美洲商陸毛狀根誘導及其離體培養的影響因素

為了探討利用美洲商陸毛狀根生產其藥用成分的可能性，研究了美洲商陸毛狀根誘導及其離體培養的影響因素。結果表明,美洲商陸葉片外植體被發根農桿菌ATCC 15834感染約18 d后，從其葉片外植體形態學下端葉脈切口處產生毛狀根，其中以預培養1 d，農桿菌感染20 min，共培養4 d時的毛狀根誘導率最高，達到70%。PCR擴增和硅膠薄層層析結果顯示，發根農桿菌Ri質粒的rol C基因以及冠癭堿合成酶基因已在美洲商陸毛狀根基因組中整合和表達。所獲得的美洲商陸毛狀根系都能在無外源激素的MS固體培養基上快速自主生長;其中以毛狀根根系2的生長速度最快、分生側根能力最強和根表面的根毛密度最高；毛狀根根表面呈紫紅色或呈白色。在供試的MS、1/2MS、B5和6,7-V液體培養基中，以無外源激素的MS培養基最適合美洲商陸毛狀根根系生長。與無外源激素的MS培養基相比，6,7-V培養基更有利于毛狀根中商陸皂苷甲的合成與積累。本文所建立的美洲商陸毛狀根誘導及其離體培養的適宜條件為今后利用其毛狀根株系的規模培養來生產其藥用有效成分商陸皂苷甲奠定了實驗和技術基礎。
To use hairy roots for producing medicinal ingredients of Phytolacca americana L. we studied the factors influencing the induction and in vitro culture. Hairy roots could be incited from the veins of cut surface(morphological lower) of P. americana L. leaf explants around 18 days after infection with the strain of Agrobacterium rhizogenes ATCC15834. The highest rooting rate, 70%, was obtained when leaf explants were pre-cultured for 1 day, infected for 20 min, and co-cultured for 4 days. The transformation was confirmed by PCR amplification of rol C of Ri plasmid and silica gel thin-layer chromatography of opines from P. americana L. hairy roots. All the hairy root lines could grow rapidly on solid exogenous phytohormone-free MS medium. Among the 9 hairy root lines, the hairy root line 2 had most rapid growth, most branched lateral roots and most intensive root hair; the root surface of some hairy root lines seemed purple or red, while that of the other hairy root line appeared white. Among liquid media MS, 1/2MS, B5 and 6,7-V tested, the best growth for hairy root lines was attained in liquid exogenous phytohormone-free MS medium. Compared with exogenous phytohormone-free MS medium, 6,7-V medium was better for synthesis and accumulation of esculento side A in hairy roots. The established optimal conditions for induction and in vitro culture of P. americana hairy roots had laid an experimental and technological foundation for production of medicinal constituents esculento side A from large scale culture of hairy roots. Copyright © 2017 中國科學院微生物所.