何首烏之組織培養

Abstract

本試驗之目的為：﹝1﹞建立何首烏之芽體培養大量繁殖系統；﹝2﹞建立何首烏之癒合組織培養系統；﹝3﹞利用農桿根群菌建立何首烏之毛狀根誘導系統；﹝4﹞進行何首烏有效成分-大黃素（emodin）及大黃素甲醚（physcion）之分析，以探討利用植物組織培養方式，生產植物二次代謝物之可能性。 將試驗結果摘錄如下： ﹝1﹞將何首烏試管苗之莖節培養於含有0.5 - 2.0 mg/l BA、0.2 mg/l NAA、3﹪蔗糖及1﹪agar之MS固體培養基中，可快速增殖何首烏之芽體（可得4－5個芽體）；將快速增殖所得之芽體，培養於添加0.01 mg/l IBA與1/2MS或WPM的基本鹽類培養基時，可有效提高組培苗馴化後移植到溫室的存活率。 ﹝2﹞將何首烏之節間培養於含有1 mg/l 2,4-D、3﹪蔗糖及1﹪agar之MS固體培養基中，癒合組織之誘導率及所誘導產生的癒合組織生長最佳（可得466 mg/callus）；癒合組織繼代培養也以含有0.5 - 2.0 mg/l 2,4-D之培養基較佳。 ﹝3﹞在毛狀根的誘導與確認方面，發現以農桿根群菌BCRC15722的菌系感染何首烏葉片培植體的誘導效果較佳。而轉殖毛狀根的鑑定，則應用農桿根群菌的rol B及rol C基因序列為引子，進行PCR反應後，可確認所產生的根是否為轉殖成功的毛狀根。 ﹝4﹞運用薄層色層分析（TLC）與高效液相層析（HPLC）的分析方式，得知組織培養之芽體與田間種植株（莖與根部）含有豐富的大黃素及大黃素甲醚；但分析馴化後培養於溫室的何首烏組織培養苗時，卻發現其莖部與根部均無大黃素及大黃素甲醚的累積，其可能原因有待進一步探討。

The main objectives of the present investigation were (1) to standardize a protocol for rapid multiplication of P. multiflorum plants using nodal explants; (2) to standardize a protocol for establishment of callus culture using the internode explants; (3) using Agrobacterium rhizogenes mediated transformation to establish a hairy root system; (4) to analyze and compare the amount of anthraquinones (emodin and physcion) present in the in vitro grown shoots, tissue cultured raised plants and crude drugs (aerial and underground parts of the plants) by HPLC and TLC. Establishing methods for the rapid production of higher amounts of anthraquinones using tissue culture technology. The results obtained on the studies carried out are as follows: 1) A simple and rapid protocol of complete in vitro plant regeneration using nodal explants has been standardized. Among the various media tested maximum number of nodal explants responded and produced (4-5 shoots) after six weeks of culture on MS medium supplemented with 0.5 - 2.0 mg mg/l BA, 0.2 mg/l NAA, 3% sucrose and 1% agar. Maximum percentage of shoots rooted on half MS or WPM basal medium supplemented with 0.01 mg/l IBA or NAA successfully acclimatized on transfer to soil and maintained under greenhouse conditions. 2) Various explants were tested for the induction of callus. Induction and proliferation of callus (466 mg per explants) from the internode explants has been achieved on MS basal medium containing 0.5 —2.0 mg/l 2,4-D, 3% sucrose and 1% agar after six weeks of incubation in dark at 25 ± 1°C. The callus cultures are been successfully maintained by subculturing 200 mg of calli on fresh media every six weeks. 3) Thirty-seven percentage of the leaf pieces (8 ´ 8 mm) induced hairy roots after co-culturing with Agrobacterium rhizogenes BCRC15722. The hairy roots were induced after one month of culture on MS basal medium in dark. The hairy roots were further proliferated in the MS liquid medium. The rol B and rolC gene in the hairy roots were confirmed by PCR analysis. 4) The anthraquinone contents in the in vitro grown shoots, greenhouse plant (root and stem), field plants (root, stem leaf and flower) and marketed crude drug (processed underground or stem parts) were determined by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). HPLC analysis revealed that the major medicinal compounds - emodin and physcion in the in vitro grown shoots and field plants (root and stem) were higher than the marketed crude drug (processed underground or stem parts) and greenhouse plant (root and stem).