Optimizing Geneticin Lethal Dose for Effective NPT-II Selection in Transgenic Sugarcane (Genotype CoC671)

Abstract

Establishing a kill curve is an essential preliminary step in the genetic transformation of plants, including sugarcane. This process helps determine the optimal selection pressure required to differentiate between transformed and non-transformed tissues, ensuring that only genetically modified cells survive under antibiotic or herbicide treatment. In the context of sugarcane, the kill curve is particularly critical due to the plant's recalcitrant nature and genotype-specific responses to transformation protocols. In this study, the sugarcane genotype CoC671, which is extensively cultivated in India, especially in Maharashtra, was selected to develop a kill curve using geneticin (G418) as the selection agent. The sensitivity of different tissue types—callus and in vitro regenerated shoots—was tested across a gradient of geneticin concentrations: 0, 20, 30, 40, 50, and 60 mg/L. The impact of geneticin was evaluated based on parameters such as callus proliferation, regeneration potential, and shoot survival. The results revealed a concentration-dependent response to geneticin. At the highest concentration of 60 mg/L, complete inhibition of callus growth was observed, with 100% tissue mortality. A concentration of 50 mg/L also showed a strong inhibitory effect, severely affecting callus regeneration and resulting in the death of most tissues. At 40 mg/L, about 70% of the calli exhibited bleaching, and regeneration was limited. Among the few regenerated calli, approximately 53% of the shoots showed bleaching symptoms, indicating compromised viability. In shoot-based assays, similar trends were noted. At 50 mg/L, no shoot development occurred within a month, even with regular subculturing every 14 days. The concentration of 40 mg/L geneticin emerged as the most suitable for selection, as it resulted in 76% shoot mortality while still allowing the survival of a small fraction of shoots, critical for the identification and propagation of successfully transformed tissues. Based on these observations, 40 mg/L geneticin was established as the optimal selection pressure for conducting genetic transformation in sugarcane genotype CoC671. This optimized kill curve provides a reliable foundation for transformation protocols involving CoC671, whether via Agrobacterium-mediated methods or biolistic particle delivery. It ensures effective discrimination between transformed and non-transformed tissues, improving the efficiency and precision of sugarcane genetic engineering efforts.