Asymmetric Somatic Hybrid Plants Research Articles

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments--cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.

Transfer of bacterial blight resistance from Oryza meyeriana to O. sativa L. by asymmetric somatic hybridization

Asymmetric somatic hybrid plants were produced between cultivated rice (Oryza sativa L.) and wild species [O. meyeriana (Zoll. etMor. exSteud.)] with high resistance to rice bacterial blight. X-ray-irradiated protoplasts of the wild species were used as donor and chemically fused with iodoacetamide-inactivated protoplasts of rice cv. 02428 to produce hybrids. Seventy-two plants were regenerated from 623 calli based on metabolic complementation. The morphological characters of the plants closely resembled that of the rice. Simple sequence repeats were employed to identify their hybridity. Cytological analysis of root-tips revealed that their chromosome number varied in the range of 27–38. The somatic hybrids were inoculated with strains of Xanthamonas oryzae pv. oryzae at adult growth stage and demonstrated the resistance to bacterial blight introgression from the O. meyeriana.

Cytogenetic analysis of interspecific sunflower hybrids and molecular evaluation of their progeny

Meiotic cells of transgenic asymmetric somatic hybrid (ASH) plants obtained by fusion of microprotoplasts of the donor species Helianthus giganteus or Helianthus maximiliani and recipient protoplasts of Helianthus annuus were investigated. Over 85% of the ASH meiocytes showed regular bivalent chromosome pairing; however, several anomalies like anaphase bridges, laggard chromosomes, univalent and multivalent pairing were also observed. Pollen viability of the ASH plants ranged from 79.2 to 95% with a strong negative correlation to chromosome number which varied between 34 and 42. Molecular investigation of ASH progeny using RAPD markers revealed the presence of donor genotype markers in 68% of the offspring. These results suggest that asymmetric somatic hybridization offers an efficient alternative method to overcome sexual barriers for gene flow and the genetic improvement of H. annuus by introgression of economical important traits from wild Helianthus species.

Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L.

In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with γ-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either ”in vitro” or ”in situ.” Cybrid plants grown in the field produced seeds when free-pollination occurred.

Characterization and molecular analysis of transgenic plants obtained by microprotoplast fusion in sunflower

Asymmetric somatic hybrid (ASH) plants were obtained by PEG-mediated mass fusion of microprotoplasts from perennial Helianthus species and hypocotyl protoplasts of Helianthus annuus. The formation of micronuclei in perennial sunflower cell cultures was induced, at early log phase, by addition of the herbicides amiprophos-methyl or oryzalin. Sub-diploid microprotoplasts were isolated by high-speed centrifugation and the smallest enriched by sequential filtration through nylon sieves of decreasing pore size. Fusion products were cultured and the regenerated plants phenotypically, genetically and cytologically characterized. DNA analysis using RAPD markers revealed that 28 out of 53 regenerated plants were asymmetric hybrids. Subsequent nuclear-DNA flow cytometric analysis showed that these plants had a higher DNA content than the receptor H. annuus, suggesting that they represented addition lines. Cytological investigation of the metaphase cells of 16 hybrids revealed an addition of 2–8 extra chromosomes in these plants. The phenotype of most ASH plants resembled H. annuus. These results indicate that micronuclear induction and asymmetric somatic hybridization represent a potent tool for partial genome transfer aimed at the specific transfer of economically important traits in breeding programs.

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.

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

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.

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.

T-DNA-tagged chromosome 12 in donor Lycopersicon esculentum × L. pennellii is retained in asymmetric somatic hybrids with recipient Solanum lycopersicoides

Asymmetric somatic hybrid plants were recovered after fusing irradiated mesophyll protoplasts of donor Lycopersicon esculentum × L. pennellii (EP) interspecific hybrid with callus-derived protoplasts of recipient Solanum lycopersicoides. EP plant A54 had been previously transformed by an agrobacterium vector, and the T-DNA insert mapped to the L. esculentum chromosome 12. The T-DNA insert conferred kanamycin resistance to EP that was subsequently used to select cell fusion products and recover asymmetric hybrid plants that retained tagged chromosome 12. Doses of 50- and 100-Gy irradiation promoted the elimination of only a few donor chromosomes. At 200 Gy, the regenerated plants had ploidy levels higher than tetraploid. However, the T-DNA tagged chromosome 12 was always retained in the asymmetric hybrid plants tested. Likewise, all plants from the 100-Gy series, with the exception of number 160, were mixoploid in the root-tip cells. Such mixoploid asymmetric somatic hybrids could be stabilized by inducing adventitious shoots on leaf strips cultured on shoot regeneration medium containing kanamycin. The asymmetric hybrid plants did not produce viable seed when self-pollinated or backcrossed to tomato or S. lycopersicoides.

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P1, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.

ASYMMETRIC PROTOPLASM FUSION IN TOMATO

Sexual hybrid plants of Lycopersicon esculentum × L. pennellii (E × P) have been transformed and the T-DNA inserts genetically mapped. Donor protoplasts of E × P were isolated from leaves, and subsequently irradiated with 0, 5, 10 and 20 krad of a 60Co. They were then fused with suspension-derived protoplasts of S. lycopersicoides using the PEG-CA++-high pH technique. The protoplasts were cultured in medium 8E at 1.5 × 106 protoplasm/ml. Selection of heterokaryon-derived macrocalli was facilitated by the inability of irradiated donor protoplasts to divide and by plating p-calli on regeneration medium containing kanamycin - an antibiotic for which the resistance gene NPTII is part of the T-DNA. Morphological characteristics of the resulting asymmetric somatic hybrid plants indicate that 10 and 20 krad irradiation eliminates a larger port ion of the genome than does 5 krad. This has been confirmed with isozyme analysis and chromosome counts which generally show the 5 krad asymmetric somatic hybrid plants to differ little from symmetric hybrids although they contain some significant exceptions. Isozyme data reveals the 10 and 20 krad plants to have received much less of the donor genome. Exact quantification is continuing using isozyme markers, chromosome counts and cDNA probes.