Studies On Methodology Of Agrobacterium Tumefaciens-mediated Transformation For Sugarcane

Agrobacterium-mediated transformation is a more preferable approach in transgenic research due to less copy of introduced genes and high stability of inheritance in comparison with other ways. Sugarcane is on the top list of biosafety crops. However, there is not a repeatable protocol for Agrobacterium-medmted transformation in sugarcane so far. In this study, following verification of infection ability of five strains of Agrobacterium tumefaciens with tobacco, Agrobacterium-mediated transformation with the five strains were optimized for sugarcane cultivars ROC22, FN15, FN28, FN30, FN39based on their calli, apical young leaf rolls and suspension cells.The main results were summarized as follows:1. The infection experiments with tobacco leaves verified the infection ability of five strains of Agrobacterium tumefaciens, namely, EHA105/pCAMBIA2301, LBA4404/pCAMBIA2301, AGL1/pCAMBIA2301, GV3101/pCAMBIA2301and A281/pCAMBIA2301, and the culture protocol for transformation.2. The optimized protocols for Agrobacterium-mediated transformation with sugarcane embryonic calli indicated that species specificity was still true for strains of Agrobacterium tumefaciens in sugarcane transformation. A high infection efficiency was observed in sugarcane cultivar ROC22up to71percent in an infection protocol, of which the calli were induced for25days, cultured on differential medium for6days, immersed in MS medium with AS for6hours, and then transferred into a liquid MS inoculation medium plus glucose at68.5g/L, arginine at174mg/L, glutamine at876mg/L, sucrose at30g/L and AS at100μmol/L(pH5.4) with EHA105/pCAMBIA2301at OD600value of1.0for50min. Thereafter, the calli were moved to a co-culture medium and kept for5days at25℃. Lower infection efficiencies were presented in sugarcane cultivar FN15with GV3101/pCAMBIA2301and FN30with EHA105/pCAMBIA2301. No GUS expression was observed to FN39in all of the testings.3. Experiments on Agrobacterium-mediated transformation with apical young leaf rolls showed the following conditions contributed to improve transformation efficiency of sugarcane cultivar FN30. The strain was EHA105/pCAMBIA2301with an OD600value of1.0. The liquid MS inoculation medium was supplemented with glucose at68.5g/L, arginine at174mg/L, glutamine at876mg/L, sucrose at30g/L and AS at100μmol/L(pH5.4). It was also necessary to have infection duration of50min and co-culture duration of5days at25℃. Lower infection efficiencies were presented in testing sugarcane cultivar FN15, FN28and FN39. No GUS expression was observed in apical young leaf rolls from ROC22.4. Growth characteristics of sugarcane suspension cell lines were investigated. Three phases were found in the growth of the sugarcane suspension cell lines, including a lag phase (0-4d),logarithmic phase (5-8d) and stationary phase (9-14d), indicating the best subculture cycle of sugarcane suspension cells was around6to8days. The results of Agrobacterium-mediated transformation for sugarcane suspension cell lines indicated that the following conditions promoted transformation efficiency of sugarcane cultivar ROC22when the sugarcane suspension cell lines were sub-cultured for7days and the concentration of strain EHA105/pCAMBIA2301was1.0at OD600. The liquid MS inoculation medium was the same as that above. It was necessary to maintain infection duration of50min and co-culture duration of3days at25℃. No GUS expression was observed in suspension cell lines from FN15.