Department of Biology and Pharmaceutical Botany, Medical University of Lodz, Muszynskiego 1, 90-151, Lodz, Poland.
Department of Pharmacognosy, Medical University of Lodz, Muszynskiego 1, 90-151, Lodz, Poland.
J Biotechnol. 2020 Jul 20;318:10-19. doi: 10.1016/j.jbiotec.2020.05.002. Epub 2020 May 6.
This study was to obtain stable transformed roots of Salvia bulleyana using A. rhizogenes strain A4 and then evaluate their phytochemical profile and selected the most productive clone. Our results indicated that the type of explant and medium used for bacterium and explant incubation had an influence on the frequency of hairy root formation. The best response was obtained on leaves infected with bacteria cultivated on YMB medium supplemented with acetosyringone. Of the four selected transformed root clones, after five-week cultivation in Woody Plant (WP) medium, the highest growth indexes were demonstrated for line C1: i.e. 13 for fresh and 15 for dry weight (81.4 and 8.2 g/l fresh and dry weight, respectively). The qualitative analysis of hydromethanolic extracts of hairy roots of S. bulleyana using UPLC-PDA-ESI-MS/MS method showed the presence of 10 polyphenolic compounds including predominant rosmarinic acid (RA), its derivatives (hexoside and methyl rosmarinate), caffeic acid, its derivatives and several salvianolic acids: K, E and F. Their production varied among the four root clones studied; the highest RA (39.6 mg/g dry weight) and total polyphenol (48.9 mg/g dry weight) level were found in the roots of C4 clone. These values were significantly higher than those of the roots of plants grown for several years under field conditions. The transformation of the obtained root cultures was confirmed by polymerase chain reaction using aux1, aux2, rolB, rolC and rolD primers.
本研究利用发根农杆菌 A4 获得了丹参稳定的转化根，然后评估了它们的化学成分谱，并选择了最具生产力的克隆。我们的结果表明，外植体的类型和用于细菌和外植体孵育的培养基对毛状根形成的频率有影响。在 YMB 培养基中培养的细菌感染的叶片上获得了最佳的反应，该培养基中添加了乙酰丁香酮。在选择的四个转化根克隆中，在 Woody Plant（WP）培养基中培养五周后，C1 系表现出最高的生长指数：即鲜重 13，干重 15（分别为 81.4 和 8.2 g/L 鲜重和干重）。使用 UPLC-PDA-ESI-MS/MS 方法对丹参毛状根的水-甲醇提取物进行定性分析，结果表明存在 10 种多酚类化合物，包括主要的迷迭香酸（RA）、其衍生物（己糖苷和甲基迷迭香酸）、咖啡酸、其衍生物和几种丹酚酸：K、E 和 F。它们的产量在研究的四个根克隆中有所不同；C4 克隆的根中发现 RA（39.6mg/g 干重）和总多酚（48.9mg/g 干重）含量最高。这些值明显高于在田间条件下生长多年的植物根中的含量。通过使用 aux1、aux2、rolB、rolC 和 rolD 引物的聚合酶链反应证实了获得的根培养物的转化。
