Molecular-cytological analysis of plant fertillization and early-embryogenesis by using in vitro fertilization system and micromanipulation techniques

利用3种托伦尼亚和2种凡得利亚花粉管吸引信号，发现了花粉管吸引信号的种特异性。在这些植物中，胚囊从胚珠中突出，通过激光烧蚀证实了吸引信号来自于协同细胞。当托伦花的胚珠与其他物种的胚珠一起培养时，每个物种的花粉管都倾向于向同一物种的胚囊生长。还发现这种物种特异性可能是体内生殖障碍的一个原因。与物种特异性一致，先前的候选引诱剂如Ca^<2+>和GABA被证明不是来自协同细胞的引诱剂。推测其引诱剂可能是在协同细胞中合成的某些分子。我还开发了一个活配子体细胞的可视化系统。通过化学荧光探针染色和灵敏摄像系统，揭示了两个精子细胞的行为和动力学。制备了利用绿色荧光蛋白的转化系统。将融合了拟南芥ACT11启动子的GFP基因轰击后，在Torenia胚囊内观察到明亮的荧光。这里建立的这些技术对以后的工作很重要。

Species specificity in the attraction signal of the pollen tube was found by using three species of Torenia and two species of Vandellia. In these plants, the embryo sac protrudes from the ovule and it was confirmed that the attraction signal is derived from the synergid cell by laser ablation. When ovules of Torenia were cultivated with those of other species, pollen tubes of each species tended to grow toward the embryo sac of the same species. It was also found that the species specificity might be a cause of reproductive barrier in vivo. Consistently with the species specificity, previous candidates for the attractant such as Ca^<2+>and GABA were shown not to be the attractant from the synergid cell. It was suggested that the attractant might be some molecules synthesized in the synergid cell.I also developed a visualization system of living gametophytic cells. By staining with chemical fluorescent probes and using the a sensitive camera system, behavior and kinetics of two sperm cells was revealed. Trnasformation system to use GFP was also prepared. When a GFP gene fused with ACT11 promoter of Arabidopsis was delivered with bombardment, bright fluorescence was observed in the embryo sac of Torenia. these techniques established here would be important for future works.