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Induction, Culture, and Taxane Production of Crown Galls and Hairy Roots of Taxus sumatrana (Miq) de Laub
南洋紅豆杉之腫瘤細胞及毛狀根誘導、培養與紫杉烷類生產
摘要
Taxus sumatrana (Miq) de Laub crown galls and hairy roots were induced by infecting stem and leaf segments with Agrobacterium tumefaciens strain A281, and A. rhizogenes strains AR1600 and ATCC15834. Results showed great variations in the transformation efficiencies among different Agrobacterium strains and T. sumatrana clones. Strain A281 had better transformation ability than strains AR1600 and ATCC15834 (42.3 vs. 15.1~19.2%). Among all 7 clones, clones E7 and NJR had the highest transformation efficiency of both crown gall and hairy root formation, whereas the lowest transformation rates occurred with clones E2 and J85 which developed no hairy roots when inoculated with strain AR1600. Some hairy roots formed callus tissues, like crown galls, which grew rapidly and produced taxanes when cultured on plant growth regulator-free medium. The integration of Agrobacterium iaaM and rol B genes into the crown gall and hairy root genome was verified with a PCR. After 40 d of culture, growth indexes (ratio of net fresh weight of cultures to initial weight) of crown galls, hairy roots, and hairy root calli (3.9~5.5) were higher than those of untransformed calli (2.5) and roots (0.31). Transformed crown galls and calli formed from hairy roots produced more 10-deacetyl baccatin III, baccatin III, and paclitaxel than did hairy roots. After 30 d of culture in liquid medium, growth indexes of crown galls and hairy roots were 8.13 and 6.03, respectively, which were better than those on solid medium, suggesting that this system has the potential for large-scale bioreactor culture in the future.
並列摘要
本研究係利用農桿菌A281與農桿叢根菌AR 1600與ATCC15834建立南洋紅豆杉(Taxus sumatrana (Miq) de Laub)莖段與葉片的腫瘤細胞與毛狀根培養。農桿菌的轉殖率在不同菌種與單株間有很大的差異，以A281誘導產生腫瘤的轉殖率(42.3%)高於AR1600與ATCC15834的毛狀根轉殖率(15.1~19.2%)。比較7個不同單株的轉殖率，不論在腫瘤或毛狀根，都以E7與NJR最高，E2與J85最低且於接種AR1600無法產生毛狀根。有些毛狀根會產生癒合組織，此癒合組織和腫瘤細胞一樣，可於不含植物生長調節劑的培養基快速生長與生產紫杉烷類化物。以PCR確認農桿菌的iaaM基因及農桿叢根菌的rol B基因已轉移至腫瘤與毛狀根。腫瘤細胞、毛狀根與毛狀根癒合組織在MS固體培養基培養40天，生長指數(培養結束之培植體淨生長鮮重對開始培養鮮重比)為3.9~5.5，高於未轉殖癒合組織(2.5)與根(0.31)。調查10-DB, baccatin III與paclitaxel的含量，以腫瘤細胞與毛狀根產生的癒合組織較高。將腫瘤細胞與毛狀根移入液體培養基中培養30天，其生長量為8.13與6.03，高於固體培養基培養，顯示具有利用生物反應器放大培養的潛力。