| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Culture conditions for pollen germination of Lilium longiflorum on a synthetic medium were established: 1 mM CaCl_2, 1mM H_3BO_3 and 0.3 M sucrose were required for an agar medium, 0.1 mM Ca(NO_3)_2, 0.5mM H_3BO_3 and 0.3 M sucrose for a liquid medium, with a pollen density of 0-3 mg/ml. Microanalyses of calcium and boron using X-ray energy spectrometer were not successful because of difficulty in pollen tube sample preparation.
EDTA and EGTA, a chelator, and H-7 as protein kinase C inhibitor, stimulated pollen germination at the early stage of germination, whereas protein synthesis inhibitor cycloheximide (CHI) inhibited the germination. However, treatment of pollen with CHI followed by EDTA treatment stimulated the germination rather than inhibited it. This suggests that protein actions required for the germination can be replaced with EDTA and that calcium may interact with cell wall and/or plasma membrane.
Fatty acid composition of phosphatidylinositol (PI) changed during the pollen germination, distribution of unsaturated fatty acids such as 18:2 and 18:3 reduced while percentage of 16:0 increased. This change with PI was different from that of phosphatidylcholine. Although detection of inositol triphsphate (IP_3) has not been successful in the pollen germination system in our experiment, the above result suggests that PI-mediated calcium regulation system may play an important role during pollination.
To examine calcium effect on cell wall loosening, cell wall degrading enzgme activities were assayed. Significant activities of polygalacturonase and beta-1, 3-glucanase were detected. However, addition of calcium to the medium neither stimulated nor inhibited these enzyme activitie. Now we successfully purified beta-1, 3-glucanase from L. longiflorum leaves to administrate to rabbit for preparation of anti-beta-1, 3-glucanase serum and immuno-histological investigation of pollen termination in connection with calcium and boron action.
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