In Vitro Adventitious Shoot Regeneration through Direct and Indirect Organogenesis from Seedling-derived Hypocotyl Segments of Ficus religiosa L.: An Important Medicinal Plant

Abstract

Ficus religiosa is an important industrial, medicinal, and ornamental plant, so in vitro regeneration is of high paramount in this valuable germplasm. Two efficient protocols were developed for indirect and direct shoot organogenesis through hypocotyl explants. In the first experiment, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and indole butyric acid (IBA) (0.5, 1.0, and 1.5 mg·L−1) in combination with 6-benzyl amino purine (BAP) (ratio 10:1, respectively) were used for callus formation. Two types of callus were obtained from different concentrations of plant growth regulators (PGRs). Also, 2,4-D produced yellow-brownish and friable callus (Type I), whereas the green and compact callus (Type II) was achieved in IBA. The highest callus fresh weight (2.43 g) was observed in Murashige and Skoog (MS) medium containing 0.5 mg·L−1 2,4-D plus 0.05 mg·L−1 BAP. In the later experiments, various concentrations of thidiazuron (TDZ), 6-furfuryl amino purine (KN), and BAP (0.25, 0.5, 1.0, and 1.5 mg·L−1) in combination with IBA (ratio 10:1, respectively) were applied for shoot regeneration (direct and indirect organogenesis). In shoot regeneration from callus, the highest regeneration frequency (86.66%) and shoot number per callus (4.13) were achieved in MS medium supplemented with 1.5 mg·L−1 BAP plus 0.15 mg·L−1 IBA from type I calli. However, no regeneration was observed in type II calli. In direct shoot regeneration, the highest regeneration frequency (96.66%) and shoot number (6.26) were obtained in the medium mentioned previously. In root induction experiment, different concentrations of naphthalene acetic acid (NAA) and IBA alone or in combination were applied, and MS medium containing 2.0 mg·L−1 IBA along with 0.1 mg·L−1 NAA was the best hormonal balance for root induction. The rooted plantlets’ survival rate was more than 95% in the acclimatization stage. These results demonstrated that the direct regeneration method provides more shoot regeneration frequency and take a less time for shoot organogenesis than the indirect regeneration method. Based on our knowledge, this study is the first report of direct and indirect shoot organogenesis of F. religiosa via hypocotyl from in vitro–grown seedling.