應用花粉管法於蝴蝶蘭基因轉殖之研究

Studies on Genetic Transformation of Phalaenopsis via Pollen Tube Pathway

本研究主要是以花粉管法（pollen tube pathway）進行蝴蝶蘭之基因轉 殖，希望建立效率高且穩定的轉殖系統，以應用於蝴蝶蘭育種。本試驗主要分為兩部分；（一）花粉管生長及受精之觀察：蝴蝶蘭之子房和大孢子母細胞，於授粉後才開始發育。花粉粒在授粉後三天已發芽長出花粉管。大約在授粉後50天，大孢子母細胞開始進行減數分裂，胚囊之形成應在授粉後60天左右，而受精約在授粉後70天發生，在授粉後100天，胚已大致或型。（二）花粉管法之基因轉殖：以含有GUS和NPT Ⅱ基因之pRT99gus質體進行轉殖，幼胚及幼苗以組織化學法分析GUS活性，已獲得部份的轉殖証據，但由於蝴蝶蘭具有類似GUS酵素的活性，使得分析工作倍感困難。經進一步，抽取花粉管法處理的群體幼苗DNA，進行聚合酶連鎖反應以及南方氏核酸雜交分析，證明花粉管法確實可達到基因轉殖之目的，而且在授粉後第30天左右注射DNA，可得到較佳的轉殖效率。

The purpose of this study was to establish an efficient and stable gene transfer system for Phalaenopsis via pollen tube pathway. For this purpose, the growth and development of ovary and megaspore mother cell in Phalaenopsis was microscopically examined. Pollens germinated readily 3 days after pollination and megaspore mother cells underwent meiotic division at about 50 days after pollination. Ten days post meiosis, megaspore gave rise to the one-nucleate embryo sac. The zygote was observed 70 days after pollination and fertilization presumably occurred at this time. Immature embryos were found 100 days after pollination. Tlie pRT99gus plasmid containing GUS and NFT Ⅱ genes was injected into Phalaenopsis ovary at different times after pollination. Transformed seedlings as identified by polymerase chain reaction using primers specific for GUS gene were found when the time for DNA injection was between 25 and 40 days after pollination. An intrinsic GUS-like enzyme activity was found when the seedlings were 30-day-old. Stable integration was demonstrated by Southern blot analysis of genomic DNA isolated from pooled seedlings.