Production and genetic characterization of somatic hybrids between leaf mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Inter-specific somatic hybrids of leaf mustard (Brassica juncea) and broccoli (Brassica oleracea) were established to introduce cytoplasmic male sterility (CMS) and Verticillium dahliae Kleb. resistance from broccoli to leaf mustard. Protoplasts isolated from hypocotyls and cotyledons of inbred lines of leaf mustard and broccoli were fused using 40% (w/v) polyethelene glycol and then cultured in modified k8p medium supplemented with 0.2 mg/l 2,4-dichlorophenoxi-acetic acid, 0.5 mg/l 6-benzyladenine (BA), 0.1 mg/l naphthaleneacetic acid (NAA), and 0.1 mg/l kinetin (Kin), and 0.4 M mannitol as osmoticum. At the eight- to ten-cell stage, divided cells were transferred to Kao’s basal medium supplemented with 0.3 M sucrose as carbon source and 0.1% agarose, 2 mg/l BA, 2 mg/l zeatin (ZEA), 1 mg/l NAA, and 0.5 mg/l Kin for callus proliferation. After 35 d, when small calli reached 2–3 mm in diameter, calli were transferred to regeneration medium containing 5 mg/l ZEA and 2 mg/l indole-3-acetic acid. The chromosome numbers of putative somatic hybrids varied from 46 to 54. A total of ten plants showed a 0.5-kb, CMS-specific band, while two regenerated plants showed a missing band similar to that of leaf mustard by polymerase chain reaction amplification using Ogura CMS-specific primers. Hybrid state was confirmed by random amplified polymorphic DNA analysis. Regenerated plants had normal petals and stamens, but only two plants produced pollen and set seed.     

Targeted cybridization in citrus: transfer of Satsuma cytoplasm to seedy cultivars for potential seedlessness

CMS (cytoplasmic male sterility) can be controlled by the mitochondrion genome in higher plants, including Satsuma mandarin. Somatic fusion experiments in citrus combining embryogenic callus protoplasts of one parent with leaf protoplasts of a second parent often produce cybrid plants of the leaf parent, a phenomenon occurring most often with interspecific fusion combinations. In an attempt to practically exploit this cybridization phenomenon, we conducted somatic fusion experiments combining embryogenic suspension-derived protoplasts of Satsuma mandarin, Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a male-sterile cultivar, with leaf protoplasts of other seedy types—Hirado Buntan Pink pummelo (HBP) [Citrus grandis (L.) Osbeck], Sunburst mandarin (C. reticulata Blanco), Orie Lee hybrid (C. reticulata cv. Clementine × Murcott tangor), and Murcott tangor [C. reticulata × C. sinensis (L.) Osbeck], respectively—in an attempt to generate seedless cybrids by the targeted transfer of CMS. The genetic identities of regenerated plants from all four parental combinations were determined by flow cytometry, SSR, CAPS (or PCR-RFLP), RFLP, and chloroplast-SSR analyses. Regenerated plants from the first three parental combinations were diploids, and the cybrid nature of G1 + HBP with the mitochondrion genome from G1 and the chloroplast genome from HBP was confirmed, whereas the cybrid nature of the remaining two combinations was difficult to confirm because of the close phylogenetic relatedness of both fusion parents, as expected. Plants from G1 + Murcott were confirmed as tetraploid somatic hybrids. This is the first report of targeted citrus cybrid production by symmetric fusion with male-sterile Satsuma as the callus parent and other seedy cultivars as the leaf parents.Abbreviations CAPS: Cleaved amplified polymorphic sequence - CMS: Cytoplasmic male sterility - cp-SSR: Chloroplast simple sequence repeat - PEG: Polyethylene glycol - SSR: Simple sequence repeat - RFLP: Restriction fragment length polymorphism Communicated by G.C. Phillips     

Synthesis of Brassica oleracea/Brassica napus somatic hybrid plants with novel organeile DNA compositions

Summary Broccoli (Brassica oleracea L. italica) hypocotyl protoplasts were fused with mesophyll protoplasts of two B. napus lines, one carrying the Ogura (ogu) cms cytoplasm, and the other carrying a hybrid cytoplasm consisting of ogu mitochondria combined with triazine-tolerance-conferring chloroplasts from ctr cytoplasm. Two male-sterile somatic hybrids were recovered from the fusion of broccoli protoplasts with those of ogu/ctr cybrid B. napus. The ogu mtDNAs and ctr cpDNAs were not altered in these hybrids. Four male-sterile plants were recovered from the somatic hybridization of broccoli with ogu cms B. napus. Three of these possessed mitochondrial genomes that appeared to have resulted from recombination between the ogu and normal B. oleracea (ole) mtDNAs, while the fourth possessed an unrearranged ogu mtDNA. All four of these plants had B. oleracea cpDNA, and none displayed the seedling chlorosis associated with ogu chloroplasts. Most of the plants recovered from these fusions had the chromosome number expected of B. oleracea + B. napus hybrids (2n = 56). The novel cytoplasms may prove to be useful for the molecular analysis of Brassica cms and for the production of hybrid Brassica.     

Protoplast fusion-derived Ogura male sterile cauliflower with cold tolerance

Cauliflower (Brassica oleracea L. ssp. botrytis) protoplasts with Ogura male sterile and fertile B. oleracea cytoplasms were fused, producing plants with an array of organellar types. Plants with Ogura mitochondria were male sterile; those with B. oleracea chloroplasts were cold tolerant. In some fusions, unfused parental protoplasts were eliminated by double inactivation with iodoacetate and gamma-irradiation; in others, fused protoplasts were physically isolated by micromanipulation or by cell sorting. Double inactivation fusions produced the most plants, including many which were male sterile, female fertile, cold tolerant and diploid.Abbreviations IA iodoacetate - FDA fluorescein diacetate - CMS cytoplasmic male sterility - mtDNA mitochondrial DNA     

Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion

The donor-recipient protoplast fusion method was used to produce cybrid plants and to transfer cytoplasmic male sterility (CMS) from two cytoplasmic male-sterile lines MTC-5A and MTC-9A into a fertile japonica cultivar, Sasanishiki. The CMS was expressed in the cybrid plants and was stably transmitted to their progenies. Only cytoplasmic traits of the male-sterile lines, especially the mitochondrial DNAs, were introduced into the cells of the fertile rice cultivar. More than 80% of the cybrid plants did not set any seeds upon selfing. Sterile cybrid plants set seeds only when they were fertilized with normal pollen by hand and yielded only sterile progenies. This maternally inherited sterility of the cybrid plants showed that they were characterized by CMS. The CMS of cybrid plants could be restored completely by crossing with MTC-10R which had the single dominant gene Rf-1 for restoring fertility. These results indicated that CMS was caused by the mitochondrial genome introduced through protoplast fusion. The introduced CMS was stably transmitted to their progenies during at least eight backcross generations. These results demonstrate that cybrids generated by the donor-recipient protoplast fusion technique can be used in hybrid rice breeding for the creation of new cytoplasmic male-sterile rice lines.     

Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.)

We previously used Southern blot analysis to detect restriction-length polymorphisms between male fertile and cytoplasmic male sterile (CMS) cytoplasms at the coxII and atp6 loci of the mtDNA of Capsicum annuum L. Two copies of atp6 were found in each male fertile and CMS pepper lines. Interestingly, one of the copies of atp6 in CMS pepper was a 3'-truncated pseudogene. The open reading frame of the coxII gene was the same in the fertile (N-) and CMS (S-) lines. However, the nucleotide sequence in the S-cytoplasm diverged from that in the N-cytoplasm 41 bp downstream of the stop codon. To develop CMS-specific sequence-characterized amplified region (SCAR) markers, inverse PCR was performed to characterize the nucleotide sequences of the 5' and 3' flanking regions of mitochondrial atp6 and coxII from the cytoplasms of male fertile (N-) and CMS (S-) pepper plants. Based on these data, two CMS-specific SCAR markers, 607 and 708 bp long, were developed to distinguish N-cytoplasm from S-cytoplasm by PCR. The CMS-specific PCR bands were verified for 20 cultivars containing either N- or S-cytoplasm. PCR amplification of CMS-specific mitochondrial nucleotide sequences will allow quick and reliable identification of the cytoplasmic types of individual plants at the seedling stage, and assessment of the purity of F1 seed lots. The strategy used in this report for identifying CMS-specific markers could be adopted for many other crops where CMS is used for F1 seed production.     

Analyses of mitochondrial DNA structure and expression in three cytoplasmic male-sterile chicories originating from somatic hybridisation between fertile chicory and CMS sunflower protoplasts

Cytoplasmic male-sterile (CMS) chicories have been previously obtained by somatic hybridisation between fertile industrial chicory protoplasts and CMS sunflower protoplasts. In this study, we compared three different CMS chicory cybrids that originated from three different fusion events. The cybrids were backcrossed with different witloof chicories in order to transfer the three male-sterile cytoplasms from an industrial chicory nuclear environment to a witloof chicory nuclear context. Southern hybridisation, using different mitochondrial genes as probes, revealed that the three cybrid mitochondrial genomes were different and that they were stable throughout backcrossing generations regardless of the pollinator. However, pollinators were found to influence floral morphologies – with one being able to restore fertility – showing that nuclear context can affect the sterility of the cybrids. PCR and RFLP analyses revealed that the orf522 sequence, responsiblefor CMS in PET1 sunflower, was present in two out of the three cytoplasms studied, namely 411 and 523, but was absent from the other cytoplasm, 524. We thus concluded that orf522 is not responsible for CMS in the 524 cybrid. Although the orf522 gene is present in the 411 and 523 cytoplasms, it is probably not responsible for the sterile phenotype of these cybrids. Received: 3 June 1998 / Accepted: 30 April 1999     

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.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Identification of Cytoplasmic Male Sterility in Chinese Radish Following PCR Analysis of Mitochondrial DNA

In order to understand the molecular characteristics of the Chinese radish, the mitochondrial DNA structure and sequence were analyzed in Chinese wild radish and cultivated varieties. A total of four male-sterile lines, four maintainer lines, 17 cultivars, and a single Chinese wild radish were used, along with 25 male-sterile individuals and 159 fertile plants. We found that the cytoplasm of Chinese radishes could be classified into two types: the normal type and the Ogura type. The Ogura-type cytoplasm was detected in 25 male-sterile plants. The 159 fertile plants had normal cytoplasm. Both the Ogura cytoplasm and the normal cytoplasm were detected in the male-sterile ??RA??. The orf138 gene in mitochondrial DNA was detected in cultivated Chinese radish cultivars but not in the wild radish. The Chinese radish orf138 nucleotide sequence was determined in four male-sterile lines and displayed complete homology to the known orf138 type A nucleotide sequence. Three types of mitochondrial orfB (type 1, type 2 and type 3) were found in Chinese radishes. Type 1 was only present in the male-sterile lines. Chinese cultivated radishes were divided into type 2 and type 3, while the Chinese wild radish only had type 3 cytoplasm.     

Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids

Summary Brassica cybrids were obtained after fusing protoplasts of fertile and cytoplasmic male sterile (CMS) B. napus lines carrying the original b. napus, and the Ogura Raphanus sativus cytoplasms, respectively. Iodoacetate treatment of the fertile line and X-irradiation of the CMS line prevented colony formation from the parental protoplasts. Colony formation, however, was obtained after protoplast fusion. Hybrid cytoplasm formation was studied in 0.5 g to 5.0 g calli grown from a fused protoplast after an estimated 19 to 22 cell divisions. Chloroplasts and mitochondria were identified in the calli by hybridizing appropriate DNA probes to total cellular DNA. Out of the 42 clones studied 37 were confirmed as cybrids. Chloroplast segregation was complete at the time of the study. Chloroplasts in all of the cybrid clones were found to derive from the fertile parent. Mitochondrial DNA (mtDNA) segregation was complete in some but not all of the clones. In the cybrids, mtDNA was different from the parental plants. Physical mapping revealed recombination in a region which is not normally involved in the formation of subgenomic mtDNA circles. The role of treatments used to facilitate the recovery of cybrids, and of organelle compatibility in hybrid cytoplasm formation is discussed.     

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

Transfer of wild abortive cytoplasmic male sterility through protoplast fusion in rice

Wild abortive cytoplasmic male sterility has been extensively used in hybrid seed production in the tropics. Using protoplast fusion between cytoplasmic male sterile and fertile maintainer lines; we report here, transfer of wild abortive cytoplasmic male sterility to the nuclear background of RCPL1-2C, an advance breeding line which also served as maintainer of this cytoplasm. In total, 27 putative cybrids between V20A and RCPL1-2C and 23 lines between V20A and V20B were recovered and all of them were sterile. DNA blots prepared from the mitochondrial DNA of the cybrid lines from both the sets were probed with orf155 that is known to exhibit polymorphism between the mitochondrial DNA of the male-sterile and fertile maintainer lines. Hybridization of orf155 to 1.3 kb HindIII-digested mitochondrial DNA fragment of the cybrids showed transfer of mitochondrial DNA from wild abortive cytoplasmic male-sterile line to the maintainers, viz. RCPL 1-2C and V20B. Expression of male sterility was confirmed by the presence of sterile pollen grains and the lack of seed setting due to selfing in all the cybrid lines. These cybrids, on crossing with respective fertile maintainers set seeds that in turn, produced sterile BC1 plants. DNA blots from HindIII-digested mitochondrial DNA of these BC1 plants when probed with orf155 again exhibited localization of orf155 in wild abortive cytoplasm-specific 1.3 kb HindIII-digested mitochondrial DNA fragments. This demonstrated that the cytoplasmic male sterility transferred through protoplast fusion retained intact female fertility and was inherited and expressed in BC1 plants. Fusion-derived CMS lines, on pollination with pollen grains from restorer, showed restoration of fertility in all the lines. The results demonstrate that protoplasts fusion can be used for transferring maternally inherited traits like cytoplasmic male sterility to the desired nuclear background which can, in turn, be used in hybrid seed production programme of rice in the tropical world.     

Plant regeneration from protoplasts of cytoplasmic male sterile lines of rice (Oryza sativa L.)

This study compared plant regeneration from protoplasts isolated from suspension cultures of threeJaponica rice (Oryza sativa L.) lines with different male sterile cytoplasms. More than 180 green plants were regenerated from protoplasts from 5–8 month old suspensions of IR58024A, a line with the WA type of cytoplasmic male sterility (CMS). About 40% of the calli recovered from protoplasts produced green plants. ShuangbaiA (BT type of CMS) and Tai2A (Dian I type of CMS), both from Zhejiang province of China, responded less well in culture. ShuangbaiA produced green plants from 6.6% of calli, although initial protoplast yield per gram fresh weight was higher than for IR58024A. Tai2A showed lower protoplast yield, and only 1.1% of the calli produced green plants. Flow cytometric analyses of nuclear DNA content indicated that many of the regenerated plants were tetraploid. The percentage of tetraploids varied in the different lines. The male sterile characteristics of the original lines were maintained in the regenerated plants. Pollen abortion occured earliest in IR58024A and latest in Tai2A. IR58024A is a promising rice genotype for use as a recipient in direct gene transfer experiments.Abbreviations BAP 6-benzylaminopurine - CMS cytoplasmic male sterility - 2,4-D 2,4-dichlorophenoxyacetic acid - IRRI International Rice Research Institute - LS Linsmaier and Skoog's (1965) medium - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - WA wild abortive     

A unique cytoplasmic male sterility (CMS) determinant is present in three Phaseolus species characterized by different mitochondrial genomes

Previous results have shown that cytoplasmic male sterility (CMS) in lines from Phaseolus coccineus and Phaseolus vulgaris contain the same CMS-specific sequence, raising the question of whether this sequence rearrangement arose before divergence of the two species or afterward with subsequent transfer by introgression. Hybridization patterns of total DNA from eight P. vulgaris lines with cytoplasm from P. coccineus and three P. vulgaris lines were examined in order to analyze the mitochondrial DNA (mtDNA) diversity within each species and to determine differences between CMS lines derived from the two species. Three restriction enzymes and 17 heterologous mtDNA sequences were used. The analysis of the different hybridization patterns revealed a considerable diversity in mtDNA organization particularly within P. coccineus. We obtained distinctive hybridization patterns for the five CMS lines tested. The resulting classification showed that mitochondrial genomes from P. coccineus CMS lines group with those of fertile P. coccineus but not with CMS lines from P. vulgaris. The groupings concur with the taxonomic classification of these lines. The results support the hypothesis of a single ancient origin of the CMS determinant and exclude the transfer of cytoplasm by introgression from P. vulgaris to P. coccineus and P. coccineus ssp polyanthus.     

Intra- and inter-specific variations in the mitochondrial gene orf138 of Ogura-type male-sterile cytoplasm from Raphanus sativus and Raphanus raphanistrum

In order to gain a better understanding of the evolution of Ogura male-sterile cytoplasm in radish, a large-scale sequence analysis of mitochondrial orf138 was conducted using 107 Japanese wild radishes, 29 cultivated radishes and seven Raphanus raphanisturum. A single approximately 0.8-kb fragment containing the orf138 locus was amplified from each plant by PCR, and the nucleotide sequence of an entire coding region of orf138 was determined by direct-sequencing procedures. An identical sequence to the published orf138 (Type A) was identified in Japanese wild radish, including a single plant in a population near Kagoshima prefecture where Ogura (1968) first found ’Ogura male-sterile radish’. Thus, it was confirmed that the ’Ogura male-sterile cytoplasm’ was derived from Japanese wild radish, with a Type A orf138 sequence, growing in this area. A total of six nucleotide changes and a single insertion/deletion (indel) were found in orf138 from both wild and cultivated radishes. By a combination of mutations, the orf138 sequences of the 143 radish plants were classified into nine types. Based on the pattern of mutations and the distribution of orf138 variants, it was concluded that the orf138 variants are derived from Type B or C, after Ogura-type cytoplasm was introduced from R. raphanistrum into Japanese wild radish. Received: 19 December 2000 / Accepted. 26 January 2001     

Production of new CMS Brassica oleracea by transfer of `Anand' cytoplasm from B. rapa through protoplast fusion

New types of cytoplasmic male sterility (CMS) in Brassica oleracea would be useful for F₁ hybrid seed production. The `Anand' cytoplasm derives from the wild species B. tournefortii. Rapid cycling stocks of B. rapa and B. oleracea were used in cybridization experiments as donor and recipient of `Anand' (=`tour') CMS, respectively. Prior to fusion with PEG, donor protoplasts were inactivated with 30 krad γ-rays and recipient ones with 3 mM iodoacetate, respectively. No calli were obtained from the pre-treated protoplasts. The frequency of shoot regeneration was 21–43% in untreated B. oleracea controls, but only 0–0.5% in `Anand' B. rapa. Putative cybrids were regenerated from about 3% of the calli from fused protoplasts. Regenerated plants were analyzed for nuclear DNA content, plant and flower morphology, pollen production, female fertility, cold tolerance, and organelle composition. Eighty-one percent of the regenerated controls and 63% of fusion-derived plants were diploid. The rest showed DNA contents corresponding to 2x–4x, 4x, or higher ploidy levels, presumably due to somatic doubling in vitro and/or fusions in which the donor nucleus was not completely eliminated. Sixty-four percent of the cybrids had stamens and petals varying in size and shape and were male-sterile, with indehiscent anthers. Their phenotype was otherwise similar to that of B. oleracea. The remaining plants had normal flowers and were male-fertile. Data from crosses with fertile pollinators indicated good female fertility in some of the sterile lines, both after hand and insect pollinations in cages. Mitochondrial (mt) segregation in the cybrids was slightly biased towards `Anand' mitochondria, and the presence of `Anand' mtDNA fragments was strongly associated with male sterility. Evidence of mtDNA rearrangements was obtained in some cybrids. Segregation of chloroplasts was slightly biased towards B. oleracea. The presence of `Anand' chloroplasts with a B. oleracea nucleus did not result in cold temperature chlorosis, as seen in `Ogura' CMS plants. Received: 22 February 1996 / Accepted: 10 May 1996     

GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage

Somatic hybridization was performed via electrofusion between embryogenic suspension-derived protoplasts of transgenic green fluorescent protein (GFP) Satsuma mandarin (Citrus unshiu Marc. cv. Guoqing No. 1) (G1) callus and mesophyll protoplasts of calamondin (Citrus microcarpa Bunge), and three embryoids expressing GFP under UV light were obtained after 60 days of culture. The three embryoids were considered not as diploid cybrids but true allotetraploid somatic hybrids, as it was based on: (1) citrus heterokaryons are generally more vigorous and have higher capacity for embryogenesis as compared with unfused and homo-fused embryogenic callus protoplasts; (2) the callus line of G1 Satsuma mandarin has lost the embryogenesis capacity; and (3) citrus diploid cybrids produced by symmetric fusion always possess nuclear genome of mesophyll parent, and calamondin without GFP gene was used as leaf parent in this study. Subsequent flow cytometry, simple sequence repeat and cleaved amplified polymorphic sequence analysis of one regenerated callus mass and three resulting plants validated this supposition, i.e., the callus was derived from transgenic G1 callus protoplasts, and the three plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from callus parent. The potential of transgenic GFP citrus callus as suspension parent in citrus somatic fusion to study the mechanism of cybrid formation, create new citrus cybrids, and transfer organelle-encoded agronomic traits was also discussed.     

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.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat