PhD

The present investigations on in vivo and in vitro screening of Sida spp. For ephedrine content were carried out at the experiment field and Plant Tissue Culture Laboratory of the Department of plantation Crops and Spices and Biochemistry laboratory of All India Co-ordinated Research Project on Medicinal and Aromatic Plants, College of Horticulture, Kerala Agricultural university, Vellanikkara, Thrissur, from September 1993 to march, 1998. The objectives of the study were to screen four commonly occurring Sida spp. Viz S. rhombifolia ssp. Retusa, S. acuta, S. rhombifolia ssp. Rhombifolia and S. cordifolia for ephedrine under in vivo and in vitro conditions and to explore the possibilities of upgrading the content of this alkaloid in vitro.
Leaf, stem and root callus cultures of the four experimental species were established in vitro. Half strength MS medium supplemented with 2, 4-D 1 mg 1-1 was observed ideal for initiation and proliferation of calli. Kinetin at 0.3 and 1.0 mg 1-1 enhanced the callus inducing property of NAA. Among the species tested, S. acuta recorded significantly superior performance registering high callus index values of 319.60 and 311.19 respectively for leaf and stem cultures on half MS medium incorporated with 2, 4-D 1.0 mg 1-1 . The auxin synergist, phloroglucinol at levels of 100.0 mg 1-1 and 125.0 mg 1-1 was singularly effective in promoting callusing in Sida spp. Incubating leaf and stem cultures under illuminated conditions at 27 + 10C was significantly superior to incubation in dark or at 10-110C. Root explants were inferior to leaf and stem explants in inducing and proliferating calli.
Successful regeneration of roots from leaf and stem calli of the experiment species was achieved with NAA, 1.0 mg 1- . Stimulatory effects of the growth factor combination, NAA and kinetin at 1.0 mg 1-1 and 0.3 mg 1-1 were reflected in number of roots regenerated. The promotive effects of 2 ip in root regeneration were more evident in stem cultures. NAA and kinetin at 0.5 mg 1-1 and 1.0 mg 1-1 respectively initiated shoots in leaf (20.10 per cent) and stem (26.93 per cent) callus cultures. Substituting sucrose with maltose in proportions of 1:2 in half strength MS media fortified with NAA and kinetin each at 1.0 mg 1-1 initiated embryoids in S. rhombifolia spp rhombifolia and S. cordifolia.
Half MS media supplemented with NAA and kinetin, each at 1.0 mg 1-1 was standardised as the production medium which recorded positive response in leaf calli of S. cordifolia with respect to synthesis of ephedrine in qualitative and chromatographic tests. Butanol – glacial acetic acid – water at 4:1:1 was identified as the appropriate solvent system with ninhydrin as the localizing spray. Incorporation of yeast extract at 2.0g 1-1 and the precursor phenyl alanine at 50.0 mg 1-1 and 100.0 mg 1-1 elicited synthesis of ephedrine in leaf and stem calli of S. cordifolia. Methionine, another precursor failed to elicit synthesis of alkaloid in callus cultures of Sida spp. Addition of osmoregulant, polyethylene glycol at 2.0 per cent exerted a favourable influence on synthesis of ephedrine in leaf and stem calli of S. cordifolia. Definite presence of ephedrine in in vitro cultures of S. cordifolia was confirmed by eliciting the cultures with autoclaved mycelia of Pythium aphanidermatum at 500.0mg 1-1, 2.0g 1-1 and 5.0 g 1-1. Supplementing elicitation with precursor feeding was particularly beneficial to synthesis of ephedrine, wherein apart from S. cordifolia leaf callus cultures of S. rhombifolia ssp. Rhombifolia synthesised ephedrine. Immobilization or irradiation of calli failed to produce the alkaloid.
Success in establishment of hairy root cultures from in vitro calli of S. cordifolia was dependant on the efficiency of the strain of Agrobacterium rhizogenes employed Strain A4 induced hairy roots in 50 per cent cultures each. In leaf and stem calli of S. cordifolia and 16.67 per cent cultures in root calli of the same species.
Sucessful liquid suspensions of the experimental species could be established with a critical cell density of 2 g calli in 50 ml culture media, subcultured at an interval of 17 days with an inoculum ratio of 1:4. S. cordifolia was most effective with respect to proliferation in liquid suspensions registering an increase in packed cell volume of 200 per cent and 150 per cent respectively in leaf and stem calli, at 17 days subculture.
Estimation of content of ephedrine in positively responding in vitro systems revealed that elicitation coupled with precursor feeding produced highest content of 0.0208 per cent and 0.0107 per cent respectively in leaf and stem calli of S. cordifolia. Barring cultures fed with phenyl alanine, static cultures synthesized higher amounts of ephedrine as compared to suspensions. Total free amino acid content of alkaloid producing fresh calli exceeded that of unproductive fresh and aged calli while total phenol content registered low values in alkaloid producing calli.
Studies on yield parameters of field grown Sida spp. Revealed that total yield per plant and mean root yield varied significantly with stages of harvest, the maximum values being obtained at harvest at 9 months after planting. However stages of harvest did not influence the content of crude extractables of the experimental species significantly. S. rhombifolia ssp. Retusa ranked superior with respect to total biological yield and shoot yield per plant. Leaf extracts of S. cordifolia recorded comparatively higher content of ephedrine (0.0089) when harvested at 7 months after planting.