Genetic transformation in Artemesia annual L. for hairy root induction and enhancement of secondary metabolites
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Date
2007
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
The present study entitled “Genetic transformation in Artemesia annua L. for hairy root induction and enhancement of secondary metabolites” was carried out at the Centre for Plant Biotechnology and Molecular Biology of the College of Horticulture, Vellanikkara. The study was undertaken to standardize the in vitro regeneration protocol in Artemesia annua from different explants, to standardize the genetic transformation using A. rhizogenes, to standardize the biochemical techniques for the estimation of secondary metabolites in Artemesia annua and also to enhance the secondary metabolite production in the hairy root cultures of Artemesia annua.
An efficient method for in vitro plant regeneration was developed in Artemesia annua. Different explants such as leaves, petiole, shoot tip, nodal segments, inflorescence segments and roots of Artemesia annua were used for the study.
Maximum regeneration from leaf explants was in MS with 0.5 mg l-1 BAP. Shoot buds produced from leaf explant showed good multiplication in the same media. MS was found to be the best basal media for regeneration from leaf explants.
The leaf originated callus produced regeneration in MS with 3 mg l-1 BAP. The shoot tip and inflorescence bits showed maximum regeneration in 0.2mg l-1 NAA and 0.5 mg l-1 BAP. Nodal segments showed maximum regeneration in MS with 0.1 mg l-1 with NAA and 0.2 mg l-1 BAP.
The roots taken from in vitro rooted plantlets showed high callusing but failed to regenerate by direct organogenisis. However, a green coloured structure was obtained from root callus in MS media supplemented with BAP 3 mg l-1 and NAA 0.1 mg l-1.
The shoot/ shoot buds showed good elongation in MS media with GA3 0.2 mg l-1. The shoots were successfully rooted in half MS with 0.5 mg l-1 IBA and 2 per cent sucrose. The plants were successfully hardened and transferred to large pots in the green house.
Genetic transformation was carried out in A. annua. using three different A. rhizogenes strains like A4, ATCC 15834 and MTCC 2364 for inducing hairy roots. The explants such as 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 used, ATCC 15834 produced the highest transformation efficiency. Acetosyringone (100 M) enhanced the transformation percentages in all the strains.
The hairy root cultured in hormone free basal media showed high lateral branching. Among the four liquid media tested, B5 with 3.0 per cent sucrose was found to be superior in promoting hairy root growth followed by B5 with 2.0 per cent sucrose, MS and half MS.
The confirmation of transformation was obtained by opine detection and PCR.
A Thin Layer Chromatographic method was employed for artemisinin estimation. Artemisinin obtained from Sigma Chemicals, USA was used as the standard in estimation studies. Silica gel60 F254 plate with hexane- diethyl ether (1:1) as the solvent system was used. The pink spot was observed by immersing the plates in a pool of freshly prepared glacial acetic acid: conc. sulphuric acid: anisaldehyde (50:1:0.5) followed by drying in a chromatographic oven at 110˚C for 15 min. No artemisinin was detected in roots taken from plants grown in the field, in vitro roots and hairy roots induced by MTCC 2364. Rooting of in vitro shoots enhanced the artemisinin content.
Enhancement of secondary metabolite production was studied using techniques such as addition of osmoregulants, precursor feeding and elicitation. The artemisinin content in the hairy root biomass and the culture medium were estimated. The osmoregulant PEG (2.0 % and 5.0 %) and the precursor methionine (1 mM) and yeast extract (2.5g l-1) failed to enhance the artemisinin content. However, the biotic elicitor Aspergillus homogenate (250l /125 ml) elicited a positive influence on the biosynthesis of artemisinin in the hairy root cultures.
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