Abstract
In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO3, NaNO3, and CaCl2. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO3 compared to other salts and the in vitro shoots appeared healthy at 50 mM concentration of NaCl and KNO3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl2 produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Agastian P, Kingsley SJ, Vivekanandan M (2000) Effect of salinity on photosynthesis and biochemical characteristics in mulberry genotypes. Photosynthetica 38:287–290
Alia P, Saradhi PP, Mohanty P (1991) Proline enhances primary photochemical activities in isolated thylakoid membranes of Brassica juncea by arresting photo inhibitory damage. Biochem Biophys Res Commun 181:1238–1244
Balasundram N, Sundram K, Samman S (2006) Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem 99:191–203
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207
Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287
Bohnert HJ, Nelson DE, Jensen RG (1995) Adaptations to environmental stresses. Plant Cell 7:1099–1111
Chance B, Maehly AC (1955) Assay of catalases and peroxidases. Methods Enzymol 2:764–775
Chaves MM, Oliveira MM (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55:2365–2384
Cheng AX, Lou YG, Mao YB, Lu S, Wang LJ, Chen XY (2007) Plants terpenoids: biosynthesis and ecological functions. J Integr Plant Biol 49:179–186
De Abreu IN, Mazzafera P (2005) Effect of water and temperature stress on the content of active constituents of Hypericum brasilienne Choisy. Plant Physiol Biochem 43:241–248
Dionisio-Sese ML, Tobita S (1998) Antioxidant responses of rice seedlings to salinity stress. Plant Sci 135:1–9
Elkahoui S, Hernandez JA, Abdelly C, Ghrir R, Limam F (2005) Effects of salt on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus suspension cells. Plant Sci 168:607–613
Erturk U, Sivritepe N, Yerlikaya C, Bor M, Ozdemir F, Turkan I (2007) Responses of the cherry rootstock to salinity in vitro. Biol Plant 51:597–600
Hare P, Cress WA, van Staden J (1998) Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ 21:535–553
Kovacik J, Klejdus B, Hedbavny J, Backor M (2009) Salicylic acid alleviates NaCl-induced changes in the metabolism of Matricaria chamomilla plants. Ecotoxicology 18:544–554
Ksouri R, Megdiche W, Debez A, Falleh H, Grignon C, Abdelly C (2007) Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritime. Plant Physiol Biochem 45:244–249
Kumar KB, Khan PA (1982) Peroxidase and polyphenol oxidase in excised ragi (Eleusine coracana cv. PR 202) leaves during senescence. Ind J Exp Bot 20:412–416
Kwon SY, Jeong YJ, Lee HS, Kim JS, Cho KY, Allen RD, Kwak SS (2002) Enhanced tolerance of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress. Plant Cell Environ 25:873–882
Lattanzio V, Cardinali A, Ruta C, Fortunato IM, Lattanzio VMT, Linsalata V, Cicco N (2009) Relationship of secondary metabolism to growth in oregano (Origanum vulgare L.) shoot cultures under nutritional stress. Environ Exp Bot 65:54–62
Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11:591–592
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275
Mattioli R, Costantino P, Trovato M (2009) Proline accumulation in plants, not only stress. Plant Signal Behav 4:1016–1018
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410
Molassiotis AN, Sotiropoulos T, Tanou G, Kofidis G, Diamantidis G, Therios Ι (2006) Antioxidant and anatomical responses in shoot culture of the apple rootstock MM 106 treated with NaCl, KCl, mannitol or sorbitol. Biol Plant 50:331–338
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15:473–497
Muthukumarasamy M, Gupta SD, Pannerselvam R (2000) Enhancement of peroxidase, polyphenol oxidase and superoxide dismutase activities by tridimefon in NaCl stressed Raphanus sativus L. Biol Plant 43:317–320
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
Navarro JM, Flores P, Garrido C, Martinez V (2006) Changes in the contents of antioxidant compounds in pepper fruits at ripening stages, as affected by salinity. Food Chem 96:66–73
Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 163:375–380
Oueslati S, Bouraoui NK, Attia H, Rabhi M, Ksouri R, Lachaal M (2009) Physiological and antioxidant responses of Mentha pulegium (Pennyroyal) to salt stress. Acta Physiol Plant 32:289–296
Panda SK, Upadhyay RK (2003) Salt stress injury induces oxidative alterations and antioxidative defense in the roots of Lemna minor. Biol Plant 48:249–253
Sabir F, Sangwan NS, Chaurasiya ND, Misra LN, Sangwan RS (2008) In vitro withanolide production by Withania somnifera L. cultures. Z Naturforsch 63:409–412
Sam O, Ramirez C, Coronado MJ, Testillano PS, Risueno MC (2003) Changes in tomato leaves induced by NaCl stress: leaf organization and cell ultrastructure. Biol Plant 47:361–366
Sangwan NS, Farooqi AHA, Sangwan RS (1994) Effect of drought on growth and essential oil metabolism in lemongrass species. New Phytol 128:173–179
Sangwan NS, Yadav U, Sangwan RS (2003) Genetic diversity among elite varieties of the aromatic grasses, Cymbopogon martini. Euphytica 130:117–130
Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2004) Phytochemical variability in commercial herbal products and preparations of Withania somnifera (ashwagandha). Curr Sci 86:461–465
Shewfelt RL, Purvis AC (1995) Towards a comprehensive model for lipid peroxidation in plant tissue disorders. Hort Sci 30:213–221
Shimoni M (1994) A method for activity staining of peroxidase and β-1,3-glucanase isozymes in polyacrylamide electrophoresis gels. Anal Biochem 220:36–38
Smith IK, Vierhelle TL, Thorne CA (1988) Assay of glutathione reductase in crude tissue homogenate using 5,5′-dithiobis(2-nitrobenzoic acid). Anal Biochem 175:408–413
Vijayan K, Chakraborti SP, Ghosh PD (2003) In vitro screening of mulberry (Morus spp.) for salinity tolerance. Plant Cell Rep 22:350–357
Wang B, Lüttge U, Ratajczak R (2004) Specific regulation of SOD isoforms by NaCl and osmotic stress in leaves of the C3 halophyte Suaeda salsa L. J Plant Physiol 161:285–289
Woodward AJ, Bennett IJ (2005) The effect of salt stress and abscisic acid on proline production, chlorophyll content and growth of in vitro propagated shoots of Eucalyptus camaldulensis. Plant Cell Tissue Organ Cult 82:189–200
Xiong L, Zhu JK (2002) Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ 25:131–139
Zhao X, Tan HJ, Liu YB, Li XR, Chen GX (2009) Effect of salt stress on growth and osmotic regulation in Thellungiella and Arabidopsis callus. Plant Cell Tissue Organ Cult 98:97–103
Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71
Acknowledgments
The financial grant from the NIMITLI Program of the Council of Scientific and Industrial Research, Government of India, New Delhi, is gratefully acknowledged. FS and RK are thankful to CSIR, New Delhi and UGC, New Delhi for the senior research fellowship.
Author information
Authors and Affiliations
Corresponding author
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sabir, F., Sangwan, R.S., Kumar, R. et al. Salt Stress-induced Responses in Growth and Metabolism in Callus Cultures and Differentiating In Vitro Shoots of Indian Ginseng (Withania somnifera Dunal). J Plant Growth Regul 31, 537–548 (2012). https://doi.org/10.1007/s00344-012-9264-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00344-012-9264-x