Keshavachandran, RSmini VargheseKAU2019-06-202019-06-202006http://krishikosh.egranth.ac.in/handle/1/5810109366PGThe present study entitled ‘Genetic transformation for hairy root induction and enhancement of secondary metabolites in Aswagandha (Withania somnifera (L.) Dunal) was carried out at the Centre for Plant Biotechnology and Molecular Biology and Biochemistry Laboratory of the College of Horticulture, Vellanikkara and the Biochemistry Laboratory of Aromatic and Medicinal Plants Research Station, Odakkali. The study was undertaken to standardize the in vitro regeneration protocol in W. somnifera from different explants, to standardize the genetic transformation using Agrobacterium rhizogenes, to standardize the biochemical techniques for the estimation of secondary metabolites in roots and also to enhance the secondary metabolite production in the hairy root cultures of W. somnifera. An efficient method for in vitro plant regeneration was developed in W. somnifera. Different explants such as hypocotyls, cotyledonary segments, leaf segments, shoot tips, nodal segments and roots from in vitro germinated seedlings were used for the study. The seeds showed early and high germination under dark compared to 16 h photoperiod. Multiple shoot formation was observed from hypocotyl segments placed directly and upside down. Maximum regeneration response was obtained from hypocotyls, cotyledonary segments and leaf segments in MS + BAP 1.0/ 1.5 mg l-1 + IAA 0.5 mg l-1. Shoot buds produced from hypocotyls and cotyledonary segments showed good multiplication in MS + BAP 0.4 mg l-1 + IAA 0.5 mg l-1. Cotyledonary segments derived callus regenerated in MS + BAP 2.0 mg l-1. Shoot tips and nodal segments taken from axenic seedlings showed highest frequency of multiple shoot formation in MS + BAP 2.5 mg l-1 and IAA 0.5 mg l-1. Regeneration from basal callus, produced by shoot tips and nodal segments was also obtained in the same culture cycle. The roots taken from in vitro seedlings and in vitro rooted plantlets showed high callusing but failed to regenerate by direct organogenisis. However, somatic embryo like structures were produced from seedling roots in MS + BAP 1.5 mg l-1 and IAA 0.5 mg l-1. The shoot/ shoot buds produced good elongation in MS + GA3 0.5 mg l-1. In vitro flowering of cotyledonary segment derived shoots was also obtained in this combination. The shoots were successfully rooted in half MS + 0.25 per cent activated charcoal by pulse treatment with IBA 1000 mg l-1 for five seconds. The plantlets were successfully hardened and transferred to large pots in the green house. Genetic transformation was carried out in W. somnifera using three different A. rhizogenes strains like A4, ATCC 15834 and MTCC 2364 for inducing hairy roots. The explants such as hypocotyls, cotyldonary segments, leaf segments, shoot tips and nodal segments were used for genetic transformation. Here the influence of different parameters such as type of explants, type of bacterial inoculum, co-cultivation periods and acetosyringone effects on transformation frequencies were studied. Among the three A. rhizogenes strains, the strains A4 and ATCC 15834 produced successful transformation. Of this two successful strains ATCC 15834 showed a greater potential for transformation. Among the various explants used, only the leaf segments and shoot tips produced hairy roots. Leaf segments showed a greater percentage of transformation than the shoot tips. Though A4 strain produced successful transformation in W. somnifera by direct inoculation of bacteria from single cell colonies as well as in the suspension form, the strain ATCC 15834 produced transformation only in the suspension form. A co-cultivation period of one day was found to be the best for leaf segments, whereas shoot tips responded more under two day co-culture period. The acetosyringone (100 M) enhanced the transformation percentages with A4 strain, whereas no such influence was observed with ATCC 15834 strain. The hairy root cultures established on MS + 250 mg l-1 cefotaxime showed phenotypic variations in growth habit. The hairy roots normally produced high lateral branching with plagiotropic growth habit and showed sigmoid growth pattern. Among the four liquid media tested, half MS was found to be superior in promoting hairy root growth followed by MS, B5 + 2.0 per cent sucrose, B5 + 3.0 per cent sucrose respectively The confirmation of transformation by opine detection was found to by unsuccessful in W. somnifera because of the presence of interfering substances which produced spots near the positions of agropinic acid including control. Because of the low concentration of DNA, Southern hybridization technique failed to produce band corresponding to hairy root samples. However, the transformation was confirmed in A4 and ATCC 15834 induced hairy roots by PCR and dot blot analysis. A Thin Layer Chromatographic method was employed for withanolide estimation. Withaferin A was used as the standard in estimation studies. Silica gel60 F254 plate chloroform- methanol (9.8: 0.2) was used as the solvent system. The spot was observed under UV at 254 nm and also the sensitivity was improved by using vanillin (0.05 g) + boric acid (1.0 g) + H2SO4 (2.0 ml) + Methanol (100 ml) spray reagent. Withaferin A produced magenta spot, which changed to bluish violet on further charring. Field root possess more withaferin A followed by hairy roots and in vitro roots contained the least. Enhancement of secondary metabolite production was studied using techniques such as addition of osmoregulants, precursor feeding and elicitation. The withaferin A content in the hairy root biomass and the culture medium were estimated. The osmoregulant PEG (2.0 % and 5.0 %) and methionine precursor (1mM and 2mM) failed to enhance the withaferin A content. With the addition of yeast extract (2.5 and 5.0 g l-1) a reduction in withaferin A content was observed in the root biomass. However, the biotic elicitor Aspergillus homogenate (250 and 500 l /125 ml) elicited a positive influence on the biosynthesis of withafein A in the hairy root cultures.ennullThesis