| Project/Area Number |
61560048
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
植物保護
|
|---|
| Research Institution | Tokyo University of Agriculture and Technology |
|---|
Principal Investigator |
AOKI Joji Faculty of Agriculture, Tokyo University of Agriculture & Technology, 農学部, 教授 (30014905)
|
|---|
| Project Period (FY) |
1986 – 1987
|
| Project Status |
Completed (Fiscal Year 1987)
|
| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1987: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1986: ¥1,400,000 (Direct Cost: ¥1,400,000)
|
|---|
| Keywords | Entomophthoraean fungi / Protoplast production / Protoplast fusion / プロトプラスト融合 / プロトプラスト再生 / 対昆虫強病原性糸状菌の選抜育種 / 薬剤耐性菌株の選抜 / プロトプラストの作出 / 融合 / 再生 |
|---|
| Research Abstract |
Studies on the production, fusion, and regeneration of protoplasts were conducted on several entomophthoralean fungi. The fungi were inoculated into Sabouraud dextrose broth and incubated at 25゜ on a rotary shaker for 3-4 days. Hyphal bodies were harvested by filtration through sterile cheesecloth and centrifuged at 2000 rpm for 10 minutes in 0.15 M Sorensen phosphate buffer. The washed hyphal bodies were suspended in a lytic enzyme solution consisteing of phosphate buffer, cellulase, macerozyme, driselase and an osmotic stabilizer. The hyphal bodies were gently shaken in the lytic solution for 3-24 hours at 20゜ C. Efficiency of protoplast production was determined by comparing hemocytometer counts of protoplasts. Phosphate buffer(pH 5.9) containing 10% each of the three kinds of lytic enzymes suplemented with 0.6 M mannitol as osmotic stabilizer was determined as the most effective solution for protoplast production. DNA staining of protoplasts with 4'-6-diamidino-2-phenylindole (DAPI) suggested that about 90% of the protoplasts contained a nucleus. Nuclei were also observed by electron microscopy. Many of the protoplasts fused with each other when treated with 40% polyethylene glycol containing CaCl_2 as an osmotic stabilizer. Protoplast fusion occurred within 10 minutes of treatment. Protoplasts regenerated hyphal structures in Grace's insect medium. Regenerating protoplasts stained well with DAPI. Likewise, the periphery of regenerated hyphae were stained by fluorescein isothiocyanate-labeled wheat germ agglutinin and concanabalin A. These results suggest that protoplasts possess high metabolic activity and that cell wall formation occurs rapidly when regeneration is initiated.
|
