Cybrid production based on mutagenic inactivation of protoplasts and rescuing of mutant plastids in fusion products: potato with a plastome from S. bulbocastanum and S. pinnatisectum

• Published in: Theoretical and applied genetics Vol. 88; no. 5; pp. 525 - 529
• Main Authors: Sidorov, V.A, Yevtushendo, D.P, Shakhovsky, A.M, Gleba, Y.Y
• Format: Journal Article
• Language: English
• Published: 01-07-1994
• Subjects: chloroplast DNA; chloroplasts; DNA; gamma radiation; genomics; hybrids; interspecific hybridization; irradiation; mutagenesis; mutants; N-methyl-N-nitrosourea; protoplast fusion; protoplasts; restriction mapping; Solanum bulbocastanum; Solanum pinnatisectum; Solanum tuberosum; somatic hybridization
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/BF01240913

A procedure for cybrid production, based on double treatment of donor protoplasts by physical and afterwards chemical mutagens at superlethal doses (gamma irradiation at a dose of 1000 Gy was applied for the inactivation of nuclei: 3-5 mM N-nitroso-N-methylurea was used for the efficient induction of plastome mutation) and the rescuing of mutant plastids after fusion with untreated recipient protoplasts, was developed. For identification of mutant donor-type plastids in fusion products a selection for streptomycin was performed. In two sets of experiments, in which S. tuberosum served as the recipient of foreign cytoplasm with the wild tuber-bearing species S. bulbocastanum and S. pinnatisectum as donors, a total of about 40 streptomycin-resistant colonies was isolated. Eight regenerants from the S. tuberosum + S. bulbacastanum fusion combination and four from S. tuberosum + S. pinnatisectum were further investigated using chromosome counting, analysis of esterase isoenzymes, restriction analysis of organelle DNA, and blot hybridization. All but one plant from both combinations were characterised as potato cybrids possessing exclusively foreign plastids and retaining a morphology typical of the recipient. Only in one line was rearranged mtDNA detected. The availability of potato cybrids facilitates the analysis of plastome-encoded breeding traits and the identification of the most valuable source of cytoplasm among the wild potato species. The described system for producing cybrids without genetic selectable markers in the parental material offers the possibility for the rescue of cytoplasmic mutations which are impossible to isolate by conventional approaches.