Functional analysis of the woody oil crop Plukenetia volubilis L. LEC2 homolog PvoB3-69 in promoting regeneration.

Identified a robust regeneration-related genomic sequence of PvoB3-69 from the B3 superfamily in Plukenetia volubilis. gPvoB3-69-assisted transformation of P. volubilis obtained transgenic shoots for the first time. The regenerative capacity of host cells is critical for the genetic transformation efficiency of woody plants. The B3 superfamily  is particularly involved in early embryo morphogenesis and late-stage embryo maturation. In this study, 74 PvoB3 members were identified in the genome of P. volubilis, and classified into four subfamilies: LAV, RAV, ARF, and REM. RNA-seq and RT-qPCR analyses revealed that PvoB3-69, a member of the LAV subfamily, has specific expression patterns similar with LEC2. Overexpression of gPvoB3-69 enhanced the bud regeneration capacity of transgenic Nicotiana benthamiana organs during in vitro culture. Additionally, overexpression of gPvoB3-69 significantly improved somatic embryogenesis in transgenic Arabidopsis plants, especially with the aid of 2,4-D. Transcriptome analysis in Arabidopsis thaliana revealed that PvoB3-69 may enhance somatic embryo induction efficiency by activating developmental regulators such as WUS and LEC1, while also modulating salicylic acid, ABA, and ethylene metabolism. Furthermore, overexpression of gPvoB3-69 in the cotyledons of P. volubilis increased the regeneration ability of host cells, and broke the genetic transformation barrier through the Agrobacterium-mediated method, allowing the regenerated transgenic shoots obtained for the first time. This study provides the first systematic analysis of the B3 superfamily in P. volubilis, identifying PvoB3-69 as a key regulator of regeneration. These findings establish a foundation for further comprehensive studies of PvoB3 genes and deepen our understanding of the regulatory mechanism of shoot regeneration in P. volubilis.