Summary
Protoplasts of Nicotiana tabacum (SR1), carrying a maternally-inherited streptomycin resistance mutation, were enucleated by centrifugation through a Percoll gradient. The resulting cytoplasts containing resistant plastids, were fused with sensitive Nicotiana plumbaginifolia protoplasts. The SR1 cytoplasts, having no nuclei, were unable to form calli. All resistant clones recovered after fusion-induction were therefore supposed to be derived from interspecific cytoplast-protoplast fusion.
N. plumbaginifolia plants regenerated in 17 out of the 75 resistant clones studied. Plants obtained from eight of these clones were resistant to streptomycin and inherited the resistance maternally, as expected when transferring SR1 plastids into the N. plumbaginifolia nuclear background. Plastid transfer in these plants has been confirmed by the EcoRI restriction pattern of the chloroplast DNA.
In nine clones N. plumbaginifolia plants were sensitive although obtained from initially resistant clones. This phenomenon is explained by the maintenance of plastid heterogeneity on the selective streptomycin medium, and formation of plants from sensitive sectors on the non-selective regeneration medium.
SR1 protoplasts, originally present as “contaminants” in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment. The nuclei from such protoplasts were recovered, however, in the interspecific somatic hybrids (56 clones), and in segregants having the SR1 nucleus but some cytoplasm from N. plumbaginifolia (2 clones). The majority (about 80%) of the recovered resistant clones therefore acquired the streptomycin resistance factor from the rare (2–7%) “contaminating” SR1 protoplasts. This is explained by the protoplasts being more stable during fusion induction.
Similar content being viewed by others
References
Ar-Rushdi AH (1957) The cytogenetics of variegation in a species hybrid in Nicotiana. Genetics 42:312–325
Aviv D, Fluhr R, Edelman M, Galun E (1980) Progeny analysis of the interspecific somatic hybrids: Nicotiana tabacum (CMS)+Nicotiana sylvestris with respect to nuclear and chloroplast markers. Theor Appl Genet 56:145–150
Aviv D, Galun E (1980) Restoration of fertility in cytoplasmic male sterile (CMS) Nicotiana sylvestris by fusion with X-irradiated N. tabacum protoplasts. Theor Appl Genet 58:121–127
Belliard G, Pelletier G, Vedel F, Quetier F (1978) Morphological characteristics and chloroplast DNA distribution in different cytoplasmic parasexual hybrids of Nicotiana tabacum. Mol Gen Genet 165:231–237
Ege T (1980) Fusion of cell fragments as a method in cell genetics. In: Celis JE, Graesmann A, Loyter A (eds) Transfer of cell constitutents into eucaryotic cells. Plenum Press, New York and London, pp 201–233
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127
Lörz H, Paszkowski J, Dierks-Ventling C, Potrykus I (1981) Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells. Physiol Plant 53:385–391
Maliga P, Lázár G, Joó F, H-Nagy A, Menczel L (1977) Restoration of morphogenic potential in Nicotiana by somatic hybridisation. Mol Gen Genet 157:291–296
Maliga P, Sz-Breznovits A, Márton L (1973) Streptomycin-resistant plant from callus culture of haploid tobacco. Nature New Biol 244:29–30
Márton L, Maliga P (1975) Control of resistance in tobacco cells to 5-bromodeoxyuridine by a simple Mendelian factor. Plant Sci Lett 5:77–81
Medgyesy P, Menczel L, Maliga P (1980) The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and isolation of their somatic hybrids. Mol Gen Genet 179:693–698
Menczel L, Galiba G, Nagy F, Maliga P (1982) Dose used to irradiate protoplasts influences the efficiency of chloroplast transfer by protoplast fusion in Nicotiana. Genetics, in press
Menczel L, Nagy F, Kiss Zs, Maliga P (1981) Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum+Nicotiana knightiana: Correlation of resistance to N. tabacum plastids. Theor Appl Genet 59:191–195
Nagy F, Török I, Maliga P (1981) Extensive rearrangements in the mitochondrial DNA in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana. Mol Gen Genet 183:437–439
Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78:453–455
Sidorov VA, Menczel L, Nagy F, Maliga P (1981) Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts. Planta 152:341–345
Vedel F, Quetier F, Bayen M (1976) Specific cleavage of chloroplast DNA from higher plants by EcoRI restriction endonuclease. Nature 263:440–442
Wallin A, Glimelius K, Eriksson T (1978) Enucleation of plant protoplasts by cytochalasin B. Z Pflanzenphysiol 87:333–340
Zelcer A, Aviv D, Galun E (1978) Interspecific transfer of cytoplasmic male sterility by fusion between protoplasts of normal Nicotiana sylvestris and X-ray irradiated protoplasts of male sterile N. tabacum. Z Pflanzenphysiol 90:397–407
Author information
Authors and Affiliations
Additional information
Communicated by G. Melchers
Rights and permissions
About this article
Cite this article
Maliga, P., Lörz, H., Lázár, G. et al. Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana . Molec. Gen. Genet. 185, 211–215 (1982). https://doi.org/10.1007/BF00330789
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330789