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.     

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.     

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     

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.     

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     

Transmission genetics of the somatic hybridization process in Nicotiana

Summary Callus protoplasts of a Nicotiana tabacum chlorophyll-deficient mutant were fused with mesophyll protoplasts from one of following five sources: 4 cmsanalogs of tobacco bearing the cytoplasms of N. plumbaginifolia, N. suaveolens, N. repanda, and N. undulata, respectively, as well as wild species N. glauca. In another series of experiments, callus protoplasts from the chlorophyll-deficient genome Su/Su mutant of tobacco were fused with mesophyll protoplasts of the wild species N. glauca and those of a plastome chlorophyll-deficient tobacco mutant. The screening of hybrids consisted of visual identification followed by mechanical isolation and cloning of heteroplasmic fusion products in microdroplets of nutrient medium. Studies of regenerated plants included the analyses of gross morphology of plants, leaf and flower morphology, analysis of chromosome size and morphology and chromosome numbers, studies of multiple molecular forms of esterase and amylase, analysis of chloroplast DNA restriction patterns and analyses of chlorophyll-deficiency controlled by Su and P ^– genes. The study of progeny of 41 clones representing all species' combinations demonstrated that regenarants of most (63%) clones from intraspecific (for nuclear genes) combinations were cybrid forms, whereas in the case of the fusion N. tabacum + N. glauca, the true nuclear hybrids prevailed and the proportion of cybrids did not exceed 26%. Clones regenerating both hybrid and cybrid plants from the same fusion product were also found.     

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.     

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     

Asymmetric hybridization between cytoplasmic male-sterile (CMS) and fertile rice (Oryza safiva L.) protoplasts

Summary ⁶⁰Co-irradiated protoplasts of the cytoplasmic male-sterile line A-58 CMS (Oryza saliva L.) were electrofused with iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar Fujiminori. Seven of the colonies that formed were identified as cytoplasmic hybrids (cybrids): they all had the peroxidase isozymes of the fertile Fujiminori parent, but contained four plasmid-like DNAs (Bl, B2, B3 and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. In addition, digestion of cybrid mtDNA gave a set of restriction fragments that differed from those of the parents.     

Restoration of male fertileNicotiana by fusion of protoplasts derived from two different cytoplasmic male-sterile cybrids

Summary Using the donor-recipient protoplast-fusion technique, we have recently constructed several alloplasmic-like lines ofNicotiana in which the original cytoplasms (or part of them) of eitherN. tabacum orN. sylvestris were replaced respectively, either byN. undulata or byN. bigelovii cytoplasms. These cybridizations resulted in two kinds of cytoplasmic male-sterile (CMS) cybrid plants:N. tabacum withN. undulata-like cytoplasm andN. sylvestris withN. bigelovii-like cytoplasm. Fusion of protoplasts, derived from the above two CMS types, by the donor-recipient technique, lead to the recovery of 21 cybrid calli. One of these regenerated a cybrid with fertile pollen but having shortened filaments and slighly tappered anthers. Self pollination of the latter cybrid resulted in a second generation progeny having almost normal filaments and anthers. Further selfings produced a third generation in which numerous plants had normal stamens and fertile pollen. Mitochondrial DNA (mtDNA) analysis of second and third generation progenies revealed a novel pattern which differed from each of the parental CMS cybrids and also from the mtDNA of normal, male-fertileNicotiana species. The results suggest that mtDNA recombination between different types of CMS can lead to restoration of male-fertility.Paper presented at the 1st Plant Molecular Biology Congress, Savannah, Georgia, Oct./Nov. 1985.     

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.     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

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).     

The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

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.     

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.     

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

Summary The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using ³²P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.     

Valine resistant plants derived from mutated haploid and diploid protoplasts of Nicotiana sylvestris and N. tabacum

Summary Protoplasts were derived from haploid and diploid Nicotiana sylvestris and N. tabacum. Exposure of the protoplasts to mutagenic doses of ultraviolet (U.V.) radiation prior to two selection rounds in the presence of 4 mM (or 5 mM) and 8 mM of valine, respectively, was required to obtain cell lines with persistent valine resistance. Such lines were obtained from haploid and diploid N. sylvestris protoplasts as well as from haploid protoplasts of N. tabacum but not from (1.8 × 10⁷) diploid N. tabacum protoplasts. The ratio between number of verified valine-resistant cell lines and the initial number of U.V. exposed protoplasts enabled the estimation of the following order of mutation frequency: haploid N. sylvestris > haploid N. tabacum > diploid N. sylvestris. Plants which retained the valine resistance and transmitted it to their sexual progeny were derived from the resistant cell lines.     

Somatic hybridization in Nicotiana: Restoration of photoautotrophy to an albino mutant with defective plastids

Protoplasts of a cytoplasmic albino mutant of Nicotiana tabacum L. characterized by a deficient chloroplast genome were fused with protoplasts of a nitrate-reductase deficient mutant (NR^-) of N. tabacum. Somatic hybrids were obtained where the genome of the NR^- mutant was complemented by the cytoplasmic albino mutant which could synthesize an active nitrate reductase, and the chlorophyll deficiency in the albino mutant was restored by the chloroplasts from the NR^- mutant. Cybrids were also obtained in which the deficient plastids of the cytoplasmic albino mutant were replaced by normal chloroplasts from the NR^- mutant. The system used permitted a simple selection of the hybrids and the cybrids. The NR^- mutant was excluded at the cellular level by transfer of the cells to medium deficient in reduced nitrogen. The cytoplasmic albino mutant grew well on the selective nitrate medium. However, during callus formation, clear differences in the morphology and pigmentation of the calli were found which permitted selection for photoautotrophy at the callus level. The hybrid or cybrid nature of the plants was confirmed by examination of their morphology and chromosome number. Although the fusion partners come from the same species, only one plant showed the white-green variegated pattern typical of that of the cytoplasmic albino parent, indicating that segregation of plastids occurred during development of the calli and regeneration of the plants.