Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry.

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry (Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: 'Blue Muffin' ('BM') displaying a high regeneration rate (>80%) and 'O'Neal' ('ON') exhibiting a low regeneration rate (<10%). The findings revealed differential expression of numerous auxin-related genes; notably, 'BM' exhibited higher expression of auxin signaling genes compared to 'ON'. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION (VcESR), VcWUSCHEL (VcWUS), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in 'BM' relative to 'ON'. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in 'BM' promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.