應用花粉管法轉移甘藍基因注射處理時間之探討

Investigations on the DNA Injection Time for Cabbage Gene Transfer via Pollen Tube Pathway

本試驗以花粉管基因導入法進行甘藍基因轉殖，並以螢光顯微鏡及掃描式電子顯微鏡觀察花粉管發芽及受精時間，目的即在探討甘藍應用花分管基因導入法最佳注射外源DNA之時間。結果顯示，注射外源DNA之時期會影響外源基因進入轉殖植物基因組整合重組與表現。甘藍授粉後12小時、24小時均可見花粉管發芽及伸長，但於授粉後24小時，才可達胚珠，經以針筒由柱頭注射外源DNA，　於授粉同時，授粉後12小時，授粉後24小時等三種不同時間進行處理，長角果收穫率分別為40%、30%、35%，無顯著差異，但種子收穫數則以授粉同時處理者最少，授粉後12小時及24小時處理者，平均每果莢種數差異不顯著，實際應用時以授粉後24小時處理較方便。另以盛開之花進行授粉，同一天於全花序注射DNA，探討不同花序位置注射DNA對結實率之影響。結果顯示，花序序號相當於盛開後第二天之花結實較高，即授粉後24小時之花，與不同授粉後時間注射DNA對結實率之影響的結果相同。故建議甘藍利用花粉管基因導入法以授粉後24小時處理最適宜。

The effect of different DNA injection time was investigated to find the optimum time for maximum seed set for gene transfer via pollen tube pathway. Different injection times after pollination had no effect on the percentage of siliquae harvested but affected the amount of seed set The number of seeds per siliqua achieved maximum in the injection at 24 hours after pollination. Furthermore, according to the observation with fluorescence microscope and scanning electron microscope, the pollen tubes of cabbage did penetrate into ovule at 24 hours after pollination. We therefore suggest the optimum time for injection via pollen tube pathway is 24 hours after pollination. Additionally, giving the opening flower an order number 0, up to the inflorescence axis the order number is -1; down to the inflorescence axis the order number is +1, +2, +3 When all flowers were pollinated on the opening thy, and the injection of foreign DNA was performed all at one day, the flowers at the bearing position relative to the inflorescence axis on the order number +1~+4, equivalent to 24 hours after pollination, had the largest number of seeds of harvested siliqua. This result indicated that the bearing position for cabbage flowers can be used as an indication for obtaining proper time for the injection.