Development of an appropriate protocol for micropropagation of Calotropis gigantean (L.) (Wara).

Calotropis gigantea (Wara) is one of the least concerned native plant in Sri Lanka. Because of its high pharmaceutical application and other usage, this species has been over exploited and included in the Red list since year 2012. The present study was carried out to develop a protocol to produce healthy plantlets of C. gigantea. Murashige and Skoog medium (MS) (0, ¼, ½, ¾ and full MS) were tested to select the best medium for in vitro seed germination. To determine the best explant and 6-Benzyl amino purine (BAP) concentration for micro propagation, in vitro shoot tip, stem without node, stem with node, leaf and root were tested with different concentrations of BAP (0, 1 mg/l, 2 mg/l, 3 mg/l, 4 mg/l, 5 mg/l and 6 mg/l) and 0.1 mg/l NAA. Agar and Corn flour were used to gelatinize the medium. To determine the proper maturity level of plantlet, 1, 1 ½, 2, 2 ½ and 3 months old plants were tested. Then regenerated shoots were transferred to different IBA (Indole 3-butyric acid) concentrations (0, 0.5 mg/l, 1 mg/l and 2 mg/l) for rooting. Ten replicates per each treatment were used in every experiment. Data were analyzed according to 2-factor factorial Completely Randomized Design (CRD) using SAS statistical software. The medium with agar (without MS) showed the highest seed germination of 72% while the medium containing full MS showed the lowest seed germination of 34%. Significantly higher number of shoots (2.4) was observed from MS medium with 2 mg/l BAP + 0.1 NAA + Agar within 11days, showing 100% regeneration from stem with node. When corn flour was used instead of agar for the above medium, shoots regeneration was 2.7 within 17 days. One month old explant (stem with node) showed significantly higher number of shoots (2.0) whereas three month old explant showed lowest number of shoots (0.8). The highest number of roots (3.4) was observed from 1mg/l IBA +MS + Agar. However, with corn flour instead of agar, average number of roots was 4.4. Considering these observations, it could be concluded that Agar is suitable as a medium for seed germination. Stem with node is the best explant to produce large number of healthy shoots. The best BAP concentration for shoot regeneration was 2mg/l BAP+0.1mg/l NAA, while 1mg/l IBA was good for rooting. One month old explants were better than matured explants, and corn flour was better compared with agar in increasing the growth and development of C. gigantea.