GENETIC STUDIES of ARTEMISIA SCOPARIA WALDST ET KIT SUBJECTED to SALT STRESS and ITS PROTEIN PROFILING *Aditi Singh1, Renu Sarin2, Anushree Yaduvanshi2

Review GENETIC STUDIES OF ARTEMISIA SCOPARIA WALDST ET KIT SUBJECTED TO SALT STRESS AND ITS PROTEIN PROFILING *Aditi Singh1, Renu Sarin2, Anushree Yaduvanshi2

1Department of Environmental Sciences, S.S. Jain Subodh P.G. College, Jaipur 2Department of Botany, University Of Rajasthan, Jaipur

Accepted 17 December, 2011.

Abiotic stress significantly influences survival, biomass production and crop yield. Abiotic stresses including salt stress are serious threats to the sustainability of plants. Use of modern molecular biology tools for elucidating the control mechanisms of abiotic stress tolerance, and for engineering stress tolerant plants is based on the expression of specific stress-related genes. The present investigation concentrates on the salt stress tolerance level in Artemisia scoparia Waldst et Kit, which emerges as a salinity tolerant variety at the end of the study.

Keywords: A. scoparia, DNA, protein, salinity.

INTRODUCTION

Excessive salinity is the most important environmental Singh and Sarin 2010 a, b,c). An analysis of effect of factors that greatly affect plant growth and productivity salinity on the DNA and protein content was under taken. worldwide (Munns, 2002). In area of low rainfall (Western regions of India), salts accumulate because percolating moisture is insufficient to wash out salts added by MATERIALS AND METHODS irrigation. In soils containing an excess of sodium chloride, the water available to the plant is restricted. This For the present investigation abiotic stress was given to process results in a partial dehydration of the cytoplasm. 4-6 weeks old calli of the plant. Callus was raised from Such plamolysis affects the metabolism of the cells and young nodal segments on MS medium supplemented the functions of macromolecules and, ultimately, results with2,4-D(3mg/l) ,Kinetin (0.25mg/l) and proline(100mg/l). in the cessation of growth (Le Rudulier, 2005).Plants Abiotic stress in the form of salinity was given to the growing in the regions with scanty rainfall and increased respective calli to see whether there is any change salt concentration has devised mechanisms to combat (increase/decrease) in the content of DNA and protein, the adverse effects of this abiotic stress on their growth. under stress conditions Keeping this in view a weed, Artemisia scoparia Waldst For the experimental purpose two different et Kit from the western regions of India particularly concentrations of NaCl were selected i.e. 0.5 M and Rajasthan was selected to study the effect of salts at 0.1M. 0. 1ml of each of these concentrations were varying concentrations on its growth in vitro. A. scoparia incorporated into the medium after autoclaving and it was is selected as it has proven to be economically and observed that salt at the concentrations of 0.5M rendered medicinally important species (Yeung Him Che, 1985; extra hardness to the medium. As a result of which there Duke and Ayensu, 1985; Chopra et al., 1986; was no growth of the calli on these two concentrations. Therefore, the further studies were carried out by taking the concentration 0.1M only. The calli of both the plants were incubated on 0.1M salt concentration for about 15- Corresponding Author Email:[email protected] 21 days and then taken for further studies as- DNA Singh et al. 81

Figure 1: DNA Quantification of Artemisia scoparia Waldst et Kit ( Normal callus and Stressed callus)

Abbreviations:N –Normal calli,S- Stressed Calli quantification and protein profiling. DNA extraction was medium. It was observed that salt at a concentration of performed by using the CTAB extraction method. Protein 0.5 M rendered extra hardness to the medium so the profiling was done using SDS-PAGE method. tissue died. Only the tissue in the 0.1M concentration survived but also showed blackening and showed very slow or no growth. These calli were further taken for their RESULTS AND DISCUSSIONS DNA quantification studies, The DNA analysis reflects the following results:-DNA With an objective to study the effect of abiotic stress on quantification was done by extraction of DNA from normal A.scoparia the calli of the plant was subjected to stress in and stressed calli (0.1 M NaCl) from A. scoparia. Figure 1 the form of salinity. NaCl solutions of two different shows the results of DNA quantification for normal and concentrations were prepared and incorporated into the stressed calli of A. scoparia. The following Scholarly J. Agric. Sci. 82

Figure 2: Estimation of proteins isolated from normal and the bands in the marker from top to bottom is as follows: - stressed calli of Artemisia annua. L and A. scoparia Waldst 200 KDa, 97 KDa, 65 KDa, 43 KDa, 29 KDa and et Kit 21.8KDa respectively. In the lane 2, which contained the isolated protein samples from normal callus of A. scoparia, numerous bands appeared; of which the size of the identifiable bands was 200 KDa, 97 KDa, 27 KDa and 21.8KDa respectively in top to bottom approach. In the lane 3 the stressed callus protein samples of A .scoparia were loaded which showed the banding pattern almost similar to that of normal samples.From the studies conducted in the form of DNA quantification and Protein assays it could be inferred that salt exerts a little or negligible effect on the the growth of the weed A. scoparia which was revealed after molecular studies of the normal and stressed calli and thus when DNA quantification of A. scoparia was done due to very minute amount no clear inferences could be drawn. But, after the protein profiling it can be concluded that the plant of A. scoparia is very much stress tolerant as there appeared no significant damage caused to the proteinalt at the concentration 0.1 M.Protein bands of the stressed calli appeared to very much similar as those of the normal calli and thus at the end of the study A.scoparia evolved as a salt stress tolerant variety.

ACKNOWLEDGEMENT

The authors are grateful to Head, Department of Botany, and University of Rajasthan for providing research facilities.

REFERENCES

Chopra, RN, Nayar, SL, Chopra, IC (1986). Glossary of Indian Medicinal Plants (including the supplement). Council of Industrial and Scientific Research, New Delhi. Duke, JA, Ayensu, ES (1985). Medicinal plants of China. Reference Publications. Le Rudulier, D (2005). Osmoregulation in rhizobia : The key role of compatible solutes. Grain Legume, 42:18 - 19. Munns, R (2002). Comparative physiology of salt and Water stress. Plant, cell and Environment, 25 : 239 - 250. Abbreviations: PMW- Protein molecular weight marker AscN- Singh, A, Sarin, R (2010a) Artemisia scoparia-a new source of Artemisia scoparia (Normal), AscS - Artemisia scoparia artemisinin, Bangladesh J. Pharmacol., 5:13-16 (Stressed) Singh, A, Sarin, R (2010b). Artemisinin content in Artemisia scoparia Recent research in science and technology, 2(6): 47-50. Singh, A, Sarin, R (2010c). Antimalarial artemisinin in Artemisia scoparia Waldst et Kit. J. Pharmacol. Res., 3(12):2831-2833 inferences could be drawn from figure 2 Yeung, HC (1985). A very good Chinese herbal. Handbook of Chinese The DNA bands in the tissue of A. scoparia are very herbs and Formulas Institute of Chinese medicine Los Angeles. faint. A very faint band appeared in the normal calli but almost a negligible band appeared in the stressed calli of A. scoparia. From the band patterns obtained no clear inferences could be drawn. Hence, further studies including protein assay was undertaken. Further effect of stress was studied with the help of protein assay, which was done by extracting the proteins out of the stressed and normal calli of A. scoparia (fig 2). In the lane 1 a protein molecular weight marker was loaded. The size of