A Method for Genetic Transformation Using Embryonic Callus of Pinus koraiensis

Pinus koraiensis is classified as a second-class protected wild plant in China, recognized for its considerable economic and ecological importance. However, progress in functional research and breeding applications for this species has been hindered by the lack of an effective genetic transformation system. The purpose of this study was to develop a reliable and efficient genetic transformation system for a Pinus koraiensis embryonic callus using somatic embryogenesis technology. The Pinus koraiensis embryonic callus and β-glucuronidase (GUS) were employed as the reporter gene in an Agrobacterium-mediated transformation to investigate critical transformation factors, including antibiotic type and concentration, Agrobacterium bacterial solution concentration, infiltration, and co-cultivation times. The findings indicated that the proliferation of the Pinus koraiensis embryonic callus was substantially inhibited by 10 mg·L−1 of Hygromycin (Hyg), and a remarkable 93.42 ± 2.13% efficiency was achieved with an OD600 absorbance value of 0.6 during transformation. Two days of optimal co-cultivation yielded a transformation rate of 82.61%, with the resistant embryonic callus exhibiting a high GUS staining rate of 88.89%. Resistant somatic embryos were effectively obtained following the optimized protocol. This research contributes to the advancement of seed resource breeding and genetic enhancement for Pinus koraiensis, establishing a solid foundation for the investigation of gene functions specific to this species.