In vitro regeneration and development of rapd- scar marker for the identification of Artemisia Absinthium L.

Abstract

Experiments were carried out to determine the effect of different plant growth regulating hormones on in vitro propagation of Artemisia absinthium L. from leaf explants. Organogenic callus was observed when explants were inoculated on the MS medium supplemented with BAP, 1.0 mg/l in combination with 2,4-D, 0.5mg/l. Maximum adventitious (28) shoots were induced on MS medium supplemented with NAA, 1.0 mg/l and KIN, 2.0 mg/l after the fifth subculture. MS medium augmented with 2 mg/l gave maximum root initiation, the average number of roots per shoot were 3.9. Individual plantlets were removed from the flasks, washed and transferred to a sterilized pot having soil:sand:decomposed coffee husk at a ratio (v/v) of 1:1:2 kept at high humidity (80-90%) for 15 days. RAPD-based SCAR marker was also developed using 25 random primers for initial screening. A 475 bp polymorphic band obtained with an OPAA-8 primer which was specific to all A. absinthium accessions. This RAPD-amplicon was eluted, sequenced and cloned and a pair of SCAR primers was designed. A single, bright, distinct band of 225 bp was obtained which shows its specificity towards the accessions of A. absinthium and thus can be useful in its authentication.