Intraspecific gametosomatic hybridisation in Petunia hybrida

Following PEG and high pH induced fusion of haploid tetrad protoplasts of a normal purple flowered variety of P. hybrida with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace, triploid gametosomatic hybrid plants were recovered. These hybrids possessed an intermediate floral morphology and the expected chromosome number of 2n=3x=21. Selection was based on the fact that pollen tetrad protoplasts failed to divide in culture and that, following complementation to chlorophyll proficiency in the gametosomatic hybrid, the hybrid cells were visualised against a background of albino cells of the variety Blue Lace. The production of such gametosomatic hybrid plants in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana and that alternative selection strategies are available.Abbreviations BAP 6-benzylaminopurine - IAA 3-indole acetic acid - NAA naphthalene acetic acid - Z zeatin - ABN bromonaphthalene - MS Murashige and Skoog (1962) - MW molecular weight - PEG polyethylene glycol     

The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Summary Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10⁶ mature pollen protoplasts were fused with 7·10⁶ mesophyll protoplasts using a PEG/Ca²⁺ method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l^-1). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l^-1). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.Abbreviations AAT aspartate aminotransferase - BCP bromocresol purple - EST esterase - MES 2-(N-morpholino) ethanesulfonic acid - AP acid phosphatase - PEG polyethyleneglycol - PER peroxydase     

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

Summary Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82–120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33–78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.     

Does pretreatment by rhodamine 6-G affect the mitochondrial composition of fusion-derived Nicotiana cybrids?

Rhodamine-6G(R6G), a lipophilic dye which degrades mammalian mitochondria, was shown to arrest the division of Nicotiana protoplasts. When albino recipient-protoplasts were treated with R6G and fused with X-irradiated (green) donor- protoplasts, only green cybrid plants were obtained. The mtDNA of the cybrids was analyzed by Southern-blot hybridization. We found that cybrids which resulted from N. rustica (donor) protoplasts, fused with R6G-treated albino protoplasts, had only parental-type mtDNA. When another donor, with N. undulata mtDNA, was used, most of the resulting cybrids contained non-parental mtDNA. Only one cybrid (out of 12) had N. undulata -type (donor) mtDNA.Abbreviations big N. bigelovii - IAA indoleacetic acid - mtDNA mitochondrial DNA - R6G rhodamine 6G - tbc N. tabacum und, N. undulata     

Somatic hybridization in the genus Solanum: S. tuberosum and S. brevidens

Somatic hybrid plants were regenerated from fused mesophyll protoplasts of an albino potato (Solanum tuberosum spp. tuberosum) variant and Solanum brevidens, a non-tuber bearing species which is sexually incompatible with S. tuberosum. These somatic hybrid plants represent the first example of direct hybridization between potato and members of the taxonomic group Etuberosa, and offer the potential for introgressing valuable germplasm from Solanum species outside the sexually compatible range into a worldwide crop species.     

Interspecific hybridization between Brassica juncea and B. spinescens through protoplast fusion

Hypocotyl derived protoplasts of B. juncea cv. RLM-198 were fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids. Fusion products could be microscopically identified by characteristics of the protoplasts of both parents in the hybrid cells; they are colourless and vacuolated like the hypocotyl protoplasts and possess chloroplasts of the mesophyll protoplasts. The heterokaryotic fusion frequency was around 5%. However, the frequency of calli regenerating hybrid shoots was more than 10% of the regenerating calli. Putative somatic hybrids had morphological features characteristic of both the parents. Twelve plants analysed cytologically, possessed 52 chromosomes (26_II) at meiosis representing the complete genomes of B. juncea (18_II) and B. spinescens (8_II). For esterase isozymes, the hybrids had bands of Doth the parents. Hybrid nature of some of the plants was confirmed by their close resemblance to B. juncea, chromosome number and isozyme bands of B. spinescens as in Rsp-19. Somatic hybrids had rudimentary, non-dehiscent anthers and completely sterile pollen. However, on back crossing with B. juncea, 10 out of 12 plants produced seeds and about 100 plants were realized.Abbreviations PEG Polyethylene glycol     

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae)

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.ABBREVIATIONS BSA bovine serum albumin - EDTA ethylene diamine tetra acetic acid - FDA fluorescein diacetate - f.wt. fresh weight - IAA indole 3-acetic acid - MS Murashige and Skoog (1962) medium - TEMED N,N,N,N-Tetra methyl ethylene diamine - TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)     

Chromosome stability of cell suspension cultures of Nicotiana spp.

Cell suspension cultures of Nicotiana were initiated using conditions designed to selectively favor stable chromosome number. These conditions included use of leaf explants to initiate cultures, growth of cells in culture medium containing 2,4-D, and transfer of cells with short subculture intervals. Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number. Each line could be regenerated to recover plants that retained the somatic chromosome number during culture. Establishment of haploid and diploid cell lines with stable chromosome number is important for mutant isolation and protoplast fusion.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon

Intergeneric somatic hybrid plants between Hamlin sweet orange [Citrus sinensis (L.) Osbeck] and Flying Dragon trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. Hamlin protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with Flying Dragon protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the Hamlin protoplasts with the subsequent expression of the trifoliate leaf character of Flying Dragon. Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.Abbreviations MDH malate dehydrogenase - CTV citrus tristeza virus - MT Murashige and Tucker basal medium - BH3 protoplast culture medium, Grosser and Chandler, 1987 - PEG polyethylene glycol - GA3 giberellic acid - BA N-(phenylmethyl)-1 H-purin-6-amine - HCl hydrochloric acid Florida Agricultural Experiment Station Journal Series No. 7972     

Transmission genetics of the somatic hybridization process in Nicotiana

Summary Callus protoplasts of a Nicotiana tabacum chlorophyll-deficient mutant were fused with mesophyll protoplasts from one of following five sources: 4 cmsanalogs of tobacco bearing the cytoplasms of N. plumbaginifolia, N. suaveolens, N. repanda, and N. undulata, respectively, as well as wild species N. glauca. In another series of experiments, callus protoplasts from the chlorophyll-deficient genome Su/Su mutant of tobacco were fused with mesophyll protoplasts of the wild species N. glauca and those of a plastome chlorophyll-deficient tobacco mutant. The screening of hybrids consisted of visual identification followed by mechanical isolation and cloning of heteroplasmic fusion products in microdroplets of nutrient medium. Studies of regenerated plants included the analyses of gross morphology of plants, leaf and flower morphology, analysis of chromosome size and morphology and chromosome numbers, studies of multiple molecular forms of esterase and amylase, analysis of chloroplast DNA restriction patterns and analyses of chlorophyll-deficiency controlled by Su and P ^– genes. The study of progeny of 41 clones representing all species' combinations demonstrated that regenarants of most (63%) clones from intraspecific (for nuclear genes) combinations were cybrid forms, whereas in the case of the fusion N. tabacum + N. glauca, the true nuclear hybrids prevailed and the proportion of cybrids did not exceed 26%. Clones regenerating both hybrid and cybrid plants from the same fusion product were also found.     

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120. This revised version was published online in June 2006 with corrections to the Cover Date.     

Examination of genome stability in cultured Lycopersicon

The genetic stability of plants regenerated from either mesophyll protoplasts or leaf slices of the F1 hybrid between Lycopersicon esculentum and L. pennellii was assayed by comparing the ploidy level, leaf morphology and isozyme patterns of the regenerants with their somatic parents. Regenerants from protoplasts were predominantly tetraploid, regenerants from leaf slices were predominantly diploid; both classes of regenerants had isozyme patterns identical to those of the parent plant. Callus was analyzed that grew up from cultures containing fused protoplasts from either irradiated or untreated protoplasts of L. esculentum and L. pennellii. The L. pennellii cell line used was 18 months old and could no longer regenerate. Out of 75 calli scored at 3 isozyme loci, 51 were heterozygous at only one or two of the loci. Irradiation of the two parental lines was not necessary to produce fusion products exhibiting asymmetric expression of parental genes.Abbreviations Got-2 glutamate oxaloacetate transaminase-2 - Pgi-1 phosphoglucoisomerase-1 - Pgm-2 phosphoglucomutase-2     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

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.     

Production of a triple mutant,chlorophyll-deficient,streptomycin-, and kanamycin-resistant Nicotiana tabacum,and its use in intergeneric somatic hybrid formation with Solanum melongena

Summary In order to produce a triple mutant, sexual crosses between a chlorophyll-deficient, streptomycin-resistant mutant of Nicotiana tabacum (SA) and a kanamycin-resistant transformant of N. tabacum (KR.) were carried out. From the offspring of this cross, a triple mutant (KR-SA) was selected. In N. tabacum KR-SA, chlorophyll deficiency is due to recessive mutation in the nuclear genome, streptomycin resistance is due to a dominant mutation in the chloroplast genome, and kanamycin resistance is shown to be a dominant nuclear marker. Cell suspension protoplasts of N. tabacum KRSA were fused with callus protoplasts of Solanum melongena by dextran treatment. Somatic hybrid plants were selected for streptomycin resistance and the ability to produce clorophyll in regenerated plants. By using this selection system, green plants were recovered from two colonies. When these green plants were then tested for kanamycin resistance, all analyzed plants carried this trait. In addition, the hybrid nature of these plants was confirmed by investigation of the peroxidase isozyme. The present results show that the use of N. tabacum KR-SA in studies of somatic hybridization makes it possible to select somatic hybrid plants easily and provides information of the N. tabacum genome.Chemical Regulation of Biomechanism, The Institute of Physical and Chemical Research, Wako 351-01, Japan     

Somatic hybridization of sexually incompatible petunias: Petunia parodii,Petunia parviflora

Summary Somatic hybrid plants were regenerated following the fusion of leaf mesophyll protoplasts of P. parodii with those isolated from a nuclear-albino mutant of P. parviflora. Attempts at sexual hybridization of these two species repeatedly failed thus confirming their previously established cross-incompatibility. Selection of somatic hybrid plants was possible since protoplasts of P. parodii would not develop beyond the cell colony stage, whilst those of the somatic hybrid and albino P. parviflora produced calluses. Green somatic hybrid calluses were visible against a background of albino cells/calluses, and upon transfer to regeneration media gave rise to shoots. Shoots and the resultant flowering plants were confirmed as somatic hybrids based on their growth habit, floral pigmentation and morphology, leaf hair structure, chromosome number and Fraction 1 protein profiles. The relevance of such hybrid material for the development of new, and extensively modified cultivars, is discussed.     

Somatic hybridisation of Daucus carota and D. capillifolius by protoplast fusion

Summary Protoplasts isolated from cultured cells of albino carrot (Daucus carota) and normal green D. capillifolius were fused by polyethylene glycol. Selection of somatic hybrid plants was based on the restoration of photosynthetic function in hybrids. Green plantlets selected from embryo cultures were characterized on the basis of leaf morphology. The interspecific protoplast fusion resulted in green plants with leaves which were intermediate between those of the parents. The somatic hybrids between orange rooted carrot variety and D. capillifolius with long white roots produced long, white and fleshy roots. The cytological analysis of parasexual hybrids revealed that the chromosome number ranged from 34 to 54. The most frequent chromosome number was 2n = 36. Hybrids were also found with 34 and 35 chromosomes. The somatic hybrid showed the same isoenzyme pattern of leaf peroxidase as D. carota.