2001 Fiscal Year Final Research Report Summary
Confocal laser microscopic observation of the effects of culture environment on somatic embryo formation
| Project/Area Number |
10460114
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物環境
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| Research Institution | THE UNIVERSITY OF TOKYO |
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Principal Investigator |
KURATA Kenji Graduate School of Agricultural and Life Sciences, THE UNIVERSITY OF TOKYO, Professor, 大学院・農学生命科学研究科, 教授 (90161736)
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| Co-Investigator(Kenkyū-buntansha) |
IBARAKI Yasuomi Yamaguchi Univ., Faculty of Agriculture, Associate Prof., 農学部, 助教授 (50242160)
KANEKO Keiko Graduate School of Agric. and Life Sciences, THE UNIVERSITY OF TOKYO, Associate Prof., 大学院・農学生命科学研究科, 助手 (50332599)
FUJIWARA Kazuhiro Graduate School of Agric. and Life Sciences, THE UNIVERSITY OF TOKYO, Associate Prof., 大学院・農学生命科学研究科, 助教授 (30211535)
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| Project Period (FY) |
1998 – 2001
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| Keywords | somatic embryos / carrot / calcium / pH / confocal laser microscope |
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| Research Abstract |
Concentrations of Ca^<2+> during carrot somatic embryogenesis was measured based on the ratiometric analysis of fluo-3 and Fra Red fluorescence using a confocal laser microscope. Similarly, pH was also ratiometrically measured using carboxy-SNARF-1 as a fluorescence probe, because Ca^<2+> -ratio varied with pH. The following points were elucidated.
1. Changing the culture medium pH did not alter pH in pro-embryogenic cell masses (PEMs) nor in somatic embryos, but influenced the Ca^<2+>-ratio. Also the temperature affected the Ca^<2+>-ratio.
2. Stable pH (about 6.8) was observed after transferring PEMs to embryo differentiation medium up to day 9 except for day 1, on which pH of 6.0 was observed, where day 0 is the transferring day.
3. [Ca^<2+>] gradually decreased from 0.1 μmolL^<-1> (day 0) to below 0.04 μmolL^<-1> (day 5-7) and sharply increased to 0.4 μmolL^<-1> on day 9 on which globular embryos were first observed.
4. In the case of cell masses which have lost embryogenic potential due to repeated subculturing, pH and [Ca^<2+>] were always below the values observed in the case of embryogenic cell masses. No prompt increase in [Ca^<2+>] was observed.
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