Effective Surface Sterilization Method using Plant Preservatives Mixture and Shoot Multiplication of Clinacanthus nutans

Abstract

Effective surface sterilization and shoot regeneration protocols for Clinacanthus nutans are described. Nodal segments of C. nutans were sterilised by single-and double-stage sterilisation methods. In the double-stage method, explants were soaked in Plant Preservative Mixture (PPM) at various concentrations (0, 1.25, 2.50, 5.0 µL/mL). For direct shoots regeneration, MS media were supplemented with TDZ, BAP, or KN in single or in combination (BAP+NAA or TDZ+NAA). For indirect shoot regeneration, single application (BAP or KN) and combination treatment (TDZ+BAP or TDZ+KN) of cytokinins were evaluated. The use of PPM (1.25 µL/mL) resulted in survival rate of explants at 82 ± 4.5 %. Direct shoot multiplication was the highest on the medium with 1.0 mg/L BAP + 0.1 mg/L TDZ where explants produced 20 ± 1.5 % primary, 57 ± 6.2 % secondary and 77 ± 6.6 % tertiary shoots respectively. The medium supplemented with BAP at 0.5 mg/L induced the highest number of shoots indirectly (3.5 ± 0.1 shoots/explant) while BAP at 1.5 mg/L induced the highest percentage of explant forming callus (84.4 ± 5.1 %). Therefore, double sterilisation method using Clorox and PPM at 1.25 µl/ml was optimum for surface sterilisation. A protocol for establishment of C. nutans culture is established.