Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC. A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC. In this research, a vector pA5containing a chitinase signal sequence fused to gfp (green fluorescent protein) and an HDEL motive was used to target and immobilize into Agrobacteriurm strain EHA105 to establish a transformation platform for Vitis vinifera L. cv. Chardonnay NEC. It was determined that NEC 10 d after subculture was the best target tissue; 30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone (AS) was optimized as protocol. The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency. Kanamycin at 200 mg L-1 was picked for positive expression selection. The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques (RT-PCR) and fluorescent microscopy after three sub-cultures of the selected cell line. Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC. This transformation platform could facilitate the verification of candidate somatic embryogenesis (SE) decisive genes, and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products, as NEC not only proliferates fast, but also keeps in a rather stable condition.