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.
Aims: Catunaregam spinosa is an exotic plant in Sri Lanka. Fruits of this plant contain saponins, steroids, flavonoids possessing piscicidal property. Since years ago C. spinosa employs in the fishery industry, especially in rural areas. This study was established to evaluate the toxicity and the teratogenic effects of fruits of C. spinosa on D. rerio embryos. Methodology: Semi-static renewal method was conducted to determine the median lethal concentration. Concentrations of 15.0, 17.0, 19.0, 21.0, 23.0 and 25.0 mg L-1 were tested with twenty embryos per treatment. Each concentration was triplicated. Dilution water and 3, 4- Dichloroaniline at 4.0 mg L-1 were tested for negative and positive controls respectively. Four apical observations and teratogenic effects were examined at 24, 48, 72 and 96 h time intervals. Results: Embryos exposed to 19.5 mg L-1 concentration exhibited 50% mortality at p = 0.05 significance level. Embryos exposed to high concentrations exhibited more teratogenic deformities with a high mortality rate. Negative control recorded >90 % survival rate and positive control 95.0% lethality after 96 h exposure. Hatchability was negatively correlated with the concentration of the extract. Conclusion: Methanolic fruit extract of C. spinosa showed concentration-dependent mortality and the teratogenic effects on D. rerio embryos. It could be concluded that the fruits of C. spinosa show moderate piscicidal activity.
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