Tissue Culture Independent Agrobacterium tumefaciens Mediated In Planta Transformation Method for Tropical Maize (Zea mays.L)

Tropical maize is recalcitrant to tissue culture regeneration because of its poor response to in vitro regeneration after transformation. In this context, the present study has developed the tissue culture independent in planta transformation protocol for tropical maize by transferring plumular meristem cells of germinating seeds of tropical maize genotype through Agrobacterium tumefaciens infection. The protocol was developed by using Agrobacterium strain EHA105 containing vector pCAMBIA3301 carrying cry1Ab, gus and bar genes. The expression of transgene gus in T0 plants was confirmed by measuring the hydrolysis rate of the fluorescent substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) assay whereas the presence of cry1Ab gene was confirmed by polymerase chain reaction and T0 plants were allowed to grow in glass house into whole plant until maturity and were selfed to produce seeds of T1 generation. The presence of transgene and its segregation was studied in T1 generation through Southern and enzyme-linked immunosorbent assay confirming the presence of transgene and its expression respectively. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 75–90 days.