Establishment of an efficient callus transformation system in Pyrus sinkiangensis for identifying PbPRX3 mediated lignin biosynthesis under BR and MeJA induction

• A transformative pipeline enabling gene functional analysis and lignin phenotyping in pear fruit callus is presented. • EBR and MeJA induce lignification by activating signaling pathways and biosynthetic genes, including PbPRX3 . • Golgi-localized PbPRX3 positively regulates EBR/MeJA-induced lignification and secondary cell wall thickening in pear callus. Functional studies of lignin–related genes in pear are often hampered by low transformation efficiency and the weak lignification capacity of dedifferentiated callus, which limits phenotypic readouts. Here, we established an Agrobacterium –mediated transformation system for fruit-flesh-derived callus of ‘Kuerle Xiangli’ ( Pyrus sinkiangensis Yu), achieving a transformation efficiency of 53.06% using the non-destructive RUBY reporter gene (encoding a betalain biosynthesis pathway). To overcome the “weak lignification” bottleneck, we developed a hormone-induction module and found that 24–epibrassinolide (EBR) and methyl jasmonate (MeJA) markedly enhanced lignin accumulation in callus, as indicated by Wiesner staining and acetyl bromide-based quantification. qRT–PCR analysis showed that EBR and MeJA treatments activated corresponding signaling components and coordinately upregulated multiple phenylpropanoid/lignin pathway genes. Notably, the class III peroxidase gene PbPRX3 was consistently induced under lignification-promoting conditions. Using this platform, we generated PbPRX3 -overexpressing callus and demonstrated increased lignin accumulation, elevated peroxidase activity, and thickened cell walls compared with empty-vector controls. A PbPRX3 –eGFP fusion signal co-localized with a Golgi marker in Nicotiana Benthamian , suggesting that PbPRX3 may traffic through the secretory pathway to support lignin accumulation. Collectively, our study provides a reusable toolkit that couples efficient callus transformation with inducible lignification phenotyping for pear functional genomics, and identifies PbPRX3 as a promising target for manipulating lignin-associated traits.