Precision microneedle-assisted wounding enhances regeneration and enables efficient Agrobacterium-mediated transformation in carnation (Dianthus caryophyllus L.)

Abstract

Carnation (Dianthus caryophyllus L.) is an important ornamental crop, but its molecular breeding is limited by low regeneration competence and inefficient Agrobacterium-mediated transformation. This study aimed to establish a reproducible transformation framework for carnation by integrating optimized regeneration conditions with precision microneedle-assisted wounding and infection strategies. Significant genotype-dependent variation in regeneration capacity and auxin responsiveness was observed among six cultivars. Targeted microneedle-assisted wounding at the leaf-base vein junction markedly enhanced direct shoot regeneration, resulting in a 2.27-fold increase in shoot number per explant and earlier regeneration compared with non-wounded controls. Among three infection strategies tested, immersion-wounding with sterile microneedles followed by bacterial immersion produced the highest regeneration frequency (60.0%) and shoot formation. Optimization of non-ionic surfactants revealed that Tween 20 (0.01%) provided a broad and stable operational window, whereas Triton X-100 showed strong concentration-dependent phytotoxicity. Using the optimized protocol, five independent acdS-transgenic carnation lines were successfully recovered, corresponding to a PCR-verified transformation efficiency of 16.67% on an explant basis. PCR and qRT-PCR analyses confirmed stable transgene integration and expression without adverse effects on plant morphology. These results demonstrate that precision microneedle-assisted wounding is a key enabling factor for efficient regeneration and facilitates Agrobacterium-mediated transformation in carnation, providing a practical platform for molecular breeding of regeneration-recalcitrant ornamental crops.