Reciprocal transfer of male sterile and normal plasmons in Petunia

Summary The goal in this experiment was to achieve direct plasmon transfer via cell fusion. Two lines were used — a normal fertile line of P. hybrida, and a cytoplasmic male sterile (cms) line with the nuclear background of P. parodii. Two plants phenotypically similar to the original male sterile line were developed from protoplasts, but instead of being cms they were male fertile. On the other hand, two plants typical of the original normal line developed from protoplasts, but they were cms instead of fertile. Chromosome counts were done and in all cases the expected diploid number (=14) was found. Genetic analysis showed that sorting out of cms and fertile segregants was evident in the first and second backcross of the cms cybrids. The fertile type cybrids were stable fertile for several generations of selfing and proper backcrossing. These results are discussed in the light of an earlier fusion experiment in which these two parental lines were involved.Contribution from the Department of Plant Genetics and Breeding, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 991-E, 1984 series     

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     

CMS due to tapetal failure in cybrids between Nicotiana tabacum and Petunia hybrida

Cytoplasmic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Petunia hybrida were constructed. Three green plants were obtained after fusion of leaf protoplasts from a cytoplasmic chlorophyll deficient mutant of tobacco, with iodoacetamide inactivated protoplasts of P. hybrida. All regenerated plants were phenotypically similar to tobacco, but male and female sterile. Chromosome and isoenzyme analyses of the nuclear genome, and restriction and blot hybridization analyses of the organelle composition revealed that the regenerated cybrids possessed nuclear genome of N. tabacum, chloroplasts from P. hybrida and recombinant chondriomes. In vitro culture of ovules from one cybrid plant pollinated by N. tabacum resulted in the regeneration of cytoplasmic male sterile progeny plants. Cross-section of anthers from these CMS plants showed that male sterility was due to a failure of tapetum development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts

X-irradiated protoplasts of a Brassica napus line carrying the Ogura Raphanus sativus male sterile cytoplasm were fused to protoplasts of male fertile B. napus cv. Olga. Plants were regenerated from six out of 34 randomly selected clones. In one clone, Bn(RS)26, a plant with male sterile flowers was obtained. Mitochondria of this plant are non-parental as revealed by DNA-DNA hybridization using a species specific probe. Its chloroplasts, however, derive from the fertile parent which results in loss of the sensitivity to low temperatures associated with R. sativus plastids in the male sterile parent. The novel cytoplasm of the Bn(RS)26 cybrid was transmitted through seed.Abbreviations CMS cytoplasmic male sterile - PEG polyethylene glycol - mtDNA mitochondrial DNA - cpDNA chloroplast DNA     

Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系。以下3种方法则可能通过目标不育系而获得其同型保持系：一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系。它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平。在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值。     

Hydroxycinnamic acid amides in fertile and cytoplasmic male sterile lines of maize

Hydroxycinnamic acid (HCA) amides in fertile and cytoplasmic male sterile lines of maize were determined in reproductive organs, developing grains and cobs. HCA amides occurred in large amounts in the anthers of fertile plants (line F7N) and were absent from the anthers of cytoplasmic male sterile lines (lines F7T and F7C). Restoration of fertility was associated with the production of these compounds (line FC31). Considerable variations were observed in the concentrations of HCA amides at different stages of growth and grain maturation. Changes of HCA amides in the grains which were to produce sterile plants followed a pattern similar to that obtained with the grains which were to produce fertile plants. Accumulation of HCA amides was substantially higher in fertile lines whatever their genotype (F7N, FC31 and F7T x FC31) than in sterile lines. Marked changes occurred in the HCA amide content of embryo and endosperm during grain development. Many changes in HCA amides were observed in cobs during development and maturation, but no substantial differences could be observed between fertile and sterile lines.     

Several nuclear genes control both male sterility and mitochondrial protein synthesis in Nicotiana sylvestris protoclones

Summary Male sterile plants appeared in the progeny of three fertile plants obtained after one cycle of protoplast culture from a fertile botanical line and two androgenetic lines ofNicotiana sylvestris. These plants showed the same foliar and floral abnormalities as the cytoplasmic male sterile (cms) mitochondrial variants obtained after two cycles of culture. We show that male sterility in these plants is controlled by three independent nuclear genes,ms1, ms2 andms3, while no changes can be seen in the mitochondrial genome. However, differences were found between thein organello mitochondrial protein synthesis patterns of male sterile and parent plants. Two reproducible changes were observed: the presence of a new 20 kDa polypeptide and the absence of a 40 kDa one. Such variations were described previously in mitochondrial protein synthesis patterns of the cms lines. Fertile hybrids of male sterile plants showed normal synthesis patterns. The male sterile plants are thus mutated in nuclear genes involved in changes observed in mitochondrial protein synthesis patterns.     

A new fertility restorer locus linked closely to the Rfo locus for cytoplasmic male sterility in radish

In this study, we have investigated a new fertility restorer (Rf) locus for cytoplasmic male sterility (CMS) in radish. We have obtained a CMS-Rf system consisting of sterile line '9802A1', maintainer line '9802B1' and restorer line '9802H'. F(1) plants from cross between sterile line '9802A1' and restorer line '9802H' were all male fertile, self pollination of F(1) plants produced an F(2) segregating population consisting of 600 individuals. The segregating population was found to fit a segregation ratio 3:1 for male fertile and sterile types, indicating that male fertility is restored by a single dominant gene (termed Rfo2) in the CMS-Rf system. Based on the DNA sequence of Rfo/Rfk1 (AJ535623), just one full length gene in the sterile line '9802A1', in the restorer line '9802H' and in the male fertile line '2006H', was cloned, respectively. The three sequences correspond to the same gene with two alleles: Rfob in '9802H' and rfob in '9802A1' and '2006H'. These two alleles differ from Rfo/Rfk1 and rfk1 (AJ535624) alleles by two synonymous base substitutions, respectively. Based on the differences between the Rfob and rfob genes, one PCR-based marker was developed, and designated Marker 1, which is identical to the corresponding region of Rfob by sequence analysis. In the F(2) segregating population described above, the Marker 1 was present in 5 sterile plants and in 453 fertile plants, absent in 4 fertile plants and in 138 sterile plants, and was found to fit a segregation ratio 3:1 indicating that Rfob was single copy in '9802H'. Linkage analysis showed that the Rfo2 locus for our CMS-Rf system was distant from the Rfo locus by about 1.6 cM. The sterile line '9802A1' was pollinated by the male fertile line '2006H' and the resulting F(1) plants were all male fertile. These results indicated that the male fertility of radish CMS can be restored by a new Rf locus, which linked tightly to the Rfo locus.     

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.     

Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

Summary Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.     

Construction of rice cybrid plants

Summary The mitochondrial genomes of rice cells were transferred to a fertile rice variety (N8) from a cytoplasmic male sterile variety (CMS) by asymmetric protoplast fusion based on metabolic complementation. Protoplasts derived from CMS were X-irradiated (125 krad) and electrofused with protoplasts which had been treated with iodoacetamide. Metabolic complementation, presumably between nuclear and cytoplasmic compartments, enabled fused protoplasts to form colonies at high efficiency. Restriction digest analysis of mitochondrial DNA (mtDNA) indicated that hybrid cells carried mtDNA derived from both parents. Of the plants regenerated from hybrid calli, 68% carried a diploid chromosome set (2n=24) and the rest of them carried 48 chromosomes. All of them expressed the aryl acylamidase I deficient phenotype encoded by the recessive allele of the fertile N8 parent. These results indicate that the novel somatic hybrid plants regenerated were cybrids, deriving their nucleus from the iodoacetamide treated parent and their mitochondria from both parents.     

Identification of a Sterility-Inducing Cytoplasm in a Fertile Accession Line of Phaseolus Vulgaris L

Previous investigations into the genetic mechanism of fertility restoration in cytoplasmic male sterile Phaseolus vulgaris suggested that this is a particularly interesting system for the study of nuclear-mitochondrial interactions. This study was conducted to investigate the nature of nuclear-mitochondrial compatibility in fertile accession line G08063, the reported progenitor to the cytoplasmic male sterile line. Results from genetic analysis indicated that fertile line G08063 carried a sterility-inducing cytoplasm with a fertility restoring nuclear genotype. Mitochondrial DNA analysis indicated that the mechanism of fertility restoration by line G08063 was different from that conditioned by Fr, a previously described restorer gene. A mitochondrial DNA sequence associated with sterility and lost upon fertility restoration by nuclear gene Fr was present in the mitochondrial genome of fertile line G08063; this sequence was not carried within the mitochondrial genome of any other P. vulgaris accession line tested.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

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.     

植物细胞质雄性不育系育种的反向核置换技术分析

通过不育细胞质为选择背景,在田间事先鉴定出杂种后代的优异完全不育株,用花药培养或诱导孤雌生殖使其纯合,测定其配合力,可以筛选到优良的目标不育系.以下3种方法则可能通过目标不育系而获得其同型保持系:一是通过体细胞变异(花药培养)产生;二是在不育系孕穗期高温或低温处理使其转换成可育,选择仍具有不育保持能力的作为保持系,或作为轮回亲本,将其细胞核换到可育细胞质中;三是用原生质体融合的方式向不育系导入已杀死细胞核的可育细胞质而获得配套保持系.它可以使杂种优势利用变得有预见性,可能提高现有杂种优势水平.在创造雄性不育新质源,排除微效可育基因,进行不育系的定向改造,选育高配合力不育系,以及加速育种进程等方面具有重要价值.