Genetic transformation of Primula sieboldii using Agrobacterium rhizogenes and whole-plant regeneration from transgenic hairy roots

Primula sieboldii E. Morren is a widely cultivated ornamental plant with horticultural and pharmacological value. However, the lack of a developed transformation system has limited genetic studies and biotechnological applications of this species. In this study, we established a transformation method using Agrobacterium rhizogenes for the genetic manipulation of Primula sieboldii. The protocol consists of two stages: initial hairy root transformation and subsequent whole-plant regeneration from transgenic hairy roots through somatic embryogenesis. Comprehensive molecular analyses confirmed the stable integration and expression of various transgenes from the root-inducing plasmid (Ri) and the binary vector carrying the RUBY reporter in independent transgenic lines, as well as the stable germline transmission of the transgene to progeny. The protocol is effective, with 5% of treated explants successfully regenerating into transgenic plants. The established method provides a valuable tool for genetic and molecular studies of heterostyly and the self-incompatibility system in the genus Primula, while also offering practical applications in molecular breeding and plant biotechnology. Additionally, hairy root cultures provide a platform for metabolic engineering and the exploration of biologically active secondary metabolites with pharmacological applications.

Siebold报春花（Primula sieboldii E. Morren）是一种广泛栽培的观赏植物，兼具园艺与药用价值。然而，成熟遗传转化体系的缺失限制了该物种的遗传学研究与生物技术应用。本研究建立了一套以发根农杆菌（Agrobacterium rhizogenes）为介导的Siebold报春花遗传转化方法。该转化流程包含两个阶段：首先进行初始发根转化，随后通过体细胞胚胎发生技术，从转基因发根中再生获得完整植株。全面的分子生物学分析证实，发根诱导质粒（root-inducing plasmid，简称Ri质粒）与携带RUBY报告基因的双元载体所携带的各类外源基因，可在独立转基因株系中稳定整合并表达；同时外源基因可通过生殖系稳定传递给子代。该转化体系效率可观，经处理的外植体中有5%可成功再生为转基因植株。本研究建立的方法为报春花属植物的花柱异长与自交不亲和系统的遗传学及分子生物学研究提供了宝贵工具，同时也为分子育种与植物生物技术领域提供了实用应用途径。此外，发根培养体系可为代谢工程研究以及具备药用应用潜力的生物活性次生代谢产物的探索提供技术平台。