Structural analysis of male sterility cytoplasmic gene and induction of male sterility by gene engineering

Research Project

Project/Area Number	61480031
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	Breeding science
Research Institution	Gifu University
Principal Investigator	NISHIKAWA Kozo Gifu University,Professor, 農学部, 教授 (50021671)
Co-Investigator(Kenkyū-buntansha)	KAWAI Keiichi Gifu University,Professor, 農学部, 教授 (00002064) FURUTA Yoshihiko Gifu University,Professor, 農学部, 教授 (20021719) HORITSU Hiroyuki Gifu University,Professor, 農学部, 教授 (60021680)
Project Period (FY)	1986 – 1988
Project Status	Completed (Fiscal Year 1988)
Budget Amount *help	¥6,600,000 (Direct Cost: ¥6,600,000) Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000) Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000) Fiscal Year 1986: ¥5,100,000 (Direct Cost: ¥5,100,000)
Keywords	Callus / Cell fusion / mt-DNA / Cloning / Protoplast / Cytoplasm substitution line / 形質転換 / タバコ / イネ / マイクロインジェクション / 細胞質雄性不稔遺伝子 / mt-DNA / E.coli JM83-pUC-12系
Research Abstract	With the purpose of establishing the gene bank of cytoplasmic factor of male sterility and inducing the male sterile plants by gene engineering, the following attempts have been made with the results described below. Calli of rice varieties, Akenohosi and Siyu 6 developed roots and green spots, from which no complete plant could be regenerated. Fusion products between protoplasts from in vitro cultured cells were obtained, but could not regenerate. From mt-fraction (300mg/30g), which was isolated from in vitro cultured cells, about 30ug of mt-DNA was extracted. The apparent molecular weight amounted to 30 to 50Kb for both varietees. Among 20 to 25 restriction fragments of EcoRI and XhoI were separated by agarose electrophoresis, the largest fragment was about 20Kb and the smallest 0.5Kb, total size of these fragments being 95Kb and 120Kb for Akenohoshi and Siyu 6, respectively. Using these mt-DNA fragments and E. coli JM80-pUC12, six transformed clones were developed. There were two dwarf palnts among 20 regenerated from 63 calli of Burley 21 (a tobacco variety). Protoplasts which were isolated (yield: 106/1g) by treating leaves from the aseptically grown plants with enzyme solution, were grown on the modified MS medium to colonies of about 20 cells after two weeks. From 96 colonies thus obtained 70 normal-looking plants were regenerated. Protoplasts from ms (male sterile) Burley 21 were irradiated by UV emitted from a sterilizer lamp 25 cm apart for 3 min. to inactivate the nucleus and subjected to asymmetrical cell fusion with untreated protoplasts of normal Burley 21 by PEG method. Fusion products on the suitable medium started cell division, but no cytoplasm substitution plant was so far found. Attempts to introduce the cytoplasmic gene of male sterility have been made without success by microinjection.