褐脈少花龍葵毛狀根的誘導、培養及其澳洲茄胺的產生

利用發根農桿菌的遺傳轉化和液體培養技術，研究了褐脈少花龍葵 (Solanum nigrum L. var. pauciflorum) 毛狀根的誘導和離體培養及其澳洲茄胺的產生以及液體培養過程中培養基中N源和鈣的消耗變化。結果表明，發根農桿菌ATCC15834感染褐脈少花龍葵葉片外植體5 d後產生毛狀根，感染25d后，約90%的葉片外植體產生毛狀根。毛狀根能在無外源生長調節劑的MS固體和液體培養基上自主生長。PCR擴增結果顯示發根農桿菌Ri質粒的rolB和rolC基因已在少花龍葵毛狀根基因組中整合並得到表達。所產生的毛狀根能產生藥用次生物質澳洲茄胺，其含量約為非轉化植株根的1.3倍，達到582.05μg/g干重。少花龍葵毛狀根液體培養0-5 d內處于生長遲滯期、5-15 d為快速生長期、15d後進入生長平台期。培養基的硝態氮和銨態氮在毛狀根液體培養過程中被逐漸吸收和消耗，至培養15 d時銨態氮被消耗殆盡，而硝態氮仍剩余44.7%;培養基中鈣的濃度在培養過程中雖逐漸降低，但在培養25d時仍未被完全消耗，其濃度約為起始濃度的43.5%。該結果為今後設計合適的培養基來規模培養褐脈少花龍葵毛狀根生產藥用次生物質澳洲茄胺提供了可能性。
By using genetic transformation of Agrobacterium rhizogenes and liquid culture, induction and culture conditions for Solanum nigrum L. var. pauciflorum Liou hairy roots and its solasodine production and consumption changes of N resource and calcium in the medium during liquid culture were investigated. The results showed that hairy roots could be initiated from the cut edges of leaf explants 5 days after inoculation with the strain of A.rhizogenes ATCC15834. The percentage of rooted leaf explants 25 days after infection was more than 90%. Hairy roots could grow rapidly on solid or liquid growth regulator-free MS medium. The PCR amplification of rol genes and virC gene showed that rol genes of Ri plasmid of A. rhizogenes were integrated in the genome of transformed hairy roots of S. nigrum L var pauciflorum. The hairy roots could produce medicinal secondary metabolites solasodine and the amount of solasodine in the hairy root cultures reached a level of 582.05μg/g dry weight and was 1.31 times as much as those in the untransformed roots. The hairy roots grew very slowly in 0-5 days in the liquid medium, then, very fast from 5 to 15 days. During liquid culture, NO³⁻ and NH in the medium were gradually absorbed and utilized by hairy roots. NH⁴⁺ ⁻N of the medium was used up at day 15 of the culture, while NO³⁻ in the medium was not used up, the content of which was 44.7% of the initial amount. Ca²⁺ of the medium was gradually absorbed and utilized during liquid culture and it was not used up at day 25, the content of which was still 43.5% of the initial amount. The results presented here had provided the possibilities on how to prepare optimum medium for large scale cultivation and production of solasodine from S. nigrum L. var. pauciflorum hairy roots. Copyright © 2008 分子細胞生物學報編輯部.