TDZ-induced plant regeneration in Brassica oleracea L. var. botrytis: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

Abstract

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB5) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB5 medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB5 medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB5 medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB5 medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA3). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.