Plant regeneration from callus of Cuscuta reflexa – an angiospermic parasite – and modulation of catalase and peroxidase activity by salicylic acid and naphthalene acetic acid

Callus induction and plant regeneration system for an obligate parasite, Cuscuta reflexa (family Cuscutaceae), has been established. Callusing was induced from explants of basal portion of seedlings as well as from shoots (vines) in a modified Murashige and Skoog (MMS) media supplemented with 1 mg·L–1 benzyl adenine (BA) and 3 mg·L–1 naphthalene acetic acid (NAA) (MMS-1). The MMS media containing 1 mg·L–1 BA (MMS-2) was found to be better than the MMS-1 media for callus proliferation and long term maintenance. Use of glucose in place of sucrose enhanced callus induction and growth. The browning of callus was substantially reduced by supplementing 0.1 mg·L–1 gibberellin to the MMS-1 media. Shoot regeneration from the green callus was achieved in the MMS media containing 3 mg·L–1 NAA and 1 mg·L–1 kinetin. Cuscuta callus was characterised by having several folds higher peroxidase activity than those of culture-raised and field-grown vines. Catalase activity also revealed a pattern similar to that of peroxidase except for the fact that it could not be detected in field-grown vines. Developmental profiles of peroxidase and catalase, during the growth of the callus in the absence and presence of NAA, revealed inhibition of both the enzymes by NAA, though inhibition of peroxidase was stronger as compared to that of catalase. The inhibitory effect of NAA was also evident when in-gel activity staining of the two enzymes were performed. In callus, three acidic and four basic isoperoxidases were present, while in field-grown vines, two acidic and one basic isoperoxidases were present. Investigation of the effect of salicylic acid on catalase and peroxidase revealed inhibition of both enzymes in a concentration dependent manner during seedling growth. The inhibitory effect of salicylic acid was also evident when in-gel enzyme activity staining of the two enzymes was done. The results are discussed in light of the proposed mode of action of salicylic acid.