Escaping endogenous miRNA post-transcriptional silencing of JrGRF4b enhanced transformation efficiency in woody plants

The stable expression of transgenes was critically influenced by post-transcriptional regulatory mechanisms in transgenic plants. In this study, we investigated the influence of endogenous miRNA-mediated silencing on heterologous gene expression by introducing walnut (<i>Juglans regia</i> L.)-derived Growth-Regulating Factors 4 (<i>JrGRF4b</i>), disrupting miR396-mediated silencing of replace-<i>JrGRF4b</i> (<i>rJrGRF4b</i>), and <i>Jr-miR396a</i> into birch (<i>Betula platyphylla</i> Suk.). While <i>JrGRF4b</i> overexpression showed no significant improvement in transformation efficiency due to <i>Bp-miR396</i>-mediated suppression, transgenic lines expressing <i>rJrGRF4b</i> exhibited a 2.53% increase in transformation efficiency, along with significantly enhanced callus diameter, adventitious bud height, root elongation, cellular expansion, and shoot primordia proliferation compared to control (<i>**p</i><0.01). In contrast, <i>Jr-miR396a</i>-overexpressing plants displayed growth inhibition through suppression of endogenous <i>BpGRFs</i>. The results showed that escaping endogenous miRNA regulation by targeted site modification of <i>rJrGRF4b</i> significantly improved transgene performance in woody plants. Thus, comprehensive evaluation of post-transcriptional epigenetic regulation between transgenes and endogenous miRNAs in recipient plants was demonstrated to be important, and targeted escape from such miRNA-mediated suppression was shown to ensure stable and high-efficiency transgene expression.