Efficient transformation of Actinidia arguta by reducing the strength of basal salts in the medium to alleviate callus browning

An efficient transformation system for high-throughput functional genomic studies of kiwifruit has been developed to overcome the problem of necrosis in Actinidia arguta explants. The system uses Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pART27-10 to inoculate leaf strips. The vector contains neomycin phosphotransferase (nptII) and ${\beta}$-glucuronidase (GUS) (uidA) genes. A range of light intensities and different strengths of Murashige and Skoog (MS) basal salt media was used to overcome the problem of browning and/or necrosis of explants and calli. Callus browning was significantly reduced, resulting in regenerated adventitious shoots when the MS basal salt concentration in the culture medium was reduced to half-strength at low light intensity ($3.4\;{\mu}mol\;m^{-2}\;s^{-1}$) conditions. Inoculated leaf strips produced putative transformed shoots of Actinidia arguta on half-MS basal salt medium supplemented with 3.0 $mg\;l^{-1}$ zeatin, 0.5 $mg\;l^{-1}$ 6-benzyladenine, 0.05 $mg\;l^{-1}$ naphthalene acetic acid, 150 $mg\;l^{-1}$ kanamycin and 300 $mg\;l^{-1}$ $Timentin^{(R)}$. All regenerated plantlets were deemed putativ transgenic by histochemical GUS assay and polymerase chain-reaction analysis.