丹參為常用之中草藥,其主要活性成分有丹參酮I、丹參酮IIA、隱丹參酮以及丹參酚酸B。由於市面上丹參來源、品質及成分皆不一致且仰賴進口,故基源穩定、強壯且具高活性成分含量之丹參植株及其繁殖系統的建立便有其必要性。本試驗室曾利用主動誘變技術獲得一個在外觀型態上較為健壯之植株SH41,經本研究利用PCR技術確定為轉殖株後,進行其外觀型態及活性成份上的偵測和繁殖系統之建立。在外觀形態上,一年大之馴化植株的葉片數量雖只有WT的1/3,但在葉長、葉寬和株高皆較WT好(分別為其1.3、1.7和1.7倍),其葉片顏色較深、表面較為皺摺,利用SEM觀察到每平方毫米葉片上的毛茸組織為WT的1.9倍。在活性成分分析方面,結果顯示SH41根部之丹參酚酸B、迷迭香酸、丹參酮I及IIA明顯高於WT(分別為1.4、1.3、3及2倍),故值得利用微體繁殖系統進行大量培養。在組織培養大量繁殖系統中,不定芽繁殖率以添加1 mg/L BA處理45天後最佳,WT可達5棵,而SH41可達3棵。另外在不同培養介質(agar及gelrite)、和不同營養及荷爾蒙(MS、蔗糖和kinetin)添加的部分不定芽平均數約2棵。在丹參發根試驗中則以1 mg/L NAA處理最佳,其發根率可達100%,不過在發根培養介質(agar及gelrite)上,SH41之發根效果皆低於WT(前者少20%,後者少40%),且蔗糖及IBA處理後亦得到類似結果(皆比WT少20%)。綜合上述,SH41植株在外觀健壯度上與活性成分皆優於WT,在不同大量繁殖條件下,SH41需在添加1 mg/L BA的1X MS、3%蔗糖及0.9% agar培養條件下,其芽體繁殖率最佳。經本研究所確認SH41為較高活性成分之SH41植株及所建立大量繁殖系統,將可在GMO田間試驗後,提供業界生產大量丹參植株,以滿足市場之需求。
Salvia miltiorrhiza Bunge is a well known oriental medicinal herb. Roots of S. miltiorrhiza, generally known as Dan-shen, contain bio-active compounds such as tanshinone I, tanshinone IIA, cryptotanshinone and salvianolic acids B. On the basis of an previously obtained activation-tagged S. miltiorrhiza, SH41 is morphologically different from wild type. After the molecular verification, SH41 was identified as transgenic plant and was observed with larger and more trichomes and cell tissues. Moreover, the content of salvianolic acid B, rosmaric acid, tanshinone I and tanshinone IIA in SH41 are 1.4, 1.3, 3 and 2 times higher than wildtype. This transgenic S. miltiorrhiza, which is strong and contains high active compounds, is worthy to establish its plant regeneration system. Results showed that SH41 has weaker budding and rooting ability comparing to wild type (in different gelrite agents’s MS medium and under different sugar concentration, and different growth hormones treatment): with a higher propagation rate at 1X MS agar medium supplemented with 1 mg/L BA, 3% sucrose and a better rooting ability at 1X MS agar medium supplemented with 1 mg/L NAA, 3% sucrose. After GMO evaluation, SH41 and its regeneration system established in this study could be used for industrial purpose in the future.