Optimization of Different Parameters for Transformation of Elite Maize Inbred Lines

Four elite corn inbred lines were evaluated for susceptibility to Agrobacterium infection by investigating different parameters such as embryo physiological age, co-cultivation period, addition of L-cysteine in co-cultivation medium, heat shock to immature embryos prior to infection, resting stage and Agrobacterium strains that deliver T-DNA into plant cell from which whole plant can be regenerated. The results indicated that optimal embryo physiological age is 15 and 18 days after pollination and transient transformation efficiency decreased at 22 days after pollination. Enhanced transient beta glucuronidase (GUS) expression with average frequency of 40.56% was noticed with three days of co-cultivation period while two days of co-cultivation period resulted 24.07% transient GUS expression. Addition of 100 mgL^-1 L-cysteine in co-cultivation medium increased the transient transformation by 33.9% compared to control treatment. It has also been observed that cysteine enhanced the expression of GUS in the basal scutellar regions of immature embryos compared to apical and embryo axis region. Heat shock of 46 ˚C for 3 minutes increased the transformation efficiency of immature embryos by 26.96%. Growth of infected immature embryos on resting medium after co-cultivation period enhanced the survival of kanamycin resistant calli on selection medium by 93.97%. Agropine strain (EHA101) of Agrobacterium was found 25.67% more effective than octopine strain (LBA4404). The intensity of GUS expression was also enhanced with EHA101 compared to LBA4404. The maize genetic transformation protocol developed in this study will possibly improve the efficiency to produce new transgenic maize lines expressing desirable agronomic characteristics.