Table_1_One-Step Regeneration of Hairy Roots to Induce High Tanshinone Plants in Salvia miltiorrhiza.DOCX

Salvia miltiorrhiza is a traditional Chinese medicinal plant of Labiatae, which has been widely utilized to treat a variety of cardiovascular and cerebrovascular diseases. However, due to the long growth cycle, low content of active ingredients, and serious quality deterioration of S. miltiorrhiza, the use of biotechnology to improve S. miltiorrhiza to meet the growing demand for clinical applications has become a research hotspot. In this study, a novel one-step hairy root regeneration method was developed, which could rapidly obtain hairy roots and regenerated plants with high tanshinone content. By optimizing the parameters of Agrobacterium rhizogenes transformation in S. miltiorrhiza, it was finally established that the explants were infected in Ar.qual (OD600 = 0.6) for 10 min, co-cultured for 3 days, and then screened on the screening medium containing 7.5 mg/l hygromycin, the maximum transformation frequency can reach 73.85%. GFP and PCR detection yielded a total of 9 positive transgenic hairy root lines and 11 positive transgenic regenerated plants. SmGGPPS1 was successfully overexpressed in positive transgenic regenerated plants, according to the results of qRT-PCR. The content of tanshinone IIA and cryptotanshinone were dramatically enhanced in transgenic regenerated plants and hairy roots by Ultra Performance Liquid Chromatography analysis. Based on the Agrobacterium-mediated transformation of S. miltiorrhiza, this study developed a new method for regenerating plants with transgenic hairy roots. This method provides a foundation for the breeding of S. miltiorrhiza and the sustainable development of medicinal plant resources, as well as provides a useful reference for the application of other species.

丹参（Salvia miltiorrhiza）是唇形科（Labiatae）传统药用植物，被广泛用于治疗多种心脑血管疾病。然而，由于丹参生长周期长、有效成分含量偏低且品质退化严重，利用生物技术改良丹参以满足临床应用日益增长的需求，已成为当前研究热点。本研究开发了一种新型一步法发根再生技术，可快速获得高丹参酮（tanshinone）含量的发根及再生植株。通过优化丹参的发根农杆菌（Agrobacterium rhizogenes）转化参数，本研究最终确定最优转化体系为：外植体在OD₆₀₀为0.6的发根农杆菌菌株Ar.qual菌液中侵染10分钟，共培养3天后，转接至含7.5 mg/L潮霉素（hygromycin）的筛选培养基中进行筛选，此时最高转化效率可达73.85%。经绿色荧光蛋白（Green Fluorescent Protein, GFP）与聚合酶链式反应（Polymerase Chain Reaction, PCR）检测，共获得9株阳性转基因发根株系与11株阳性转基因再生植株。实时荧光定量聚合酶链式反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）结果显示，SmGGPPS1基因在阳性转基因再生植株中成功实现过表达。超高效液相色谱（Ultra Performance Liquid Chromatography, UPLC）分析结果表明，转基因再生植株及发根中的丹参酮IIA（tanshinone IIA）与隐丹参酮（cryptotanshinone）含量均显著提升。本研究基于农杆菌介导的丹参转化技术，开发了一种可获得转基因发根再生植株的全新方法。该方法为丹参育种及药用植物资源的可持续发展奠定了理论基础，同时也为其他物种的相关研究与应用提供了重要参考。