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

利用蓝猪耳属的三种植物和范德利亚属的两种植物，发现花粉管吸引信号具有种特异性。在这些植物中，胚囊从胚珠中伸出，并且通过激光消融证实了吸引信号来自助细胞。蓝猪耳胚珠与其它种胚珠共培养时，各种的花粉管倾向于向同一种的胚囊生长。此外，还发现种属特异性可能是导致体内生殖障碍的原因之一。与物种特异性一致，先前的引诱剂候选物如Ca^<2+>和GABA被证明不是来自助细胞的引诱剂。作者还开发了一套配子体活细胞可视化系统。通过化学荧光探针染色和灵敏的照相系统，揭示了两种精子细胞的行为和动力学。还制备了使用GFP的转化系统。将拟南芥ACT 11启动子与GFP基因融合后，用基因枪轰击蓝猪耳胚囊，在胚囊中观察到明亮的荧光。这些技术的建立将是重要的，为今后的工作。

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.