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 (OD600 = 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.