The miR319-LsTCP4 module regulates de novo shoot regeneration in lettuce.

miR319-targeted LsTCP4 negatively regulates de novo shoot regeneration, suggesting a role for the miR319-LsTCP4 module in lettuce regenerative competence. Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome evidence for miR319-guided cleavage. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with regeneration efficiency increased by 67.3% and shoot production increased by 79.4% relative to WT, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with regeneration efficiency decreased by 49.6% and shoot production decreased by 66.4%. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency by 61.8% and shoot production by 91.6% relative to WT, showing a regeneration-enhancing phenotype similar to that of OX319. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. These results support LsTCP4 as a major miR319-responsive negative regulator of de novo shoot regeneration and suggest that the miR319-LsTCP4 module contributes to regenerative competence in lettuce.