In vitro regeneration of gamma irradiated callus of artemisia annua and evaluation of increase artemisinin content by HPLC analysis

Abstract

Gamma radiations are the efficient ionizing radiations which have potential to induce mutations in plants. They also modify physiological characteristics of plant to create new mutants for large scale production of commercially important secondary metabolites. In present study, the effect of various low doses gamma irradiations (5, 10, 15, 20, 25, 30 and 35Gy.) on growth, regeneration and elicitation of Artemisinin content from Artemisia annua was studied. Callus culture was established on Murashige and Skoog medium supplemented with phytohormones Napthalein acetic acid and N6-benzylaminopurine (0.5mgL− 1). Effect of gamma doses on callus growth and elicitation of artemisinin content were investigated from M1T1, M2T2, M3T3, M4T4 and M5T5 generation of in vitro callus culture. In current study, it was found that low dose of gamma irradiations are effective strategy to enhance the yield of artemisinin in A. annua. The radiation sensitivity test was performed which is based on the reduction in survival rate of callus culture after treatment with gamma irradiation at different doses from 5 to 35Gy which showed the negligible effect till 25Gy but at 30 and 35Gy, it inhibited the callus growth and callus regeneration. The detection and quantification of artemisinin content was performed by HPLC analysis. The mobile phase for artemisinin elution was acetonitrile: water (50:50v/v) at a flow rate of 1ml min−1 and UV detection at 254nm. Artemisinin concentration of unknown sample was calculated by using the Y equation y=20266x - 5664.R²=0.914 generated from standard curve. The retention time of artemisinin in above solvent system was observed at7.3min. The higher accumulation of artemisinin content was observed in 15Gy dose 7.04µg/gm dry callus weight which is tenfold higher than non treated 0.700µg/gm dry callus weight. The present study confirms that gamma radiation induced mutational changes in callus culture and it enhanced the artemisinin content.