| Ginkgo biloba L. is one of the oldest existing relict plants now. It has wide cultivated value. But its ploidy breeding is a worldwide problem. It is proved that there were 2n male gametes in the final product of G. biloba's meiosis when the microsporangium of G. biloba was induced by colchicine. But compared with the successful triploid-breeding experience of other species, some problems need to be solved in the breeding process of triploid G. biloba. In this paper, we used the high performance liquid chromatography, the general cytology staining, the spectrometric analysis, the immunofluorescence staining and 2-DE gel electrophoresis to study the mechanism of the lower mutation rate of 2n male gamete when the microsporangium of G. biloba was induced by colchicine. The discoveries in the experiment results can be described as follows.1) By high performance liquid chromatography, the research found that there was not the colchicine in the normal microsporocyte of G. biloba, and the colchicine liquid can still pass easily through the microsporangium's wall and microsporocyte wall of G. biloba, and the protoplasm of G. biloba can't break the chemical structure of colchicine. So these factors cann't lead to the lower mutation rate of 2n male gamete when the G. biloba's microsporocyte was induced by colchicine.2) By the general cytology staining, it was found that the colchicine can induced the 2n male gamete in the G. biloba, its major induced period was the second division period of the meiosis. And the colchicine-induced microsporocytes of G. biloba grew very slowly and even they stop growing in the meiosis when the microsporangium of G. biloba was induced by colchicine.3) By the ultraviolet and fluorescent spectrometric analysis, the result was that the total protein of G. biloba's microsporocyte and the colchicine can instantly form an association complex it had a stable structure when they interact, and the molecular conformation of the total protein of G. biloba's microsporocyte was changed.4) By immunofluorescence staining, it was testified that some parts of colchicine-induced microsporocytes can't form the spindle when the G. biloba's microsporocyte was induced by colchicine.. But the colchicine was also affected by the own factor of G. biloba, so it could only partially inhibit the process that the microtubules were polymerized by tubulins and the abruption of sister chromosomes in the colchicine-treated microsporangiums.5) The research used 2-DE gel electrophoresis to study the proteins expression pattern of G. biloba microsporocyte under colchicine treatment. Comparing the gel images of colchicine-treated microsporangiums with the controls, two new protein spots appeared, while one original spot disappeared, the expression of one protein was increased and another two were reduced. The change of G biloba's total protein may be affecting the effect of colchicine when the microsporangium of G. biloba was induced by colchicine, and lead to the lower mutation rate of 2n male gamete. |
