Agrobacterium-mediated Transformation of Yunzhe 05-51

PurposeThe regeneration system of tissue culture and appropriate mass concentration of antibiotics are the basis of plant genetic engineering. The establishment of the regeneration system of sugarcane Yunzhe 05-51 (YZ05-51) and its tolerance to G418 will be determined in order to providing reference for the genetic improvement of YZ05-51.

MethodThe young leaves of YZ05-51 were subjected to induce callus and the callus regeneration to plantlets on the media with MS basic medium supplemented with 2, 4-dichlorophenooxyacetic acid (2, 4-D), 6-benzylaminopurine (6-BA) and kinetin (KT) at various mass concentration gradients and different hormone combinations. In the stage of proliferation and differentiation of the embryogenic callus of YZ05-51, the suitable concentration of geneticin (G418) was determined by adding different content of G418 to the culture medium. The selection marker gene nptⅡ was introduced into the embryogenic callus of YZ05-51 exploiting the Agrobacterium-mediated genetic transformation. The G418 resistant plants were obtained after continuous screening, and the PCR and NPTⅡ ImmunoStrip assay were used to confirm the integration and protein expression of the marker gene nptⅡ.

ResultAt the stage of callus induction and callus proliferation, the suitable medium formula was the MS basic medium added with 3.0 mg/L 2, 4-D. The MS basic medium supplemented with 1.0 mg/L 6-BA + 0.1 mg/L KT were the suitable media for YZ05-51 during the stage of callus differentiation. In the experiment of G418 tolerance screening of YZ05-51, the selection concentration of G418 was 40 mg/L, the screening concentration of G418 should be reduced to 20 mg/L in buds generation stage. The PCR amplification of the G418 resistant plants showed that the marker gene nptⅡ had been successfully integrated into the genome of YZ05-51 transformants, and the NPTⅡ ImmunoStrip assay indicated that the NPTII protein expressed by nptⅡ was normal.

ConclusionThe regeneration system of YZ05-51 from spear leaves tissue culture was established, and it was clear that the appropriate G418 concentrations for selecting resistant calli and plantlets were 40 mg/L and 20 mg/L, respectively. The above established system of the Agrobacterium-mediated genetic transformation for the embryogenic callus of YZ05-51 provides a reference for the introduction of the genes of interest into sugarcane varieties.