Ethyl Methane Sulphonate (EMS) induced mutation and selection for salt tolerance in sugarcane in vitro

Sugarcane mutants against salt tolerance were derived using in vitro selection and regeneration under NaCl stress. Sugarcane cv. Co 86032 was used for callus development from meristematic leaf whorl on MS medium supplemented with 5mg/l 2,4-D + 0.2% sucrose. Four concentrations of NaCl were evaluated for salt tolerance level in sugarcane callus in vitro. NaCl 200mM did not allow any growth of calli and was adjudged most suitable selective pressure for salt tolerant sugarcane calli and shoots. Callus was treated with EMS, a potential mutagen at 0.5% for different time intervals of 1, 1.5, 2, 2.5 and 3 hours separately and regenerated on MS + 3mg/l 2- 4, D + 0.2% sucrose, fortified with 200mM NaCl. During four subcultures calli treated for 2.5 hours only could gain higher weight. Though treatment for 2 hours was capable of gaining calli weight but the growth rate was lower. Calli were regenerated on MS + 0.2mg/l BAP + 0.1mg/l NAA + 0.1mg/l Kinetin and 0.2% sucrose fortified with 200mM NaCl. Calli treated with EMS for 2 hours failed to induce any active regeneration. Maximum number of 15 healthy plants were successfully regenerated from the calli treated for 2.5 hours. Treatment of EMS for 3 hours induced regeneration at lower rate with three healthy plants only. Treatment for 2.5 hours was most ideal in deriving maximum mutants. However, lower period of time was less effective and higher was lethal. The in vitro regeneration via callus in itself causes selection and variation and treatment with EMS creates additional variation. The pressure of NaCl would be an ideal combination for development of true mutant salt tolerant sugarcane lines. The results offer cheap, less time and less labour intensive method for development of mutants for large samples.