Twenty-nine cybrids possessing an Atropa belladonna nuclear genome and a Nicotiana tabacum plastome were selected from two independent protoplast fusion experiments. In contrast to the previously described reciprocal, green and fertile cybrids with a Nicotiana nuclear genome and an Atropa plastome (Kushnir et al. 1987), the plants obtained were totally chlorophyll-deficient. An Atropa nuclear genome and a Nicotiana plastome from these chlorophyll-deficient cybrids were combined with an Atropa or a Scopolia plastome and a Nicotiana nuclear genome, respectively, in control fusion experiments. All of these nuclear genome/plastome combinations gave rise to normal, green plants. Therefore, we conclude that an N. tabacum plastome is incompatible with an A. belladonna nuclear genome.
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.
Asymmetric intergeneric hybrid plants were obtained through protoplast fusion between Orychophragmus violaceus (L.) O.E. Schulz and Lesquerella fendleri (Gray) Wats. The latter carried chloroplasts transformed with the fused aadA16gfp gene construct, conferring streptomycin-spectinomycin resistance and UV-induced green fluorescence. The somatic hybrids were selected using the properties of spectinomycin-induced plastid defects in ''albino'' O. violaceus plants (chloroplast recipient) combined with the c-irradiation-induced inactivation of nuclei in plastid donor L. fendleri. The morphology and esterase isozyme pattern of the hybrid plant as well as the results of the PCR analysis of internal transcribed spacer of nuclear ribosomal DNA proved that the regenerated hybrids carried O. violaceus nuclei, while PCR amplification of the atpB-rbcL spacer and aadA16gfp gene fragments confirmed the presence of the transformed L. fendleri chloroplasts in these plants. Expression of the fused aadA16gfp gene construct was confirmed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis and the resistance of the obtained plants to both streptomycin and spectinomycin.
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