DAB assay: A simple, rapid and reliable protocol for screening for water stress - Case study of common bean and cowpea

H₂O₂, induced due to biotic and abiotic stresses, is a strong oxidant and can initiate localized oxidative damage in leaf cells leading to disruption of metabolic function and loss of cellular integrity resulting in senescence. Staining with DAB (3,3-Diamainobenzidine), which is an efficient marker of H₂O₂ accumulation in vivo, can be reliably used for delineating stress response in crop plants. In the present paper we validated the utility of DAB assay for stress screening by evaluating 50 genotypes of common bean and 40 genotypes of cowpea under irrigated and drought conditions. In both the crops, the assay clearly differentiated the lines on the basis of darker staining of leaves under drought. The lines showing greater per cent reduction in yield parameters showed greater staining in DAB assay underlining the reliability of using this assay as a supplement to phenotyping protocols for characterizing large germplasm sets. The assay is cost effective as compared to spectophotometric methods of assessing oxidative damage, less time consuming and can be replicated across crops. The detection of cellular levels of H₂O₂ using DAB staining method and our results reveal a clear difference in degree of staining achieved in the stressed plant. It can be safely concluded that DAB staining can be an efficient biochemical tool for screening for water stress in legume crops.