Optimised Agrobacterium -Mediated Transformation and Application of Developmental Regulators Improve Regeneration Efficiency in Melons.

Melon ( Cucumis melo L.) is a protected crop in China with high economic value. Agrobacterium -mediated genetic transformation is a powerful tool to improve agronomic traits and obtain elite germplasm. However, current transformation protocols in melons are inefficient and highly genotype-dependent. To improve transformation in melon, we tested different infiltration methods for Agrobacterium -mediated transformation. Among these methods, micro-brushing and sonication for 20 s, followed by vacuum infiltration at -1.0 kPa for 90 s, resulted in the strongest green fluorescent protein signal and increased the proportion of infected explants. We transformed melon with developmental regulatory genes AtGRF5 , AtPLT5 , AtBBM , AtWUS , AtWOX5 , and AtWIND1 from Arabidopsis and estimated regeneration frequencies as the number of regenerating shoots/total number of inoculated explants in the selection medium. The overexpression of AtGRF5 and AtPLT5 in melon resulted in transformation efficiencies of 42.3% and 33% in ZHF and 45.6% and 32.9% in Z12, respectively, which were significantly higher than those of the control. AtGRF5 and AtPLT5 expression cassettes were added to CRISPR/Cas9 genome-editing vectors to obtain transgenic phytoene desaturase CmPDS knockout mutants. Using AtGRF5 or AtPLT5 , multi-allelic mutations were observed at CmPDS target sites in recalcitrant melon genotypes. This strategy enables genotype-flexible transformation and promotes precise genome modification technologies in melons.