Ascorbic Acid Controls Lethal Browning and Pluronic F-68 Promotes High-frequency Multiple Shoot Regeneration from Cotyldonary Node Explant of Okra (Abelmoschus esculentus L.)

Abstract

The regeneration frequency of okra (Abelmoschus esculentus) is greatly influenced by its genetic makeup and recalcitrant nature. Phenolic secretion, in particular, is a major problem in okra tissue culture. This study describes a reproducible, rapid, and more efficient in vitro regeneration method using cotyledonary node explants of okra. Explants were incubated on Murashige and Skoog (MS) medium containing different concentrations and combinations of various plant growth regulators (PGRs) [benzyladenine (BA), thidiazuron (TDZ), and α-naphthylacetic acid (NAA)], and regeneration enhancers [silver nitrate (AgNO3) and Pluronic F-68]. Cut ends of cotyledonary node segments rapidly turned brown and cultures failed to establish. Antibrowning additives, such as activated charcoal (AC), ascorbic acid (AA), and AgNO3 at various concentrations in PGR-free MS basal medium were tested for their ability to control phenolic secretion from explants. Among these additives, 15 mg·L−1 AA was found to be optimal for controlling phenolic secretion, resulting in healthy explants and culture establishment. The highest number of shoots (a mean of 9.3 ± 0.9 shoots per cotyledonary node explant) was obtained on MS media containing 0.5 mg·L−1 NAA + 1 mg·L−1 TDZ + 0.1% Pluronic F-68. Individual shoots were elongated on MS medium + 1 mg·L−1 BA + 0.1 mg·L−1 gibberellic acid (GA3) (shoot length 5.3 ± 0.2 cm) and rooted on ½ MS medium + 1 mg·L−1 indole-3-butyric acid (IBA) and 200 mg·L−1 AC (5.3 ± 0.2 roots per shoot). Rooted plantlets were acclimatized in plastic pots inside a plant growth chamber at 25 ± 2 °C and 70% relative humidity, with an 80% survival rate. This optimized protocol can be used for producing transgenic plants of commercial okra cultivars through genetic transformation.