Obtainment Of Marker-free Lysine-rich Transgenic Maize By Co-transformation

Immature embryos of 6 maize inbred lines including X90, XI78, X08, HC, X333, X04 and 6 crossbreeds including X04/X06 X08/X178 X178/X90 X08/X333 X333/X08 HC/X178 were cultured on the N6 medium to generate callus. The embryogenic callus induced from X08, X178, X08/X178, X178/X90 can be used in the transformation of mazie.The plant expression vector pl9SB containing SBLR gene , that encodes a lysine-rich protein and was drived by 19Z promoter of maize , had been constructed to transformed embryogenic callus by particle bombardment. The particles were coated by the mixture of pl9SB and another plant expression vector pHyg containing the gene of hygromycin phosphotransferase at molar radio of 2:1. 5061 pieces of embryonic callus of X08, XI78, X08/X178, X178/X90 were transformed and 207 regenerated plants were obtained by the seletion of hygromycin. PCR analysis showed that there were 69 regenerated plants with the intergration of hpt gene, including 11 plants with the intergration of SBLR gene. The frequency of co-transformation is 5.3% in this experiment.PCR analysis of 4 RO generation plants showed that there are 2 plants with the intergration of both SBLR and hpt genes , 1 plant with the intergration of hpt gene and 1 plant with the integration of SBLR gene. We obtained maker-free transgenic maize by co-transformation.