Research article Enhanced in vitro regeneration and change in photosynthetic pigments, biomass and proline content in Withania somnifera L. (Dunal) induced by copper and zinc ions Nigar Fatima a , Naseem Ahmad a , Mohammad Anis a, b, * a Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202 002, India b Department of Plant Production, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia article info Article history: Received 30 July 2011 Accepted 31 August 2011 Available online 16 September 2011 Keywords: Plant growth regulators Inorganic nutrients Biochemical parameters Morphogenesis Withania somnifera abstract In the present study the effect of inorganic nutrients (CuSO 4 & ZnSO 4 ) on morphogenic and biochemical responses from nodal explants in Withania somnifera L. was investigated. Incorporation of either Copper sulphate (25e200 mM) or Zinc sulphate (50e500 mM) in the optimized Murashige and Skoog (MS) medium highly influenced the shoot bud formation and subsequent elongation, which induced maximum percentage (95%) regeneration, number (61.7  0.25) of shoots with shoot length (5.46  0.16 cm) on CuSO 4 (100 mM) and maximum percentage regeneration (100%), number of shoots (66.1  0.96) with shoot length (6.24  0.21 cm) on ZnSO 4 (300 mM) after 12 weeks of culture. Healthy growing in vitro microshoots rooted efficiently on ½ MS medium supplemented with NAA (0.5 mM), which induced (16.2  0.12) roots with root length (3.30  0.12 cm) after 4 weeks. Pigment content increased with increasing concentration of Cu and Zn and the maximum Chl. a (0.47), (0.41); Chl. b (0.52), (0.42); total Chl. (0.99), (0.83)and Carotenoid (0.16), (0.16) mg/g FW contents in regenerants were found on CuSO 4 (100 mM) and ZnSO 4 (300 mM), respectively. Maximum proline content (0.17), (0.16) mg/g FW was observed on high concentrations of CuSO 4 (200 mM) and ZnSO 4 (500 mM) respectively, in the basal medium. Regenerated plantlets were acclimatized successfully in soilrite with a survival rate of 95%. No morphological variations were detected among the micropropagated plants when compared with seedling raised plants of the same age. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Withania somnifera L. (Dunal) (Solanaceae), a highly reputed plant in Ayurvedic system of medicine is widely distributed in the drier parts of India upto an altitude of 2000 m in the Himalayas [1]. The plant is reported to have an anti-microbial, anti-tumour, anti-oxidant, anti-stress, radiosensitising, adaptogenic and immu- nomodulating activities [2]. It propagates through seed but prop- agation via seed poses problems due to low germination and poor viability. Due to over-exploitation, the plant faces high risk of extinction [3]. Hence, there is a strong need for proactive under- standing for the replenishment, conservation and sustainable usage of this species using modern biotechnological tools preferably through plant tissue culture techniques. Plant, cell, tissue and organ culture techniques have emerged as an escapable tool with the possibilities of complimenting and supplementing the conventional method in plant breeding, plant improvement, biosynthetic pathways etc. Plant tissue culture plays a major role in conservation of germplasm, rapid clonal propaga- tion, regeneration of genetically manipulated superior clones, production of secondary metabolites and ex vitro conservation of valuable phytodiversity [4,5]. This technique has several potential applications in crop improvement and efficient regeneration is a prerequisite in such improvement programmes. It has been shown that the nutrient levels in the medium have profound effect on shoot bud induction and subsequent plant regeneration. Appropriate levels of nutrient may also partially substitute the requirement of PGRs in the plant regeneration medium. Inorganic nutrients are major components of MS medium and hence offer the best variable to study their effect on the morphogenetic potentials of plant under study. Proline is a protei- nogenic amino acid with an exceptional conformational rigidity and it is essential for primary metabolism. Proline accumulation is also reported to occur at low level in response to heavy metal * Corresponding author. Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202 002, India. Tel.: þ91 571 2702016; fax: þ91 571 2704225. E-mail address: anism1@rediffmail.com (M. Anis). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.08.011 Plant Physiology and Biochemistry 49 (2011) 1465e1471