Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch

Abstract

A protocol for Agrobacterium-mediated transformation was developed for embryogenic callus of an excellent climber species, Parthenocissus tricuspidata. A. tumefaciens strain EHA105 or C58 harboring the pCAMBIA2301 binary vector with the neomycin phosphotransferase (nptII) and β-glucuronidase (uidA) gene was used. Factors affecting the transformation efficiency, including the Agrobacterium strains, co-cultivation time, Agrobacterium concentration, and infection time, were evaluated. Strain EHA105 proved to be significantly better than C58, and 4 days of co-culture was critical for transformation. An Agrobacterium suspension at a concentration of 0.5–0.7 × 108 cells ml−1 (OD600 = 0.5–0.7) and an infection time of 40 min was optimal for transformation. By applying these optimized parameters, we recovered six independent transformed shoots that were kanamycin-resistant and contained the nptII gene, as verified by polymerase chain reaction (PCR) analysis. Southern blot analysis confirmed that T-DNA was stably integrated into the genome of three out of six PCR-positive lines. Furthermore, histochemical GUS assay revealed the expression of the uidA gene in kanamycin-resistant calli, somatic embryos, and leaves of transgenic plants.