Red fluorescent protein (RFP): An effective reporter gene for Agrobacterium-mediated genetic transformation of Hevea brasiliensis

Genetic transformation of the rubber tree ( Hevea brasiliensis ) is significantly hindered by low efficiency and the absence of a robust reporter system for the real-time, precise selection of transgenic events. In this study, we evaluated the efficacy of red fluorescent protein (RFP) as a superior visual marker for Agrobacterium -mediated transformation. Compared with traditional reporters, RFP exhibited minimal interference from chlorophyll autofluorescence, facilitating the unambiguous discrimination of transgenic somatic embryos in this pigment-rich species. By characterizing RFP expression dynamics across successive subcultures, we identified a critical peak induction phase at approximately 44 days. Following this period, persistent fluorescence strongly suggests stable T-DNA integration, establishing a predictable and rational timeline for screening. Whole-genome sequencing (WGS) further confirmed precise T-DNA insertion sites and revealed both single- and multicopy integration events. Notably, RFP accumulation levels did not affect regeneration capacity, magenta lines showed a 32.1% normal embryo regeneration rate, whereas light-pink lines were severely impaired (2.8%). This was attributed to magenta lines with high hygromycin resistance which could counteract the adverse effects of hygromycin on regenerative capacity, whereas light-pink lines could only neutralize a small fraction of hygromycin’s detrimental impacts. Furthermore, stable RFP expression was faithfully maintained in regenerated plants propagated by secondary embryogenesis and grafting, particularly within the specialized laticifer system. Taken together, the results of this study establish RFP as an efficient, non-destructive reporter system that streamlines the identification of stable transgenic events, providing a powerful tool for the functional genomics and precision breeding of this recalcitrant woody species.