Establishment of a single anther-induced somatic embryogenesis system and genetic transformation system in rubber tree (Hevea brasiliensis)

Hevea brasiliensis , the primary source of natural rubber (NR), faces significant production challenges owing to high disease susceptibility and inefficient conventional breeding methods. The absence of a stable and efficient genetic transformation system has hindered advances in molecular breeding and functional gene research in rubber trees. In this study, we established an optimized Agrobacterium tumefaciens-mediated transformation system using single anther–induced embryogenic callus as the transformation receptor, integrated with a temporary immersion system (TIS). We identified optimal explants—Group ii buds with longitudinal diameters of 2.81–3.20 mm by systematically correlating pollen developmental stages with floral bud morphology which resulted in a 60.6 % increase in embryogenic callus induction through single-anther inoculation. Compared to traditional solid culture, TIS significantly improved both somatic embryogenesis and regeneration efficiency. The optimized transformation protocol included a 15-day preculture of selected embryogenic calli on calcium-free medium, infection with a bacterial suspension (OD 600 = 1.0) for 20 min, followed by dark co-cultivation. Transgene integration ( GUS , GFP , HbCERK1 ) was confirmed via histochemical staining, fluorescence microscopy, and RT-PCR analysis. To address the challenge of root regeneration in transformed plants, we developed a grafting method using 4-week-old rootstocks, which established functional vascular connections within 7 days and yielded viable transgenic plants. This integrated approach—combining optimized explant selection, TIS-enhanced embryogenesis, transformation, and grafting—provided a robust platform for functional genomics and genome editing in rubber trees. • Using single anthers as explants significantly improves the induction efficiency of embryogenic callus. • Compared to conventional solid culture, the temporary immersion system (TIS) enhances embryo germination efficiency and the formation of regenerated plants. • Key parameters for genetic transformation were optimized: hygromycin concentration at 40 mg/L, bacterial concentration at OD₆₀₀ = 1.0, and infection time of 20 min. • Regeneration frequency of transformed plants was increased by grafting non-rooted transgenic shoots.