IN VITRO BIOCHEMICAL STUDIES OF SAUSSUREA LAPPA

Abstract

Saussurea lappa is an endangered medicinal plant was selected for in vitro biochemical assarys. Seeds and apical shoot explants were surface sterilized using different standard protocols. The explants were placed on semi solid MS media (1 mgL-1 2-4 D and 0.5 mgL-1 Kn) for callus induction. The three different concentrations (0.05%, 0.1% and 0.2%) of mercuric chloride (HgCl2) were used separately for surface sterilization for 8 minutes. The 0.2% concentration of HgCl2 showed best sterilization (96%). While, 0.1% used for 1 minute was found best in case of seedling apical shoot sterilization. The apical shoot explants showed better results for multiple shoot proliferation on MS medium containing 1.0 mgL-1 benzyle amino purine (BAP) and showed best results with maximum number of shoots, roots, length of shoot and root per explants. At same concentration the explants produced 90% shoots and produced 93.66% roots. About 80% plants were survived after hardening. Calli were induced from explants (root, shoot and leaf) of micropropagated plant and 3 days old sterilized seedling. It was found that seedling and root explants were more efficient compared to other explants. The highest RGR of callus was noted in seedling explants followed by root explants. The seedling derived callus RGR, relative water content (RWC) and dry weight were measured for three weeks and found that with increase of time duration a little decrease occurred in RGR. The overall results showed that 2 weeks old callus was the best to use. The highest level of proline and total soluble sugar accumulation was observed in the root callus. However, calli and root showed higher concentration of Na and K ions. Non-significant differences were observed in calcium content of all tested samples. Whereas, the content of Mg was found high in roots, petiole and lamina compared to calli. For HPLC analysis, shoot derived callus, seven days old and fifteen days old of root calli were harvested. The ethanolic extracts of calli, cultivated and wild plant roots, petiole and lamina were analysed for costunolide content using high performance liquid chromatography. Costunolide was identified at retention time 14.8 minutes in root derived calli and natural roots, not in calli derived from shoot and cultivated plant lamina and petiole. Wild root collected from Kashmir hills (root c) produced high quantity 1.257 μgmL- 1 of costunolide followed by fifteen days old (callus b) callus 1.119 μgmL-1 and seven days old callus 1.118 μgmL-1 (callus c) while the quantity from cultivated root (root b, root a) was 1.105 μgmL-1, 1.102 μgmL-1 from Koza gali, Abbottabad and Peshawar respectively.