1991 Fiscal Year Final Research Report Summary
Protoplast and pollen culture and their cell fusion of self-incompatible cruciferous vegetables
| Project/Area Number |
02660032
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
園芸・造園学
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| Research Institution | Okayama University, Department of Agriculture |
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Principal Investigator |
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| Project Period (FY) |
1990 – 1991
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| Keywords | Cruciferous vegetables / Protoplast Culture / Electric cell fusion |
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| Research Abstract |
Protoplast culture and electric cell fusion of cruciferous vegetables were studied. Protoplast culture : The most effective conditions for protoplast isolation were to shake sliced 5day-old cotyledons at 27ﾟC for 2 h in the enzyme solution containing 2% Cellulase Y-C)0.2% Pectolyase Y-23, CPW salts and 0.3 M mannitol. Isolated protoplasts were cultured on the initial plating medium, Pelettier's B medium which supplemented 1 mg/l NAA and BA, 0.25 mg/l 2, 4-D, 0.5 M mannitol and 20g/l glucose. Then C medium supplemented with 0.2 mg/l NAA, 1 mg/ BA, 0.1 mg/ 2, 4-D, 0.02 mg/l GA_3 and 2 mg/l zeatin was added every 7-10 days. Colonies were formed after 4 weeks, and transferred to E medium. Colonies developed to calli after 1 month, and they were transferred to E medium supplemented 1 mg/l BA. Regenerated shoots were excited and transferred to MS medium, and most of shoots rooted. Shoot regeneration have not achieved in all radish cultivars except 'Moriguchi' and in turnip cultivar 'Shogoin Ookabu'. Other Brassica species showed shoot formation. Root formation occurred in all cultivars.
Electrofusion : The best condition for alignment of 2 protoplasts was AC electric field from 10V for 19sec to 25V for 5sec in radish, 10V for 43sec to 25V for 15sec in cabbage and 10V for 75sec to 25V for 15sec in kairan.
The condition for membrane breakdown was to apply 2 pulses with 500V DC electric field. In these treatments, the medium was supplied with 1mM Ca.
On the other hand, pollen culture was not formed colony, since protoplasts from pollen grains showed only 2 or 3 cell divisions.
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