Optimization of factors for efficient recovery of transgenic peanut (Arachis hypogaea L.)

Abstract

De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing β-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes. Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics (incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at 21°C on shoot induction medium containing 100 mg/L l-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate of the grafted shoots was nearly 100% in glass-house conditions. The optimized protocol took around 3 months to generate healthy plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the study.