鐵炮百合癒傷組織再生系統應用於基因轉殖之研究初報

為進行鐵炮百合基因轉殖，先行建立癒傷組織再生系統，培養大量具有強再生能力之癒傷組織培植體，以供基因轉殖研究用。在誘導癒傷組織方面，由鐵炮百合自交胚珠培養，獲得一生長活力旺盛的癒傷組織細胞系「LG41」，外觀淡黃色，呈粉末狀或小顆粒狀，在含2，4-D(3.0 mg/l)及BA(0.25 mg/l)的MS培養基上，可不斷產生新的癒傷組織，形成一循環再生系統，移到不含植物生長調節劑的培養基上培養，能再生完整植株。此外，鐵炮百合花絲容易誘導產生癒傷組織，其成功率為91.8%，因此亦適合作為農桿菌基因轉殖試驗用培植體。以農桿菌轉殖法進行試驗，將鐵炮百合幼嫩花絲浸漬帶有GUS和抗hygromycin基因的農桿菌液，進行感染，花絲浸漬的時間為10，20，30分鐘三種處理，花絲與農桿菌共培養時間為1，2，3日三種處理，分別進行試驗。結果分花絲與農桿菌液浸漬的時間以30分鐘效果較佳，與農桿菌共培養的時間以2日較佳。花絲培養之後產生綠色癒傷組織，在含有40 mg/l hygromycin選擇性培養基上培養，可存活一段時間，但在培養51天之後全部褐化。另以基因槍轉殖法進行試驗，先行純化抽取含GUS及抗hygromycin基因之DNA，包覆在金粒子上，再以Bio-Rad PDS1000/He粒子傳送系統，打進鐵炮百合癒傷組織LG41之細胞內。轉殖後取癒傷組織以組織化學法檢測，結果部份細胞呈藍色反應，顯示已有GUS基因暫時性表達現象。

關鍵字

鐵炮百合；花絲培養；癒傷組織；基因轉殖

並列摘要

Two regeneration systems based on callus were established in Lilium longiflorum Thunb., which were utilized for propagating sufficient material for genetic transformation. The immature self-crossed ovules were cultured in vitro to induce callus formation. A vigorous grown callus line LG41, yellowish with friable or granular appearance, was selected thereafter on MS medium supplemented with 2, 4-Dichlorophenoxy acetic acid (3.0 mg/l) and benzyl aminopurine (0.25 mg/l). The callus could be proliferated continuously. After subcultured on a medium without plant growth regulators, the callus were able to form plantlets. Besides, the filaments were cultured in vitro to induce callus, and the successful rate was 91.8%. Since it was easy to establish a callus regeneration system, the filaments were also applicable for studying gene transformation. The Agrobacterium-mediated transformation experiments were conducted by utilizing filaments cultured system. An Agrobacterium, which contains a plasmid harboring a GUS gene together with a hygromycin resistance gene, was investigated in the experiments. Young flower filaments were immersed in Agrobacterium solution for 10, 20, 30 minutes, respectively. The co-cultured period on a semi-solid medium were 1, 2, 3 days, respectively. The best results showed that the immersion time with Agrobacterium was 30 minutes. The best co-cultured period was two days. The filaments could form green calluses on the selective medium containing 40 mg/l of hygromycin, however, they turned brown 51 days after incubation. Another transformation experiment was conducted through the PDS-1000/He particle delivery system. The purified plasmid DNA molecules were extracted and coated on gold particles in advance, then the gold particles were bombarded into friable calluses. After bombardment, these callus samples were collected to do histochemical assays for GUS expression. A part of the calluses were observed with blue-staining spots, and which appeared constantly during sub-culture period. It indicated that GUS gene could be expressed in Lilium longiflorum callus.

並列關鍵字

Easter lily ； filament culture ； callus ； genetic transformation

國際替代計量

鐵炮百合癒傷組織再生系統應用於基因轉殖之研究初報

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