| Project/Area Number |
05556001
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Breeding science
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
MIKAMI Tetsuo Hokkaido Univ, Fac.of Agr.Professor, 農学部, 教授 (50133715)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAMURE Itsuro Hokkaido Univ.Fac.of Agr.Instructor, 農学部, 助手 (90179557)
NAITO Satoshi Hokkaido Univ, Fac.of Agr.Professor, 農学部, 教授 (20164105)
TANIDA Masatoshi Hokkaido Green Bio Institute, Director, 研究部長
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥18,300,000 (Direct Cost: ¥18,300,000)
Fiscal Year 1995: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1994: ¥4,800,000 (Direct Cost: ¥4,800,000)
Fiscal Year 1993: ¥9,700,000 (Direct Cost: ¥9,700,000)
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| Keywords | Sugarbeet / transformation / Agrobacterium / Ti plasmid / leaflet culture / PCR / hygromycin resistance / Proteinase inhibitor / キモトリプシンインヒビター / 除草剤耐性 / GUS遺伝子 |
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| Research Abstract |
Sugarbeet is a species which has acquired a reputation for recalcitrance to manipulation in vitro, a characteristic which is a major limitation to the routine genetic modification of the crop. We have recently developed a reliable method for high frequency regeneration of plants from leaflet culture of sugarbeet. Seeds were aseptically germinated on solidified MS medium and seedlings (cotyledons with the upper part of hypocotyls) were then cultured on MS medium containing 0.25mg/l BA (Benzyladenine) and 0.1mg/l IBA (Indole butyric acid) . When the seedlings were 4 week old, we isolated leaf explants (5*5mm segments) from newly expanded leaves and placed them on the same medium. Leaf explants formed multiple shoots after 1 to 2 weeks of incubation, with rapid growth of shoots. A large number of newly formed shoots readily rooted on MS medium supplemented with 1.0mg/l IBA and 'could be transferred to the glasshouse where they continued normal growth. The next aim was to develop the Agrobacterium-mediated transformation system using leaflet culture strategy. Binary vectors were used, carrying both HPT-II (hygromycin phosphotransferase) and GUS (beta-glucuronidase) genes. Inoculation of cultured leaflet explants, resulted in the production of transformrd plants, determined by (1) introduced resistance to hygromycin, (2) introduced GUS activity, and (3) PCR and/or Southern blot analysis to show the integration of foreign DNA.
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