<i>In vitro</i> screening of calli of mungbean to cercosporin, a photoactivated toxin

Abstract

Mungbean or Green gram [<i>Vigna radiata</i> (L.) R. Wilczek] is an arid/semiarid pulse crop, native to India, grown mostly as a rotational crop with cereals like wheat, rice, maize, sorghum, etc. It is an affordable source of protein, carbohydrate, vitamins and minerals preferred for its nutrient digestibility, food processing properties and bioavailability. India accounts for 65% of mungbean’s world acreage and 54% of its world production. Various pests, diseases and environmental stresses have kept mungbean yield quite unstable over decades and researcher’s worldover are looking for resistant varieties to overcome these challenges. Cercospora leaf spot (CLS) caused by <i>Cercospora canescens</i> is one of the most destructive diseases of mungbean and the key polyketide toxin cercosporin plays an important role in pathogenesis. Such toxins as selective agents in the tissue culture medium can help in selecting genotype with suitable levels of resistance to the toxin and/or to the pathogen among the available germplasm. Here, we standardized the dose of cercosporin for <i>in vitro</i> selection of resistant mungbean genotypes and variable expression of peroxidase, catalase and superoxide dismutase. <em>Murashige and Skoog</em> (MS) medium supplemented with 1.0 mg L^-1 NAA and 1.0 mg L^-1 BAP was standardized for the development of callus from mungbean using hypocotyls as an explant. The calli from six cultivars of mungbean were tested in medium amended with cercosporin (0-40 µg mL^-1) and calli survived up to 20 µg mL^-1 of cercosporin. The calli from resistant cultivars survived 83.33-93.00%, and showed lower reduction in fresh weight (25.97-28.83%). Calli from the susceptible cultivars survived 50-60% and showed higher reduction in fresh weight. Callus showed browning, exposure to cercosporin (5-20 µg mL^-1). Enzymes assay from survived calli of different cultivars showed higher peroxidase activity (7.90-8.91 ∆OD min^-1 mg^‑1 callus), superoxide dismutase (0.96-1.03 ∆OD min^-1 mg^-1 callus) and a lower catalase (0.35-0.43 µ moles of H₂O₂ utilized min^-1 mg^‑1 callus) in resistant, followed by moderately resistance and susceptible cultivars. The necrosis in leaves was recorded with 200 µg mL^-1 of cercosporin, and no visible necrosis was observed below this concentration. Enzyme assayed from the controlled and cercosporin-treated (100-200 µg mL^-1) leaves of mungbean genotypes showed variable activity of peroxidase, catalase and superoxide dismutase.