Optimization of selective agents for In vitro regeneration of sugarcane transformants using NPTII gene

Abstract

In the present study, selection media was optimized, for in vitro regenerated sugarcane transformants using nptII gene as negative selectable marker, that kills non-transformed cells and ensures proliferation and growth of only transformed cells. To identify a suitable agent and its optimal dose, the embryogenic calli of sugarcane were bombarded with plasmid vector containing nptII gene, along with DREB gene. After dose optimization on untransformed calli with different selective agents and the post-bombarded transgenic calli were selectively proliferated onto selection media, composing MS+2,4-D (2.5 mg/L)+Kinetin (0.5 mg/L)+Proline (560 mg/L)+Sucrose (30 g/L) +Agar 8g/L supplemented with three promising selective agents viz. paromomycin (100 mg/L), kanamycin (50 mg/L) and geneticin (50 mg/L) for selection of transgenic calli. After two selection cycles of fifteen days each, the selected transformed putative calli were transferred onto shoot regeneration media composed of MS + IAA (5.0 mg/L) + Kin (0.5 mg/L) + BAP (0.5 mg/L), supplemented with similar selection agents. Regeneration was successful in calli carrying the nptII gene on all the regeneration media supplemented with different selective agents. Green and albino shoots with variable frequencies ranging from 13.33% to 22.58% proliferated as regenerants on different selection media. PCR analysis revealed higher number of putative transformants selected on paromomycin @ 100 mg/L, as compared to other two selection agents. Among the three different selection agents, paromomycin was observed to be the best for selection of transformed calli, using nptII gene in sugarcane.