Development of an Optimized in Planta Transformation System in Sugarcane and Its Application on Sh4CL13 in Chlorogenic Acid Biosynthesis

Sugarcane (Saccharum spp. hybrid) is the most important sugar crop. However, its genetic improvement is severely constrained by genotype-dependent regeneration recalcitrance and lengthy tissue culture cycles. Here, we established a simple, efficient, and genotype-flexible in planta transformation system using two-leaf-stage plantlets. By systematic optimization of three key parameters: Agrobacterium cell density (OD600 = 0.5), dark incubation duration (2 weeks), and infection frequency (two rounds), we achieved a maximum transformation efficiency of 51.2%. The protocol was successfully applied to eight diverse sugarcane varieties, with transformation efficiencies ranging from 33.3% to 51.2%, demonstrating broad genotype applicability. Using this optimized system, we introduced Sh4CL13 into sugarcane. Transgenic lines exhibited a 3.25-fold increase in Sh4CL13 transcript levels and a 13.7-fold elevation in chlorogenic acid (CGA) content compared to controls. Feeding bioassays with Mythimna separata larvae revealed that transgenic lines significantly prolonged larval developmental duration, reduced pupal weight, and decreased adult emergence rates. This in planta transformation system bypasses tissue culture, offers a practical platform for functional genomics and molecular breeding in sugarcane and potentially other monocot crops.