Somatic Hybrid Plants Research Articles

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.

Comparison of volatiles and waxes in leaves of genetically engineered tomatoes

The essential oils and leaf waxes of several putative somatic hybrid plants of Lycopersicon esculentum [+] L. hirsutum were compared with those of transgenic plants of L. hirsutum and non-transgenic plants of L. esculentum, and also of wild-type L. hirsutum. The latter contained β-myrcene, 3,7-dimethylocta-1,3,6-triene and a high proportion of undecan-2-one whereas the transgenic L. hirsutum and the somatic hybrids all contained similar proportions of 2-carene and β-phellandrene, but only traces of alkan-2-ones. Lycopersicon esculentum contained a very low proportion of essential oils and no alkan-2-ones. Hentriacontane ( n-C 31) and 3-methylhentriacontane ( anteiso-C 32) were the predominant components in all the leaf waxes, and a comparison of the ratio between these compounds suggested that the somatic hybrids contained at least some contributions from both the parent species.

Identification and molecular analysis of rice somatic hybrids using foreign Transgenes

Somatic fusion of rice [Oryza sativa L.] cell lines [hygr-philipino 28] and [geneticinr(G418r)-G171] was carried out using electrofusion methodology. Heterokaryons were identified in culture medium supplemented with both hygromycin B and G418. The hybrid character, of some of the putative somatic hybrid plants, was confirmed by Southern blot analysis which indicated the presence of the selectable marker genes [hpt, npt II] brought by the two parents of fusion.

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.

Donor chromosome elimination and organelle composition of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant

Morphology, the extent of elimination of donor chromosomes and the organelle composition of highly asymmetric somatic hybrid plants between a interspecific tomato hybrid Lycopersicon esculentum x L. pennellii (EP) as donor and a Solarium melongena, eggplant (E), recipient, were studied. Morphologically, the somatic hybrids most resemble eggplant but, due to polyploidy, growth is slower relative to both fusion parents. The somatic hybrids produce flowers that are characterized by abnormal styles, stigmas and by anthers which do not produce pollen. Limited amounts of donor EP genomic DNA were found in the three somatic hybrid plants (H18-1, H18-2 and H18-3), by dot-blot hybridization with probe pTHG2, equivalent to 6.23,5.41, and 5.95% EP, respectively. These percentages translated to the presence of 3.59, 2.90 and 3.19 average-size EP chromosomes in plants H1 8-1,-2 and-3, respectively. RFLP determination of L. esculentum- and L. pennellii-specific chromosomes revealed that only fragments of eight to ten out of the 24 EP chromosomes (EP has 12 L. esculentum and 12 L. pennellii chromosomes) are present in the asymmetric somatic hybrid plants. Loci of L. esculentum and L. pennellii were evenly represented in plants H18-1, -2, and -3: four to five from L. esculentum and four to five from L. pennellii. All somatic hybrid plants retained locus TG22, chromosome 4, from both EP species. Although the regeneration of plants, H18-1, -2 and-3 was from one callus, loci TG31 and TG79 of L. esculentum chromosome 2 and L. pennellii chromosome 9, respectively, were missing in hybrid plant H18-1. The three somatic hybrid plants all had chloroplast DNA fragments specific for S. melongena. The mitochondrial genome (mtDNA) in the asymmetric somatic hybrids showed predominantly the pattern of eggplant; however, some eggplant-specific polymorphic bands were not present in the three plants.

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Donor chromosome elimination and organelle composition of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant

Donor chromosome elimination and organelle composition of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant

Production of interspecific somatic hybrid plants between Asparagus officinalis and A. macowanii through electrofusion

Interspecific somatic hybridization was performed between embryogenic callus protoplasts of Asparagus officinalis cv. Mary Washington and callus protoplasts of A. macowanii by electrofusion. By utilizing iodoacetamide (IOA) inactivation treatment for A. officinalis protoplasts and the difference of cell division capacity between the two species, one callus line, which had an ability to produce somatic embryos, was selected as a putative hybrid. Normal germination of the somatic embryos was induced only when they were exposed to a period of cold treatment (14 days at 4°C). However, the regenerated plants showed intermediate but abnormal morphological characters in vitro as well as after transfer to soil. The hybrid nature of the plants derived from this callus was confirmed by isozyme, nuclear rDNA and random amplified polymorphic DNA (RAPD) analyses and counting the chromosome number.

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR(+)) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR(+) calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5-9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1-25.8% of the tomato genome. Thus, DNA contained in 3.8-13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.

Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)4 as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.

Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage

Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.

Characterization of Interfamilial Somatic Hybrids between 5-methyltryptophan-Resistant(5MT-resistant) Rice(Oryza sativa L.) and 5MT-Sensitive Carrot(Daucus carota L.); Expression of Resistance to 5MT by the Somatic Hybrids.

The production of the asymmetric hybrid plants between 5-methyltryptophan-resistant (5MT-resistant) O. sativa and 5MT-sensitive D. carota was reported previously. Al-though morphology of the somatic hybrid plants closely resembled that of the parental carrot plants, all of the somatic hybrids were resistant to 5MT. In the present study, we investigated the expression of resistance to 5MT in cells of these somatic hybrids. The calli induced from hybrid plants and their parents grew in medium that contained a high concentration of 5MT when those calli were supplied with anthranilic acid or tryptophan. However, chorismic acid was not effective in reducing the inhibition of celn growth by 5MT. The activities of anthranilate synthase and tryp-tophan synthetase of cells of the somatic hybrids and 5MT-resistant O. sativa were about 5 times and 2 times as high as those of cells of 5MT-sensitive D. carota, respectively. These results indicated that anthranilate synthase was involved in the mechanism of resistance to 5MT. However, anthranilate synthase activities of the somatic hybrids and 5MT-resistant O. sativa were quite sensitive to feedback inhibition by tryptophan, as was that of 5MT-sensitive D. carota. Northern hybridization using a gene for anthrani-late synthase as probe revealed that levels of expression of the gene in the somatic hybrids and 5MT-resistant O. sati-va were higher than that of 5MT-sensitive D. carota.

Somatic hybrids between Brassica juncea (L). Czern. and Diplotaxis harra (Forsk.) Boiss and the generation of backcross progenies

An attempt to transfer genes from droughttolerant Diplotaxis harra, a wild relative of Brassica species, to an elite oil-yielding cultivar, B-85, of mustard (Brassica juncea) was made through protoplast fusion, as the two plant systems are sexually incompatible. By following the standard protocol for PEG-mediated protoplast fusion followed by high pH, high Ca(++), DMSO treatment and appropriate cell-culture technique, 16 presumptive somatic hybrid plants could be regenerated. Chromosomal analysis of four such somatic hybrids revealed that three of them were asymmetric. Analysis of morphological characters, meiotic chromosomes, and esterase isoenzyme pattern revealed that all the somatic hybrids were different from each other. Furthermore four chromosomes of each genome could undergo homoeologous pairing at meiosis indicating the possibilities for genetic recombination and chromosomal rearrangements. Irregular distribution of chromosomes at anaphase-II at meiosis has been a consistent feature of these plants. Eventually, pollen of all the somatic hybrids showed complete infertility preventing the recovery of any selfed seed. Nevertheless, ovule fertility of one somatic hybrid was not totally impaired as it had set some seeds upon backcrossing with the B. juncea parent. The esterase isoenzyme banding pattern of 24 individual progeny plants of this backcross provided evidence for their recombinant nature. It was thus confirmed that a transfer of genetic traits from Diplotaxis harra to B. juncea had indeed taken place. Furthermore, it was conceptualised that a transfer of alien genes through the protoplast-fusion technique is primarily possible in situations where meiotic pairing of the chromosomes of the two participating genomes generates recombinant gametocytes which can pass through subsequent filial generations.

Nuclear Genome Organization of Asymmetric Intergeneric Somatic Hybrid Plants between Lycopersicon esculentum × L. pennellii and Solanum melongena

Asymmetric somatic hybrids were obtained by PEG/DMSO fusion of protoplasts of a kanamycin-resistant (KmR+) interspecific tomato hybrid L. esculentum × L. pennellii with protoplasts of S. melongena eggplant. Elimination of tomato chromosomes was directed by application of 100, 250, 500, 750, and 1000 Gy of γ-rays to the donor protoplasts. The hybrid nature of selected KmR+ calli was confirmed by RAPDs, PCR amplification of the NptII gene, and Southern hybridization. Flow cytometry revealed that asymmetric hybrid plants were regenerated only from selected somatic hybrid calli that had a ploidy level close to 4n. The amount of donor DNA in three somatic hybrid plants was quantified by dot-blot hybridization with tomato species-specific probes, and was found to be 5% to 7%. Therefore, presence of four to five tomato chromosomes in asymmetric hybrid plants has been calculated. Detection of tomato-specific chromosomes in hybrid plants by RFLP analysis will be presented.

Asymmetric somatic hybrid plants between an interspecific Lycopersicon hybrid and Solanum melongena

Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.

Colorado potato beetle resistant somatic hybrid potato plants produced via protoplast electrofusion

Leptines are natural glycoalkaloids found only in certain selections of the wild potato speciesSolanum chacoense. These rare glycoalkaloids have been identified to be phytochemical defensive agents against insect herbivores such as the Colorado potato beetle (CPB). In an attempt to introduce this CPB resistance into the cultivated potatoS. tuberosum, interspecific somatic hybrid plants were developed between a dihaploid ofS. tuberosum and a high leptine-producing germplasm selection ofS. chacoense. The somatic hybrid was fused using protoplast electrofusion and regeneration techniques. Selection of interspecies fusion cell lines was based on hybrid vigor in protoplast-callus (p-callus) growth, on shoot regeneration from p-calli, and on characteristic appearance of anthocyanin pigment. This selection system was highly efficient and 12 of 13 fully regenerated plants were identified as somatic interspecies hybrids, as determined by the analyses of morphologic biochemical, and isozyme markers. In vitro insect bioassays demonstrated that the hybrids averaged a threefold reduction in leaf consumption by the CPB when compared to cultivated potatoes.

Molecular, cytological and morpho-agronomical characterization of hexaploid somatic hybrids in Medicago

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.

Somatic hybridization of thePassifloraspecies,P. edulisf.flavicarpaDegener. andP. incarnataL.

Somatic hybrid plants have been produced following electrofusion of mesophyll protoplasts of P. edulis f. flavicarpa and P. incarnata. These hybrids exhibited morphological characteristics intermediate between those of the two parental species. Further evidence of hybridity has been provided by isozyme analysis, ploidy determinations and molecular characterization (RAPD analysis). These somatic hybrid plants have been used in first generation backcrossing experiments with both P. edulis f. flavicarpa and P. incarnata; descriptions are provided of the floral and fruit characteristics, as well as seed production.

Production of fertile somatic hybrid plants of sunflower and Helianthus giganteus L. by protoplast fusion

Protoplasts of Helianthus giganteus and Helianthus annuus were fused using polyethylene glycol. Before fusion H.giganteus protoplasts were subjected to iodoacetic acid treatment to render them unable to divide. Fused protoplasts were cultured in V-KM medium containing benzylaminopurine and naphtaleneacetic acid. Hybrid calli were identified on the basis of their ability of embryogenic development contributed by the Helianthus giganteus parent. Fifty embryogenic calli were cultured on MS based medium without growth regulators to induce further development of somatic embryos. Elongated shoots were removed, rooted and transferred into growth chambers. Overall morphology of the plants was intermediate between the two parents. Their hybrid nature was confirmed by chromosome counting and by the analysis of esterase isozymes. The plants flowered within two to three months and later died. Thus the perennial nature of H.giganteus is a recessive trait in this interspecific hybrid. Seeds were obtained from two of the regenerated plants. From these seeds normal fertile F2 plants could be grown.

Production of Somatic Hybrids between Tomato (Lycopersicon esculentum) and Night Shade (Solanum lycopersicoides) by Electrofusion

Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. cv. 'Ohgata Zuiko') and Solanum lycopersicoides were fused by electrofusion method to develop fertile tetraploid somatic hybrids. Following electrofusion the protoplasts were plated in modified TM2 (MTM2) medium in which S. Iycopersicoides protoplast could not form callus. The putative hybrid calli were regenerated on MS3ZG regeneration medium in which tomato protoplasts derived calli could not regenerate shoots. Tomato protoplasts were partially inactivated with iodoacetamide (IOA) for the easy selection of heterokaryons. Three hundred regenerated plants were grovvn to maturity in a green house. The hybridity of the plants was argued by morphological, isozyme and random amplified polymorphic DNA (RAPD) analyses. All plants were heterotic in their growth, expressing an erect stem with a prominent internode like tomato, as well as days to flowering, perennial habit, free anther without sterile tip like S. Iycopersicoides. Several traits, such as leaf margin, inflorescence, flower and fruit color were intermediate between both parents. The electrophoretic pattern of PGI (.phosphoglucoisomerase), GOT (.glutamate oxaloacetate transaminase) and PG[M (phosphoglucomutase) confirmed the hybrid nature of the nuclei of all the tested plants. RAPD markers generated from 35 randomly selected plants, using eight decamer primers reconfirmed the hybrid nature of their nuclear genomes. Cyt.ological study of 20 randomly selected plants revealed 60 %of them had euploid chromosome numbers (2x=4n= 48). Tetraploid plants produced fertile pollen and developed fruits and viable seeds upon self pollination. About two thirds of the tested somatic hybrid plants survived in the winter-cold temperature (Average minimum -1.25°C) to maximum 5.3°C).