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

<i>Primula sieboldii</i> 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 <i>Agrobacterium rhizogenes</i> for the genetic manipulation of <i>Primula sieboldii</i>. 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 (Ri) plasmid and the binary vector carrying the <i>RUBY</i> 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 forming transformed hairy roots expressing the <i>RUBY</i> reporter, from which transgenic plants were regenerated. The established method provides a valuable tool for genetic and molecular studies of heterostyly and the self-incompatibility system in the genus <i>Primula</i>, 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.