胡瓜及苦瓜基因轉殖系統之研究

Abstract

為了改良胡瓜及苦瓜基因轉殖系統及再生條件，進行培養基配方及轉殖方法之測試，轉殖質體具有過量表現或默化McPAP1基因之構築，有助於瞭解McPAP1對胡蘆科花性的影響及應用於其他胡瓜及苦瓜基因轉殖分析。由癒傷組織再生之過量表現McPAP1胡瓜轉殖株於瓶內開花，默化McPAP1轉殖胡瓜之再生芽體普遍畸形，限制轉殖植株發育出瓶，McPAP1融合綠色螢光蛋白定位構築轉殖效率低。胡瓜以農桿菌生長點注射法，取得具抗生素抗性之McPAP1基因默化及McPAP1蛋白定位擬轉殖株。將McPAP1融合綠色螢光蛋白定位基因構築置於PiggyBac 轉座子系統之載體中，以農桿菌共培養法轉殖至胡瓜癒傷組織，結果顯示轉殖率較pGKU高約2.5倍。以苦瓜品種‘月華’、‘高月’、‘小月’及‘英翠’之子葉、莖段、葉片所誘導出之癒傷組織形態均相似，分裂旺盛之細胞呈現透明鬆散狀，進行含不同濃度kinetin、TDZ、BA組合之培養基，或以不同濃度2,4-D懸浮培養，皆無法誘導芽體再生。將苦瓜種子無菌播種於添加100 µM乙醯丁香酮 (actosyringone, AS) 之培養基白化幼苗的頂端分生組織 (shoot apical meristem, SAM)，以含100 µM AS之 YEB懸浮農桿菌至OD = 0.8的注射液，進行苦瓜生長點農桿菌注射法基因轉殖，有最佳的再生及擬轉殖株抗生素抗性表現。

Not only to improve the generation and transformation efficiency in cucumber (Cucumis sativus L.) and bitter gourd (Momordica charantia L.), but also understand the relationship of McPAP1 and flower sexuality in Cucurbiteae. The effects of medium composition and delivery system were investigated using constructs for overexpression, silencing, and protein localization of McPAP1. Callus generated from cotyledon of cucumber and leaf of bitter gourd were transformed via Agrobacterium-mediated method. McPAP1-overexpressed cucumbers bloomed in vitro and McPAP1-silencing cucumbers produced abnormal shoots. Transposon PiggyBac increased 2.4 folds of transformation efficiency of Agrobacterium-mediated method in cucumber. The types of callus induced from cotyledon, stem, leaf disc of 4 bitter gourd varieties ‘Moon Shine’, ‘High moon’, ‘New moon’, and ‘F-2486’ are all whitish-green and friable. Different concentrations and combinations of kinetin, TDZ, and BA in MS medium and suspension culture in MS medium supplemented with different concentrations of 2,4-D were tested but failed to induced shoots from callus. Shoot apical meristems adapted from 5 to 7-day-old etiolated seedings were injected with Agrobacterium broth in YEB supplemented with 100 µM actosyringone and putative transgenic bitter gourds were obtained.