喜樹組織培養與喜樹鹼含量之研究

Abstract

喜樹（Camptotheca acuminate Decaisne）富含camptothecin與10-Hydroxycamptothecin等生物鹼成分。 以胚為培殖體誘導癒合組織結果顯示：胚在MS基礎培養基中添加IBA 0.5mg/l與BA 4mg/l、 Picloram 0.5mg/l與Kinetin 4mg/l、NAA 4mg/l與BA 2mg/l及單獨添加NAA 1mg/l之處理下，可以得到較佳的癒合組織生長，其中以NAA 4mg/l與BA 2mg/l組合所誘導出癒合組織較為鬆軟且生長快速，可作為懸浮培養試驗的材料。在多芽體誘導方面，以MS基礎培養基添加IBA 0.5 mg/l與BA 0.5mg/l的誘導效果最佳。根的誘導則以MS基礎培養基添加IBA 2 mg/l效果最佳。 利用喜樹癒合組織細胞建立懸浮培養系統，選擇salicylic acid、chitin、jasmonic acid、methyl jasmonate等四種誘發劑，來探討其10-hydroxycamptothecin生產的影響。添加100μM salicylic acid於MS液態培養基中，經7天培養後10-Hydroxycamptothecin產量887.5μg/g。而加入chitin懸浮3ml/25ml至MS液態培養基中時，10-Hydroxycamptothecin含量可達2502.7μg/g效果較佳。另外jasmonic acid濃度為100μM10-Hydroxycamptothecin之含量可達10246μg/g，為四種誘發劑當中效果最為顯著。而當methyl jasmonate濃度為200μM時，10-Hydroxycamptothecin之含量可達3836.6μg/g。

The happy tree (Camptotheca acuminate Decaisne) is one of the plants containing many alkloids, famous as camptothecin and 10-hydroxycamptothecin. An experiment of embryo in vito was conducted to induce callus from the happy tree. The results showed that the callus grew well under all MS medium treatments of 0.5 mg/l IBA + 4 mg/l BA, 0.5 mg/l picloram + 4 mg/l kinetin, 4 mg/l NAA + 2 mg/l BA, and 1 mg/l NAA, However, the callus obtained from the treatment of 4 mg/l NAA + 2 mg/l BA was soft and facilitated to be used as a material for the suspension culture experiment. In the multiple buds induce culture, the best result was obtained on the MS medium adding 0.5 mg/l IBA + 0.5 mg/l BA. The adventitious roots occurred best on the MS medium with 2 mg/l IBA in the culture. Four elicitor of salicylic acid, chitin, jasmonic acid and methyl jasmontate were selected to investigate 10-hydroxycamptothecin production from the happy tree using obtained callus suspension culture. The best productions of 10-hydroxycamptothecin were 887.5, 2579.5, 10246, 3836.6μg/g under the treatments of 100 μM salicylic acid, 3 ml/25ml chitin, 100 μM jasmocin acid and 200 μM methyl jasmonate, respectively, after a week incubation. Obviously, jasmonic acid has the best effect in inducing 10-hydroxycamptothecine production from the callus suspension culture.