In vitro Callus Induction of Catunaregam spinosa Using Leaves as Explant
P. K. Lawrence
Faculty of Graduate Studies, University of Sri Jayewardenepura, Sri Lanka and Department of Botany, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka.
W. T. P. S. K. Senarath *
Department of Botany, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka.
M. L. A. M. S. Munasinghe
Department of Botany, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka.
*Author to whom correspondence should be addressed.
Abstract
Aims: C. spinosa (Family Rubiaceae) is a valuable medicinal since years ago. A protocol was developed including best sterilization and best medium for callus induction of C. spinosa using leaf discs as explants.
Methodology: Sterilization protocol optimized using different concentrations of Carbendazim® (0.2,
0.3%) and Clorox (10, 15%) exposing to different time intervals (10, 15 min). Percentage survival and contaminations were calculated. Best medium was optimized using different concentrations of 6-Benzyl Amino Purine (BAP) and Naphthalene Acetic Acid (NAA) (1.0-6.0 mg L-1). Growth regulators free Murashige and Skoog (MS) medium was used as control. Completely randomized design was followed with ten replicates in each concentration. Days taken to initiate calli, morphological characteristics and mean dry weights of calli were evaluated after 3 months of incubation.
Results: Leaf discs sterilization with 0.3% Carbendazim for 10 min, 10 % Clorox for 10 min and 70% ethanol for 30 sec followed by two washings in sterile distilled water was found to be best sterilization protocol. It recorded lowest percentage contamination (13.34%) and highest percentage survival (86.66%) after 8 weeks. No observable changes were found in calli grown in growth regulators free MS medium. Calli growth and morphologies were significantly affected by the type of growth regulator and their concentrations in MS medium. Color of the calli varied from white opaque to yellow brown to green and the texture from foamy, loose, and friable to compact. Best medium with 1.0 mg L-1 BAP and 3.0 mg L-1 NAA produced green friable calli with 0.0969±0.01 g mean dry weight after 3 months. Some traits were found to be depended on synergetic effect of growth regulators and genotypic characteristics of explant.
Conclusion: The study provides a better sterilization protocol and medium for in vitro calli induction and growth of C. spinosa.
Keywords: Catunaregam spinosa, tissue culture, sterilization, plant growth regulators, callus