A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic     

Restoration of Fertility in Cytoplasmic Male-Sterile Nicotiana tabacum (Cytoplasm N. bigelovii) by Protoplast Fusion with X-Irradiated Protoplasts of N. tabacum, SR-1

The ‘donor–recipient’ fusion method was usedto investigate the intraspecific transfer of organelles andorganelle-encoded traits from donor to recipient Nicotiana speciesunder conditions which were selective for chloroplast transfer.An alloplasmic male sterile line of N. tabacum carrying thecytoplasm of N. bigelovii as the recipient and N. tabacum SR-1,a mutant with maternallyinherited streptomycin resistance, asthe cytoplasm donor were used. Organelle composition of 13 cybridplants was investigated by analysis of tentoxin and streptomycinsensitivities, chloroplast and mitochondrial DNA restrictionpatterns, and alloplasmic male sterility Chloroplast DNA analysisand the tentoxin test both showed that all 13 cybrid plantspossessed chloroplasts from the N. tabacum, SR-1 parent only.Analysis of mitochondrial DNA from second generation plantsderived from 11 of the original cybrid plants indicated verylittle heterogeneity with nine of the plants expressing pureparental-type mtDNAs. Although conditions were selective fordonor-type chloroplast transfer, the results indicate the generationof novel cytoplasmic combinations; pure donor chloroplasts incombination with either pure or recombinant-type recipient mitochondria.Our results support previous findings that the ‘donor-recipient’fusion method can be used to restore fertility of CMS linesof Nicotiana Key words: Nicotiana, cytoplasmic male sterility, chloroplast DNA, mitochondrial DNA, somatic hybridization     

Asymmetric protoplast fusion aimed at intraspecific transfer of cytoplasmic male sterility (CMS) in Lolium perenne L.

Summary Techniques have been developed for the production of cybrids in Lolium perenne (perennial ryegrass). Gamma-irradiated protoplasts of a cytoplasmically male-sterile breeding line of perennial ryegrass (B200) were fused with iodoacetamide-treated protoplasts of a fertile breeding line (Jon 401). After fusion 25 putative cybrid calli were characterized to determine mitochondrion type and composition of the nuclear genome. Analysis of phosphoglucoisomerase isozyme profiles and determination of the ploidy level by flow cytometry indicated that all of the calli tested essentially contained the nuclear DNA of the fertile line. However, the presence of parts of the nuclear DNA from the sterile line could not be excluded. Southern blotting of total DNA isolated from the parental lines and putative cybrids combined with hybridizations using the mitochondrial probes cox1 and atp6 revealed that the mitochondria of the calli originated from the fertile line (5 calli), the sterile line (5 calli) or from both parental lines (15 calli). The hybridization patterns of the mtDNA from the cybrid calli showed extensive quantitative and qualitative variation, suggesting that fusion-induced inter- or intramolecular mitochondrial recombination had taken place.     

Protoplast fusion mediated transfer of oligomycin resistance from Nicotiana sylvestris to Solanum tuberosum by intergeneric cybridization

Summary We have successfully bridged the intergeneric barriers between Nicotiana and Solanum with respect to chondriome transfer. To enable this transfer we utilized the donor-recipient protoplast-fusion procedure. Consequently protoplasts of a Nicotiana sylvestris line with putativly oligomycin-resistant mitochondria (line Oli ^R 38) were used as irradiated chondriome donors and iodoacetate-treated protoplasts of Solanum tuberosum cv. Desiree served as recipients. The plated fusion products as well as their derived colonies and calli were exposed to gradually increasing levels of oligomycin. The resulting plantlets had potato morphology and were analyzed with respect to their mitochondrial DNA and chloroplast DNA. Fifteen out of 50 regenerated plants were verified as true cybrids. Detailed analyses of one cybrid revealed chondriome components from the oligomycin-resistant donor line, Oli ^R 38, but retention of the plastome of potato. This cybrid was oligomycin-resistant as revealed by root-culture analysis. It was thus verified that due to selection, chondriome components could be transferred from a N. sylvestris donor into a cybrid having all the phenotypic features controlled by the nucleus of the recipient fusion partner (S. tuberosum).     

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.     

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

Summary Chloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F₁ and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.     

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

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

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.     

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.     

Somatic hybridization by microfusion of defined protoplast pairs in Nicotiana: morphological,genetic, and molecular characterization

Summary Somatic hybrid/cybrid plants were obtained by microfusion of defined protoplast pairs from malefertile, streptomycin-resistant Nicotiana tabacum and cytoplasmic male-sterile (cms), streptomycin-sensitive N. tabacum cms (N. bigelovii) after microculture of recovered fusants. Genetic and molecular characterization of the organelle composition of 30 somatic hybrid/cybrid plants was performed. The fate of chloroplasts was assessed by an in vivo assay for streptomycin resistance/ sensitivity using leaf explants (R₀ generation and R₁ seedlings). For the analysis of the mitochondrial (mt) DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA, with three DNA probes of N. sylvestris mitochondrial origin. In addition, detailed histological and scanning electron microscopy studies on flower ontogeny were performed for representative somatic hybrids/cybrids showing interesting flower morphology. The present study demonstrates that electrofusion of individually selected pairs of protoplasts (microfusion) can be used for the controlled somatic hybridization of higher plants.Abbreviations ac alternate current - BAP benzyl aminopurine - cms cytoplasmic male sterile - dc direct current - NAA naphthalenacetic acid - SEM scanning electron microscopy     

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.     

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.     

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.     

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.     

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.     

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     

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     

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.     

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Summary Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km⁺Sr⁺ with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts.