八角蓮再生與轉殖系統之研究

Abstract

八角蓮 Dysosma pleiantha (Hance) Woodson稀有且含有藥用的二次代謝物鬼臼素 (podophyllotoxin)，本試驗以八角蓮不同部位組織作為培植體，以含有不同生長調節劑的培養基進行培養，誘導癒傷組織以建立再生流程，進而利用該流程建立基因轉殖系統，期望應用於提高鬼臼素含量之研究。結果顯示，葉柄在暗培養條件下於添加 0.5 mg/L 2,4-D，及2.0 mg/L NAA之 B5 培養基能誘導出大量的癒傷組織。使用0.5 mg/L 2,4-D，及2.0 mg/L NAA對葉柄有 1.58 倍的增殖效果；使用0.5 mg/L 2,4-D，1.0 mg/L BA，及2.0 mg/L NAA對根有 1.78 倍的增殖效果。癒傷組織培養在含 0.5 mg/L 2,4-D，1.0 mg/L BA，2.0 mg/L NAA 的 B5 培養基上培養，體胚於癒傷組織表面生成。進一步針對不同形態之癒傷組織進行培養，葉片於 2.0 mg/L NAA與 4.0 mg/L TDZ 之 B5 培養基上培養能產生不定芽，繼續培養於 0.1 mg/L IBA 與 1 mg/L TDZ 之 MS 培養基可發育為植株。另外以紅藍綠 (7R1G1B) 組合 LED 光源照射癒傷組織，可得到較高的增殖效果，而全紅光 (9R) 照射能誘導體胚生成，全藍光 (9B) 照射可得到較多的鬼臼素含量。在轉殖系統方面，以農桿菌媒介法將含有 GUS 報導基因之質體轉殖至八角蓮組織，並以不同菌種、菌濃度、預培養天數、感染時間、與乙醯丁香酮之添加，觀察對八角蓮組織生長之影響。經以 GUS 活性組織化學染色分析結果計算轉殖率，以 A. rhizogenes ATCC15834 對癒傷組織進行感染，經感染時間 6 小時，可得到擬轉殖細胞；對葉片與組織團塊進行感染，添加乙醯丁香酮預培養三天，經感染時間2 天，葉柄轉殖率為 9%，組織團塊為 33.3%。以A. tumefaciens EHA105 對葉片、葉柄、與組織團塊進行感染，添加乙醯丁香酮預培養三天，經感染時間兩天，葉片轉殖率為 4.2%，葉柄為 12.5%，組織團塊為 53.3%，未來將繼續進行聚合酶鏈鎖反應加以確認。

Dysosma pleiantha (Hance) Woodson, a herb with creeping rhizomes, high priced for its medicinal properties, contained several compounds such as podophyllotoxin. Due to its long juvenile phase and poor in fruit setting, seed variability and slow in seedling growth, the vegetative propagation of the herb from nursery stock is very slow. This study attempts to develop a more expeditious method for mass propagation and also for somaclonal variant selection. Moreover, this study aims to establish the genetic transformation system for D. pleiantha by using a reporter gene. Calli were induced from leaf petioles on B5 medium supplemented with 0.5 mg/L 2,4-D and 2.0 mg/L NAA in the dark. Somatic embryos were regenerated from petiole-derived callus on B5 medium supplemented with 0.5 mg/L 2,4-D, 1.0 mg/L BA, and 2.0 mg/L NAA in the dark. The adventitious shoot propagation system was established using leaf segments. B5 medium supplemented with 2.0 mg/L NAA and 4.0 mg/L TDZ was found to be optimal for inducing adventitious shoots. These shoots were further developed into plantlets after transfer to the MS medium supplemented with 0.1 mg/L IBA and 1 mg/L TDZ. The leaf-derived plantlets were finally transplanted into pots covered with plastic bags to allow acclimatization before being established under greenhouse condition. When embryogenic calli were exposed to red LED light, formation of somatic embryos was enhanced. Furthermore, the highest podophyllotoxin content in callus was obtained when applying the callus with blue LED light. An Agrobacterium-mediated transformation system was developed. The Agrobacterium strains, bacterial concentrations, pre-culture duration, co-culture duration, and acetosyringone addition were optimized. The developed transformation system was validated by using a plasmid containing GUS reporter gene. A transformation efficiency of A. rhizogenes ATCC15834 was 9% and 33% when histochemical GUS staining was used to assess the putative transgenic petiole and tissue mass, respectively. Transformation efficiency of A. tumefaciens EHA105 was 4.2%, 12.5%, and 53.3% when histochemical GUS staining was used to assess the putative transgenic leaves, petiole, and tissue mass, respectively.