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Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to (32)P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled (32)P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.

Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high Ca++/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphogenetically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.

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Leaf mesophyll protoplasts of Solanum pinnatisectum (2n=24) γ-irradiated at doses of 200 Gy and consequently unable to divide were fused with untreated protoplasts of genomic chlorophyll deficient mutant IvP 841-1 (2n=24) containing the germplasms of S. tuberosum and S. phureja. Two types of plants differing in their pigmentation characteristics were selected. The regenerants of one group were identified as true somatic hybrids by using isozyme analyses of esterase and aspartate aminotransferase. The anthocyanin marker of S. pinnatisectum was phenotypically expressed in these regenerants and could be used as an additional selection trait for hybrid screening in this species combination. The regenerants of the second group were corrected for the gene controlling chlorophyll deficiency but contained species-specific isozymes of the potato cultivar only. Restriction analysis of chloroplast DNA revealed chloroplasts of the S. pinnatisectum type in all but one of the plants tested. The fusion experiments involving γ-irradiated protoplasts show that this approach in potato reconstruction has the advantage of producing a wide range of genetically novel plants.