In vitro mutagenesis and preliminary characterization of potential drought tolerant sugarcane lines

A dissertation submitted in fulfilment of the requirement for the Degree of Master of Science in the Department of Agriculture, Faculty of Science, Agriculture and Engineering, at the University of Zululand, 2020.

Drought is a major cause of yield loss in agriculture and its impacts are expected to increase due to future increases in temperature. Traditional breeding in sugarcane is constrained by sugarcane’s complex genome and an extensive breeding period. Tissue culture-based mutagenesis and selection is one of the most feasible and costeffective tools for developing and selecting for drought stress-tolerant plants. Ethyl methanesulfonate (EMS) can increase genetic variability in the sugarcane genome resulting in the initiation of advantageous traits such as drought tolerance. Therefore, its potential application was investigated in this study. Eight-week-old sugarcane calli were exposed to 16 mM EMS for 0.5, 1, 2, and 3 hours to determine the optimal incubation period. To select for osmotic tolerance, calli were cultured on media containing 0, 150, 225 and 300 mM mannitol for eight weeks. Treatment of the callus with EMS for a minimum of 1 h resulted in a significant decrease in the average number of embryonic callus clump, callus fresh weight, and relative growth rate. Based on the embryo regeneration results, an incubation period of 1 h was optimum for inducing osmotic tolerance in sugarcane. The mannitol LD50 and LD90 concentrations for selection at embryo germination stage were determined at 224 and 407 mM mannitol, respectively. Under mannitol stress conditions, inhibition of callus growth and increase in abnormalities such as hyperhydric cells with an increase in water stress were observed. Six NCo376 putative osmotic tolerant mutants were isolated from the EMS-mutagenized callus and were in vitro characterized based on their physiological response to mannitol-induced stress. Mut1 had the highest re-rooting (RR) ability under mannitol stress and Mut4 had the highest relative water content (RWC). Mut2 and Mut6 demonstrated poor RR and RWC response and were characterized by high electrolyte leakage and H2O2 activity. Only Mut1 and Mut4 were the promising drought-tolerant mutants due to their better physiological response under mannitol stress. This study provides a preliminary in vitro screening method for osmotic stress tolerance based on sugarcane callus morphology at LD50 = 224 and LD90 = 407 mM mannitol, respectively and plantlet re-rooting at 332 mM mannitol. Future work will screen selected mutant lines in pots and under field conditions to confirm tolerance as in vitro screening may not completely eliminate escapes and may alter important agronomic traits.

National Research Foundation (NRF)