Interaction of callus selection media and stress duration for in vitro selection of drought tolerant callus of wheat

Abstract

Callus culture is a novel approach addressing cultured cells as selection units independent of whole plant. Natural variations for drought tolerance existing among cell lines can be exploited in vitro in the presence of suitable concentration of osmoticum and stress duration. The study was aimed to standardized callus selection media and culture duration to select drought tolerant cell line (callus) of wheat. Calli were induced from immature embryos on Murashige and Skoog (MS) based medium supplemented with 4 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D). Proliferated calli were cultured on various callus selection medias (MS based media + 30 g/L sucrose + 6 g/L agar + 4 mg/L 2,4-D + various levels of polyethylene glycol-600 (PEG-6000) induced osmotic stress including -0.3, -0.6, -0.9 or -1.2 MPa along with control, that is, 0.0 MPa) for two, three or four weeks. Callus health, callus growth rate, callus survival (%) and regeneration (%) declined significantly on media supplemented with higher levels of PEG-6000 induced osmotic stress and with increasing stress duration. Not a single callus could survive nor regenerate on selection media comprising PEG-6000 induced osmotic stress of -1.2 MPa imposed for four weeks. The highest callus growth rate (CGR) was recorded on PEG-6000 free media when calli were cultured for four weeks (-7.32%) on the media supplemented with PEG-6000 induced osmotic stress of -1.2 MPa. Callus selection media comprising PEG-6000 which induced osmotic stress of -0.9 MPa for four weeks was found selective and sub-lethal for wheat calli with 8.63% CGR, 26.62% callus survival and 21.50% regeneration; and seemed ideal for screening drought tolerant somaclonal cell lines of wheat.

Key words: Interaction, polyethylene glycol-600 (PEG-6000), culture duration, sub-lethal, regeneration, somaclonal variation.