Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice

Twelve Japanese rice cultivars were converted to CMS by asymmetric protoplast fusion with MTC-5A, the cytoplasm of which was derived from an indica rice, Chinsurah Boro II. With the exception of the cybrids that had a nucleus from Hoshiyutaka, most of these cybrid plants were sterile. The unique sequence downstream from the mitochondrial atp6 of MTC-5A was specifically amplified in the sterile cybrid plants by PCR. All progenies of the cybrid plants carrying this unique sequence were sterile. On the other hand, in some of the sterile cybrid plants in which the unique sequence was not amplified by PCR, fertility was recovered in their progenies. Somaclonal mutation may have caused sterility in these cybrids. Only the cybrid plants that had the unique sequence detected by PCR were CMS. Thus, the CMS plants can be selected rapidly and easily by PCR, at an early stage of plant regeneration. Soon after transplanting the regenerated plants to a green house, fertile cybrids and sterile cybrids produced by somaclonal mutation can be removed. These findings also show that the unique region downstream from atp6 is tightly linked with the CMS phenotype.     

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.     

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.     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

On the mechanism of cytoplasmic male sterility in the 447 line of Vicia faba

The trait of cytoplasmic male sterility, expressed in plants bearing the 447 cytoplasm of Vicia faba, is uniquely and positively correlated with the presence of a linear double-stranded RNA molecule (dsRNA) 16.7 kb in size. Restriction enzyme digestion profiles of mitochondrial DNA isolated from fertile and cytoplasmic malesterile (CMS) lines do show a limited number of specific differences in fragment intensities and mobilities. However, mitochondria isolated from the progeny of the cross CMS × Restorer line contain DNA with an identical restriction profile as the male-sterile parent: moreover, subsequent generations are completely and permanently fertile, even upon segregation of the nuclear restoration gene. Southern hybridizations, using cDNA clones as probes, reveal homology between the CMS-associated dsRNA and the nuclear genome of both sterile and fertile lines. The regions cloned, representing approximately 22% of the total dsRNA sequence, show no homology to organelle DNA. We have not been able to stably transmit the dsRNA to fertile lines of V. faba or any other plant species, using a variety of standard virological techniques.     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F₁ seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC₃ generations.     

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.     

Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii

Cytogenetic studies were performed on four male-sterile progenies derived from four different cybrids produced between Brassica napus and B. tournefortii using the donor-recipient protoplast fusion method. The objective of these studies was to characterize the nuclear constitution of the plants. Mitotic investigation revealed that three of the four male-sterile lines had 38 chromosomes, which is equal to that of B. napus. The fourth line, C6, had variable chromosome numbers, ranging from 39 to 42 in different plants. The meiotic behavior in each progeny varied distinctly. Of the plants having 38 chromosomes, fairly high chromosome pairing, on average 18.08 bivalents per cell, was detected at metaphase-I. However, univalents with an average of 1.39 per cell, and very low frequencies of trivalents and/or tetravalents, were also observed in the lines. These results revealed that male-sterile cybrid lines were obtained with 38 chromosomes and a relatively high level of chromosome-pairing ability, indicating their potential for establishing a stable male-sterile rapeseed line. Received:15 December 1998 / Accepted:30 January 1999     

Protoplast-fusion-derived Solanum cybrids: application and phylogenetic limitations

Summary We established interspecific Solanum cybrids in order to study the intrageneric nuclear-organelle compatibility and the introgression of advantageous plasmone-coded breeding traits into potato. Cybridization was performed by the donor-recipient protoplast-fusion procedure. We found that the plastomes of S. chacoense, S. brevidens, and S. etuberosum could be transferred into the cybrids having S. tuberosum nuclear genomes; chondriome components were likewise transferred from the former species into these cybrids. The combination with S. chacoense as organelle donor and potato as recipient resulted in green fertile plants with potato morphology. By using S. etuberosum as an organelle donor and potato as recipient, male-sterile cybrid plants, most of them having pigmentation abnormalities, were obtained. The combination of S. brevidens with potato resulted in palegreen (almost albino) regenerants. The latter albino plantlets had both the chloroplast DNA and the mitochondrial DNA of the donor (S. brevidens) and did not survive the transfer into the greenhouse. An immediately applicative result of this study is the de novo establishment of male-sterile plants in a potato cultivar. Such plants should be useful as seed parents in the production of hybrid, true-potato seeds.     

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.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

Downy mildew incidence of pearl millet hybrids with different male-sterility inducing cytoplasms

The use of different sources of cytoplasmic male sterility (CMS) in hybrid seed production of pearl millet [Pennisetum glaucum (L.) R. Br.] is advocated to avoid possible disease epidemics occurring due to cytoplasmic uniformity. The effects of commercially unexploited, but potentially exploitable, sources of CMS, like A₂, A₃ and A₄, on downy mildew [Sclerospora graminicola (Sacc.) Schroet] incidence were studied by using the disease incidence of isonuclear hybrids with male-sterile and fertile cytoplasm. The mean downy mildew incidence of hybrids carrying different male-sterile cytoplasm was similar to that of hybrids retaining the fertile cytoplasm. The cytoplasm accounted for only 0.6% of the total variation and its effect was non-significant; pollinators could explain most of the variation in determining the disease incidence of hybrids. This suggested that these male-sterile cytoplasms are not linked to downy mildew susceptibility and thus can be exploited commercially to broaden the cytoplasmic base of the male-sterile lines and, ultimately, of hybrids.     

Somatic transfer of cytoplasmic traits in Brassica napus L. by haploid protoplast fusion

Summary Fusions between protoplasts from haploid cytoplasmic atrazine resistant (CATR) and haploid cytoplasmic male sterile (CMS) Brassica napus plants were used to produce a diploid CMS/CATR cybrid. The hybrid nature of the cytoplasm was confirmed by comparing the EcoRI restriction fragment patterns of chloroplast and mitochondrial DNA from the cybrid with the parental patterns. The advantages of using haploid protoplasts for fusion experiments as well as the utilization of the CMS/CATR cybrid for hybrid seed production are discussed.     

CYTOLOGICAL DIFFERENCES BETWEEN A CYTOPLASMIC MALE STERILE TOBACCO CYBRID AND ITS FERTILE COUNTERPART DURING EARLY ANTHER DEVELOPMENT

Ultrastructural differences were detected between a cytoplasmic male sterile tobacco cybrid (Nicotiana sp.) formed by protoplast fusion and normal, fertile tobacco. Cell structure was compared between anther primordia from normal, fertile tobacco and anther primordia from the cybrid using stereological methods. Particular emphasis was placed on the ultrastructure of mitochondria because of their known relationship to cytoplasmic male sterility in this cybrid and other plants. The volume density of mitochondria in cybrid anther primordia (6.3%) was significantly lower than in normal, fertile anther primordia (10.1%), although mitochondria from both plants contained similar amounts of cristae and profiles were of similar relative area. Dictyosomes and vacuoles also differed in volume density but not at a statistically significant level. Although the volume density of plastids did not differ, a larger amount of starch was stored within plastids in cybrid anther primordia than in normal, fertile anther primordia. These results are compatible with the hypothesis that an abnormally low rate of mitchondrial replication, and the resultant limit on adenosine triphosphate production, could contribute to cytoplasmic male sterility in the cybrid.     

Chondriome analysis in sexual progenies of Nicotiana cybrids

Summary We studied the chondriomes (the mitochondrial genomes) of sexual-progeny plants derived from eleven Nicotiana cybrids which resulted from donor-recipient protoplast fusions. The recipients were either N. tabacum or N. sylvestris and the donor (of the cytoplasm) was N. bigelovii. The chondriomes were characterized by the mitochondrial DNA (mtDNA) restriction-patterns. The differences in mtDNA restriction patterns were revealed after Sal I digestions and probing the respective Southern-blots with three mtDNA fragments. The hybridization patterns of mtDNAs from 35 second-generation plants (i.e. the sexual progeny derived from the cybrid plants) indicated only minor variations between plants derived from the same cybrid but pronounced variations among sibs derived from different cybrids. The mtDNA of 32 second-generation plants varied from both original fusion partners but the mtDNA of one (male-sterile) plant was apparently identical with the mtDNA of one of the original donor (N. bigelovii) and the mtDNA of two other (male-fertile) plants was apparently identical to the mtDNA of an original recipient (N. sylvestris). Generally, the mtDNAs of male-fertile, second-generation plants were similar to the mtDNAs of the original recipients while the mtDNAs of the male-sterile second-generation plants were similar to the mtDNA of the donor (N. begelovii). The analyses of mtDNAs from the thirdgeneration plants indicated stabilization of the chondriomes; no variations were detected between the mtDNAs of plants derived from a given second-generation plant.     

Direct transfer of a cold-tolerant Ogura male-sterile cytoplasm into cabbage (Brassica oleracea ssp. capitata) via protoplast fusion

Cold tolerant cytoplasmic male-sterile (CMS) cabbage (Brassica oleracea var. capitata) was produced by the fusion of leaf protoplasts from fertile cabbage and cold-tolerant Ogura CMS broccoli lines. The cabbage lines tested showed great variation in plant regeneration from unfused protoplasts; three with high regenerability were selected as the fusion partners. Several procedures for eliminating the nuclear DNA of the broccoli fusion partner were tested. Diploid cabbage plants were identified by flow cytometry and morphological characters. Gamma-irradiation (30 krad) was the most successful procedure; isolation of cytoplasts from broccoli leaf protoplasts, followed by gamma-irradiation of the cytoplast fraction, also produced diploids. UV-irradiation of the broccoli protoplasts was less effective. PCR using primers for an Ogura CMS-specific mitochondrial DNA sequence permitted the identification of cybrids likely to be CMS. Over 200 diploid plants with the CMS-specific sequence were obtained from 66 independent fusion products and three cabbage lines. Plants were ready for transfer into soil within 8 months after fusion. The plants identified as CMS by PCR produced male-sterile flowers. Our procedures permit the transfer of a desirable male-sterile cytoplasm into cabbage much more rapidly than conventional backcrossing procedures. Received: 4 June 1996 / Accepted: 2 August 1996