Establishment and optimization of particle bombardment-mediated transformation system in triploid bermudagrass

Triploid bermudagrass ( Cynodon dactylon × C. transvaalensis L.) is one of the most widely used warm-season turfgrass species. However, the low induction rate of embryogenic calli and transformation efficiency are significant barriers to the genetic transformation process in triploid bermudagrass. In the present study, the regeneration system was optimized, and particle bombardment-mediated transformation system was successfully established in triploid bermudagrass. The results demonstrated that stolons with 4–6 nodes were the optimal explant for the genetic transformation. The optimal medium for inducing embryogenic calli consists of a precise formulation: 2.0 mg/L of 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg/L of Naphthaleneacetic acid (NAA), and 0.1 mg/L of 6-Benzylaminopurine (6-BA). The addition of 2.0% mannitol and 0.5 mg/L AgNO 3 to the subculture medium could effectively increase the proportion of embryogenic calli and reduce the browning ratio. A total of four calli types including A, B, C, and D were obtained in the subculture process. Types A, C, and D calli were able to differentiate and obtain the completed regenerated plants. During the process of particle bombardment-mediated transformation, type D calli were selected and pretreated with 150 µmol/L tenoxicam (TNX) for 3 days. Plasmids mixed with PEG-4000 and CaCl 2 were used to bombard calli twice, with a target distance of 6 cm. After bombardment, ten transgenic lines were successfully obtained, and the transformation efficiency reached 7.6%. These findings would provide the theoretical foundation and technical support for genetic engineering breeding in triploid bermudagrass.