants from the asymmetric fusion experiments were established in the greenhouse. From the symmetric fusions 2 plants could be fertilised and set seeds after cross-pollination with B. napus. From the asymmetric fusions 9 plants could be selfed as well as fertilised when backcrossed with B. napus. Chromosome analysis was performed on all of the plants but 1 that were transferred to the greenhouse. Three plants from the symmetric fusions contained 50 chromosomes, which corresponded to the sum of the parental genomes. From the asymmetric fusions, 11 hybrids contained 38 chromosomes. Among the other asymmetric hybrids, plants with 50 chromosomes and with chromosome numbers higher than the sum of the parental chromosomes were found. When different root squashes of the same plant were analysed, a total of 6 plants were found that had different chromosome numbers.     

Cybridization in Nicotiana tabacum L. using double inactivation of parental protoplasts and post-fusion selection based on nuclear-encoded and chloroplast-encoded marker genes

An effective selection system preceded by double inactivation of parental protoplasts was used to transfer Nicotiana suaveolens Leh. cytoplasmic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cultivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens plants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inactivated with iodoacetamide or rhodamine 6G, while those of the cytoplasm donor were inactivated by X-irradiation. The resultant microcalli were cultured on a shoot regeneration medium containing both kanamycin and spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants, out of a total of 44 regenerated plants transferred to the glasshouse, were obtained. Intraspecific somatic transfers of the CMS trait between N. tabacum cultivars with distinctlydifferent morphologies using single inactivation and nonselective shoot regeneration medium were demonstrated. The implications of the results for practical tobacco breeding as a means of circumventing lengthy backcrossing procedures are discussed.     

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

Somatic hybrids with substitution type genomic configuration TCBB for the transfer of nuclear and organelle genes from Brassica tournefortii TT to allotetraploid oilseed crop B. carinata BBCC

Oilseed crop Brassica carinata BBCC is a natural allotetraploid of diploid species B. nigra BB and B. oleracea CC. To transfer the nuclear and organelle genes in a concerted manner from an alien species, B. tournefortii TT, to B. carinata, we produced somatic hybrids with genomic configuration TCBB using B. nigra and B. oleracea stocks that carried selectable marker genes. B. tournefortii TT was sexually crossed with hygromycin-resistant B. oleracea CC. Protoplasts isolated from shoot cultures of hygromycin-resistant F₁ hybrids of B. tournefortiixB. oleracea TC were fused with protoplasts of kanamycin-resistant B. nigra BB. In two different fusion experiments 80 colonies were obtained through selection on media containing both hygromycin and kanamycin. Of these, 39 colonies regenerated into plants. Analysis of 15 regenerants by random amplified polymorphic DNA (RAPD) markers showed the presence of all three genomes, thereby confirming these to be true hybrids. Restriction fragment length polymorphism (RFLP) analysis of organelle genomes with heterologous chloroplast (cp)and mitochondrial (mt) DNA probes showed that the chloroplast genome was inherited from either of the two parents while mitochondrial genomes predominantly showed novel configurations due to either rearrangements or intergenomic recombinations. We anticipate that the TCBB genomic configuration will provide a more conducive situation for recombination between the T and C genomes during meiosis than the TTCCBB or TCCBB type configurations that are usually produced for alien gene transfer. The agronomic aim of producing TCBB hybrids is to transfer mitochondrial genes conferring cytoplasmic male sterility and nuclear genes for fertility restoration from B. tournefortii to B. carinata.     

Chromosomes in somatic hybrids between Nicotiana plumbaginifolia and a monoploid potato

Summary Leaf mesophyll protoplasts of the monohaploid potato (Solanum tuberosum L.) clone H7322 were fused with callus protoplasts of nitrate reductase deficient (NR^–) mutants Cnx 20 and NA 36 of Nicotiana plumbaginifolia. Somatic hybrid lines were selected for nitrate reductase proficiency. All callus lines tested appeared to be stable for the retention of the potato chromosome carrying the compensating NR gene when grown for over 1.5 years in the absence of nitrate. Shoots were regenerated from six different fusion lines of Cnx 20 + H7322 24 months after fusion. Chromosomal analysis in callus cultures revealed that in both fusion combinations 40–120 N. plumbaginifolia chromosomes were present, as were 9–20 potato chromosomes. Cells with 17 potato chromosomes in combination with a relatively small number (31) of N. plumbaginifolia chromosomes were found in one line. Preferential loss of species-specific chromosomes was not observed. Analysis of regenerating tissue from three lines of Cnx 20 + H7322 revealed that after 24 months of culture intra- and intergeneric translocations, fragments and deletions were present. Elimination of the potato and N. plumbaginifolia chromosomes had taken place before and after genome doubling.     

Analysis of the plastome and chondriome origin in plants regenerated after asymmetric Solanum ssp. protoplast fusions

Protoplasts from potato cultivars used as recipient parents were fused with irradiated protoplasts from wild Solanum donor species. Regenerated plants were analysed by RAPDs to identify hybrids. Irradiation of donor protoplasts with ionizing irradiation induced a broad range of donor nuclear DNA elimination in the asymmetric hybrids. Usage of chloroplast (cp)- and mitochondrial (mt)-specific PCR markers made it possible to trace the different origins of the cp genome in seven fusion combinations, as well as the mt genomes in two fusion combinations. Regenerated plants with recipient nucleus and plastome markers from the donors were found in six of the seven analysed fusion combinations. Protoplast fusion has generated novel mt genome combinations consisting of different portions of the mt genomes from the fusion partners. Selection of heterofusion products based on fluorescence markers is an efficient method to obtain asymmetric Solanum hybrids and cy- brids from most fusion combinations. Possible models for cybrid formation are discussed. Received: 7 October 1999 / Accepted: 3 December 1999     

The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny

Genomic in-situ hybridization (GISH) was used to monitor the behaviour of parental genomes, and the fate of intergenomic chromosome translocations, through meiosis of plants regenerated from asymmetric somatic hybrids between Nicotiana sylvestris and N. plumbaginifolia. Meiotic pairing in the regenerants was exclusively between chromosomes or chromosome segments derived from the same species. Translocation (recombinant) chromosomes contained chromosome segments from both parental species, and were detected at all stages of meiosis. They occasionally paired with respectively homologous segments of N. sylvestris or N. plumbaginifolia chromosomes. Within hybrid nuclei, the meiotic division of N. plumbaginifolia lagged behind that of N. sylvestris. However, normal and recombinant chromosomes were eventually incorporated into dyads and tetrads, and the regenerants were partially pollen fertile. Recombinant chromosomes were transmitted through either male or female gametes, and were detected by GISH in sexual progeny obtained on selfing or backcrossing the regenerants to N. sylvestris. A new recombinant chromosome in one plant of the first backcross generation provided evidence of further chromosome rearrangements occurring at, or following, meiosis in the original regenerants. This study demonstrates the stable incorporation of chromosome segments from one parental genome of an asymmetric somatic hybrid into another, via intergenomic translocation, and reveals their transmission to subsequent sexual progeny.     

Genome composition of asymmetric hybrids in relation to the phylogenetic distance between the parents. Nucleus-chloroplast interaction

Summary A series of fusion experiments were performed between protoplasts of a cytoplasmic albino mutant of tomato, Lycopersicon esculentum (ALRC), and gamma-irradiated protoplasts of L. hirsutum and the Solanum species S. commersonii, S. etuberosum and S. nigrum. These species were chosen for their different phylogenetic relationships to tomato. In all fusion combinations except from those between ALRC and S. nigrum, green calli were selected as putative fusion products and shoots regenerated from them. They were subsequently analyzed for their morphology, nuclear DNA composition and chloroplast DNA origin. The hybrids obtained between ALRC and L. hirsutum contained the chloroplasts of L. hirsutum and had the flower and leaf morphology of L. esculentum. After Southern blot analysis, using 13 restriction fragment length polymorphisms (RFLPs) randomly distributed over all chromosomes, all hybrids showed L. esculentum hybridization patterns. No chromosomes of L. hirsutum were found. These results indicate that these hybrids were true cybrids.The putative asymmetric hybrids, obtained with S. commersonii and S. etuberosum, showed phenotypic traits of both parents. After hybridization with species-specific repetitive nuclear DNA probes it was found that nuclear material of both parents was present in all plants. In the case of S. nigrum, which combination has the greatest phylogenetic distance between the fusion parents, no hybrid plants could be obtained. The chloroplast DNA of all hybrid plants was of the donor type suggesting that chloroplast transfer by asymmetric protoplast fusion can overcome problems associated with large phylogenetic distances between parental plants.     

“Arabidobrassica”: Chromosomal recombination and morphogenesis in asymmetric intergeneric hybrid cells

A somatic hybrid cell line, cloned from an individual protoplast-fusion product between Arabidopsis thaliana and Brassica campestris, gave rise to formation of numerous plants differing drastically in morphology. Analysis of these various regenerants, all of which originated from one and the same heterokaryon derived from the fusion of two cells, shows the unspecific elimination of chromosomes of both parental species during the callus growth phase. Whereas the parental cells have so far not been sucessfully regenerated into plants, several of their different asymmetric hybrids are capable of morphogenesis. Furthermore, chromosomal analysis indicates extensive recombination. Most of the plants are predoinantly morphologically regular. Abnormalities are mostly limited to the flowers which tend to undergo phyllody. The results demonstrate that remote somatic hybridization may have applications although true amphidiploids may not be obtainable. The transfer of small units of genetic material between distantly related species by protoplast fusion seems to be a more realistic approach than the combination of complete, highly diverse genomes.     

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa,lucerne) and Onobrychis viciifolia Scop. (sainfoin)

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the recipient parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33–78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.     

Analysis of mitochondrial genomes amongCitrus plants produced by the interspecific somatic fusion of ‘Seminole’ tangelo with rough lemon

Interspecific somatic fusion was performed between Seminole tangelo (Citrus reticulata Blanco xC. paradisi Macf.) protoplasts isolated from embryogenic callus and rough lemon (C. jambhiri Lush.) mesophyll protoplasts. Eight plants out of ten randomly selected regenerants had 18 chromosomes and the same nuclear rDNA fragment patterns as that of the mesophyll parent. The remaining two plants showed rDNA fragment patterns from both parents and had 36 chromosomes. For the analysis of mitochondrial DNA (mtDNA),rrn26 derived from pea was used to probeBamHI digests of the regenerants. All plants showed mtDNA band patterns identical to that of the callus parent, suggesting that eight plants were cybrids and the remaining two plants were somatic hybrids. In addition to the callus parent band patterns, additional fragments from the mesophyll parent and/or a novel band fragment were revealed in some of the putative cybrids by peaatpA probe after digestion withDraI andPstI. These results suggest the occurrence of mtDNA recombination/rearrangement inCitrus cybrids produced by somatic fusion in this interspecific combination.Abbreviations mtDNA Mitochondrial DNA     

Intergeneric gene transfer mediated by plant protoplast fusion

Summary In attempts at somatic transfer of plant genomes of reduced size, X-irradiated leaf protoplasts of parsley (Petroselinum hortense, 2n=22) were fused with cell culture protoplasts of a nuclear albino mutant of carrot (Daucus carota, 2n=18). Introduction of genes from the irradiated parsley nuclei into the carrot genome was shown by the correction of the albino defect and by the appearance of parsley isoenzymes in selected green tissues and plants. The cytological studies provided information on significant deviation from the amphidiploid chromosome number. The high frequency of cells with 2n=19, 2n=38 and regeneration of plants with 2n=19 chromosomes can indicate that the elimination of parsley chromosomes is incomplete. A correlation was found between the lethality of selected tissues and differentiated or undifferentiated stages of the cells.