A new CMS source in Nicotiana developed via somatic cybridization between N. tabacum and N. alata

 Cytoplasmic somatic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Nicotiana alata were constructed. A total of 33 green regenerants were obtained after fusion of protoplasts from a tobacco cytoplasmic chlorophyll-deficient mutant and gamma irradiation-inactivated leaf protoplasts of N. alata. Twenty nine of them were male sterile and displayed an altered stamen morphology (formation of petaloid and stigmoid structures instead of stamens). Southern-blot analyses of eight CMS plants using N. alata-specific nuclear repetitive DNA and cpDNA probes revealed that they contained nuclear genetic material of N. tabacum and chloroplasts from N. alata. Restriction-enzyme analysis of mitochondrial DNAs of the cybrids in question showed different patterns consisting of an incomplete mix of mtDNA fragments from both parents, as well as new fragments. Southern-blot analysis of mtDNAs with a sunflower atpA probe gave the same recombinant hybridization pattern for all analyzed cybrids, indicating that high-frequency specific recombination occurs in the atpA region. Analysis of the progeny from three successive backcrosses of the studied cybrids with N. tabacum demonstrated a strict cytoplasmic inheritance of the male-sterile phenotype. Received: 10 May 1997 / Accepted: 31 March 1998     

Morphological changes in homeotic cytoplasmic male-sterile carrots combined with fertile cytoplasm by asymmetrical cell fusion

 The cytoplasmic male-sterile (CMS) carrot (Daucus carota ssp. sativus) with the petaloid phenotype was asymmetrically fused with eight different fertile cytoplasms to convert the CMS to a fertile state. Restoration to the fertile phenotype was successful with an over 20% efficiency. Cybrids with brown anther sterile, incomplete petaloid sterile, or "combined flower" fused on the same axis were also observed. Restricted DNA fragment patterns revealed that the mitochondrial genome organizations of the cybrids were not identical to those of their parents but were of an intermediate type. Repeated cell fusion to introduce two different foreign cytoplasms into the CMS cytoplasm was effective for obtaining fertile plants. The role of mitochondrial factors which regulate flower organ morphogenesis was demonstrated. Received: 14 December 1998 / Revision received: 7 March 1999 · Accepted: 3 June 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.     

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.     

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     

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.     

Novel 'homeotic' CMS patterns generated in Nicotiana via cybridization with Hyoscyamus and Scopolia

Cytoplasmic hybrids (cybrids) that contain nuclear genetic materialfrom Nicotiana tabacum and cytoplasms from Hyoscyamus nigeror Scopolia carniolica were constructed by protoplast fusions.Both types of hybrids exhibited cytoplasmic male sterility (CMS).Furthermore, unusual floral morphogenesis marked by ‘greenflowers’ deprived of corolla and stamens occurred in themajority of the lines. Backcrosses of these plants with wild-typetobacco demonstrated a maternal inheritance of the ‘greenflower’ trait. After repeated transfer of cytoplasm (‘donor-recipientfusion’) from cytoplasmic hybrid N. tabacum (+H. niger)to albino plastome mutant N. tabacum DSR A15, male sterile tobaccoplants with two types of flowers were recovered (‘greenflowers’ and corolla-containing flowers with transformedstamens). RFLP analysis confirmed that N. tabacum (+ H. niger)and N. tabacum (+S. carniollca) as well as their sexual progeniescontained plastids from H. niger and S. carniolica, respectively.Mitochondrial DNA within the hybrids N. tabacum (+H. niger)originated from H. niger, but was obviously altered. Repeatedparasexual transmission, cybrids in the combination of N. tabacum+N.tabacum (+H. niger), reflected similar characteristics. Cybrids,N. tabacum (+S. carniolica) and their sexual progeny, whichresulted after pollination with wild-type tobacco, containeda modified mtDNA generally originating from tobacco. Furtherhistological analysis established the dramatic difference inthe composition of ‘green flowers’ and flowers ofwild-type tobacco. Therefore, the construction of tobacco cybridswith foreign cytoplasms provides a functional method for thede nova generation of alternative CMS types. Key words: Nicotiana, Hyoscyamus, Scopolia, cybrids, CMS, homeotic patterns     

Restoration of fertility in cytoplasmic male sterile (CMS) Nicotiana Sylvestris by fusion with X-irradiated N. tabacum protoplasts

Summary Restoration of male fertility was achieved by fusing protoplasts from male sterile (CMS) Nicotiana sylvestris plants with X-irradiated protoplasts derived from fertile N. tabacum plants. The CMS N. sylvestris plants were derived from a previous somatic hybridization experiment and contained alien (Line 92) cytoplasm. About one quarter of the regenerated plants were found to be cybrids. i.e. they consisted of N. sylvestris nuclei combined with all or some components of N. tabacum cytoplasm. In one half of these cybrids male fertility was restored to different levels. The chloroplasts of the two parental donors differ in respect to tentoxin sensitivity: chloroplasts of CMS N. sylvestris are sensitive while those of N. tabacum are insensitive. It could therefore be demonstrated that there was an independent segregation of chloroplast type and male fertility/sterility: several somatic cybrids were male fertile but tentoxin sensitive and others were tentoxin insensitive yet they were male sterile. Only in about one half of the somatic cybrids was male fertility restored together with restoration to tentoxin insensitivity.     

Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus

Summary A light and electron microscopic investigation revealed that ogu cytoplasmic male sterility (CMS) in cybrids of Brassica napus is primarily a deficiency of the tapetum and clearly time and site specific. Three patterns of ogu CMS were found, and specific conclusions drawn. First, the partially male fertile cybrid 23 was highly variable. It sometimes produced heterogeneous stamens with an endothecium formed exclusively around the fertile locules, thus delineating each microsporangium as a functional unit. The second type, including cybrids 27, 58 and 85, on the contrary, was stable and completely male sterile. In the four locules of normal length, microspores were observed to die at the vacuolate polarized stage while the tapetum disappeared prematurely through excessive vacuolization by the end of meiosis followed by a rapid autolysis during the tetrad or early free microspore stage. The subepidermal layer of the locule wall failed to form characteristic thickenings. The male-sterile stamens were completely indehiscent. At the time of anthesis they contained only collapsed empty exines adhering to each other. These cybrids, 27, 58 and 85, were closest to the ogu CMS trait of radish and seemed to be the best suited for further use in plant breeding. The third pattern was found in cybrids 77 and 118, which besides showing abortion of the microsporangia also showed a feminization of the stamens. We suggest that this feminization might be due to an alloplasmic situation associating Brassica napus nuclear genes with the mitochondrial DNA of radish.     

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.     

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     

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.     

细胞融合与植物细胞质雄性不育性状改良

原生质体融合技术不仅可以实现核基因组的重组,而且可以实现胞质基因组的重组,为转移由线粒体DNA或叶绿体DNA控制的性状提供了一条捷径。本文综述了获得胞质杂种的方法及其原理,细胞融合在胞质转移及创造新的细胞质雄性不育(CMS)资源上的研究进展及其应用情况,以及CMS在有性后代中的遗传规律,探讨了此育种途径的优势及其在木本果树无籽育种上的应用。     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.