南美蟛蜞菊毛狀根誘導及其離體培養

為了探討利用南美蟛蜞菊毛狀根來改良其觀賞性狀和生產次生物質，研究了南美蟛蜞菊 Wedelia trilobata (L.) A.S.Hitche 毛狀根的誘導及其離體培養過程中培養基 N 源、碳源、磷和鈣的消耗變化。結果表明，發根農桿菌 Agrobacterium rhizogenes ATCC15834 感染南美蟛蜞菊幼嫩葉片外植體 7 d 後從其葉片切口中脈處產生毛狀根。毛狀根能在無外源激素的培養基上自主生長。PCR 擴增結果顯示發根農桿菌 Ri 質粒的 rol B 和 rol C 基因已在南美蟛蜞菊毛狀根基因組中插入、整合並得到表達。毛狀根液體培養 0~7 d 內處於生長遲滯期、7~21 d 為快速生長期、21 d 後進入生長平台期。在毛狀根液體培養過程中培養基的蔗糖、硝態氮、PO₄³⁻、Ca²⁺被逐漸吸收和消耗，培養至 7 d 時，蔗糖被消耗近50 ％；硝態氮含量只剩下起始硝態氮含量的5.8 ％；至 35 d 時，蔗糖和硝態氮含量分別約為其起始濃度的3.39 ％和1.82 ％。與Ca²⁺濃度變化不同的是，培養基的無機磷被快速消耗，培養至 7 d 時其濃度約為其起始濃度的1.76 ％；但培養至35 d 時培養基中仍殘存有占起始濃度約61.3 ％的Ca²⁺。該結果為今後利用南美蟛蜞菊毛狀根來改良其觀賞性狀和設計合適的培養基來規模培養生產其次生物質提供了可能性。
To study the possibilities for improvement of the ornamental character and production of secondary metabolites by using Wedelia trilobata hairy roots, we investigated the induction of W. trilobata L. hairy roots and its consumption changes of carbon resource, nitrogen resource, phosphate and calcium in the medium during liquid culture. The results showed that hairy roots could be incited from the cut edges of leaf explants 7 days after inoculation with Agrobacterium rhizogenes ATCC15834 and could have an autonomous growth on the medium without phytohormones. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes was integrated and expressed into the genome of transformed hairy roots. The hairy root line grew very slowly in 0–7 days, very fast from 7 to 21 days. During the liquid culture of hairy roots, sucrose, NO³⁻-N, PO4³⁻and Ca²⁺ in the medium could be gradually absorbed and utilized with time. The content of NO³⁻-N in the medium was 5.8% of the initial amount at day 7, while sucrose content was about 50% of the initial amount. At day 35, the NO³⁻-N and sucrose content in the medium was 1.82% and 3.39% of the initial amount, respectively. In combination with Ca²⁺ consumption, PO₄³⁻ of the medium was rapidly absorbed and utilized. At day 7, the content of PO₄³⁻ in the spent medium was only 1.76% of the initial amount; but even at day 35, the content of Ca²⁺ in the spent medium was still 61.3% of the initial amount. The results presented here had provided the possibilities on improvement the ornamental character and how to prepare optimum medium for large scale cultivation and production of secondary metabolites from W. trilobata L. hairy roots. Copyright © 2010 中國科學院微生物研究所《生物工程學報》.