Withania somnifera (L.) Dunal (Solanaceae), commonly known as ‘ashwagandha’ and “Indian ginseng” is a highly valued medicinal plant in Indian Ayurvedic and African traditional systems. Major withanolides like withanolide A and withaferin A present in W. somnifera have been demonstrated to possess specific therapeutic action against carcinogenesis, Parkinson’s disease and Alzheimer’s disease (Mishra et al., 2000). The requirement of dried plant material for withanolides drugs production in India has been estimated as about 9127-tonnes as against the annual production of about 5905-tonnes (Sharada et al., 2008). Moreover, field cultivation is time consuming, laborious and it is not able to meet the current Ashwagandha global market requirement (Sivanandhan et al., 2012b; 2013a): First, the plant-to-plant variation in secondary metabolites yield along with quality and second the long growing period (4-5 years) between planting and harvesting. To improve commercial cultivation of W. somnifera, biological advances must be made that should either increase the yield or reduce the time gap and assure quality (Banerjee et al., 1994).

To enhance commercial prospects for production of withanolides, an alternative choice could be the use of plant cell/organ cultures. Hairy root cultures offer many advantages over conventional cell culture systems for secondary metabolites production (Georgiev et al., 2007; Sivanandhan et al., 2013b). Hairy roots are adventitious roots derived from cells transformed by the root-inducing plasmid of A. rhizogenes and they grow in the absence of phytohormones. The hairy root harbors the T-DNA segment of plasmid Ri within its genome (Tepfer, 1990). Rao and Ravishankar (2002) suggested A. rhizogenes-mediated hairy root formation as the valuable tool for the biosynthesis of secondary metabolites, metabolic engineering studies and biotechnological production of root-derived compounds.

A. rhizogenes-mediated transformation has been previously reported in W. somnifera using various explants (leaf, cotyledon, cotyledonary node, hypocotyls, stem, and root) and various strains (A4, LBA9402, MTCC 2364, MTCC 532, ATCC 15834, LMG 150, A2/83, A20/83, R1000 and R1601) by various authors (Banerjee et al., 1994; Ray et al., 1996; Pawar and Maheswari, 2004; Kumar et al., 2005; Bandyopadhyay et al., 2007; Murthy et al., 2008; Praveen and Murthy, 2011; Sivanandhan et al., 2012a; Sivanandhan et al., 2013b). In the present study, we documented a higher number of hairy roots and transformation frequency upon inclusion of AS and MES buffer in the co-cultivation medium by optimizing different factors in W. somnifera. Hence, in the present study, two agropine type A. rhizogenes strains (R1000 and A4) have been selected to induce hairy roots in the Kolli Hills genotype of W. somnifera.

Plant material

The explants, leaf and cotyledon were prepared as per our earlier report (Sivanandhan et al., 2013b).

Agrobacterium rhizogenes-transformation

A single colony of the wild type Agrobacterium rhizogenes strains R1000 and A4 were selected and cultured in LB medium (30 ml; Himedia, Mumbai, India) in darkness at 28°C for 12 h at 180 rpm. The bacterial cells were pelleted by centrifugation followed by washing twice with liquid half strength MS medium (Murashige and Skoog, 1962). The suspension was employed for A. rhizogenes infection.

Standardization of transformation parameters

In order to induce the hairy roots with A. rhizogenes, we tested two types of explants, namely leaves and cotyledons. The explants such as leaf and cotyledon were pricked with sterile hypodermic needle (0.63×25 mm; Dispovan, Haryana, India Ltd.) and the explants were immersed in the bacterial suspension culture (OD 600≥1) for 15 min and blotted on sterile tissue paper for 10 min. After dryness, the explants were placed in half strength MS medium supplemented with different concentrations of acetosyringone (AS) (0, 50, 100, 150, and 200 µM; Sigma, St. Louis, USA), 3% (w/v) sucrose (SRL, Mumbai, India), and 0.2% (w/v) phytagel (Sigma, St. Louis, USA), at 23 ± 2°C in dark conditions. These explants were co-cultivated for different days (3, 5 and 7 days). After the co-cultivation period, the explants were washed first with sterilized-distilled water followed by half strength MS medium, which contained 300 mg/l cefotaxime (Alkime Laboratory, Mumbai, India) and transferred to 30 ml half strength MS medium supplemented with 3% (w/v) sucrose, 0.2%  (w/v) phytagel, and 300 mg/l cefotaxime. After 12-18 days of culture, the transformed roots were appeared at the wounded sites of the explants. The induced transformed roots of more than 1-2 cm in length were excised from the explants and transferred to 30 ml half strength MS medium supplemented with 3% (w/v) sucrose, 0.2% (w/v) phytagel and 300 mg/l cefotaxime until the residual bacteria have been completely killed.

Statistical analysis

All the experiments were set up in a completely randomized design and the data were subjected to Duncan's multiple range test using SPSS software version 11.5. The efficiency of hairy root formation was calculated as the percent of respective explants showed hairy root induction out of total number of explants inoculated in a particular treatment. 

Effect of co-cultivation on hairy root induction

A co-cultivation for 5 days increased the efficiency of the leaf explants with hairy root formation, as it was not observed to obtain a higher number of hairy roots than remaining co-cultivation period. A 5 day co-cultivation period induced 28.2 and 24.3 number of hairy roots/leaf explants infected with R1000 and A4 strains, respectively. In the case of cotyledon explant, the co-culture for 5 days produced 17.6 and 14.5 number of hairy roots/explants infected with R1000 and A4 strains, respectively (Figure 2). Prolonged co-cultivation to 7 days resulted to growth of the bacteria around the both explants which led to explant necrosis. Shorter co-cultivation period may result to an unsuccessful gene transfer from strain to explant. So, the transformation efficiency may differ due to different co-cultivation periods (Figure 2). Of different co-cultivation period tested, 5 day co-cultivation time duration showed dramatic improvement in hairy root induction from the leaf and cotyledon explants, infected with R1000 and A4 strains in W. somnifera.  A co-cultivation period for 2 and 3 days was found to be suitable for hairy root induction in W. somnifera, as reported by Pawar and Maheswari, (2004), Murthy et al. (2008), Banerjee et al. (1994), Kumar et al. (2005) and Bandyopadhyay et al. (2007), but Ray et al. (1996) did not mention the co-cultivation duration, whereas in the present study, 5 day co-cultivation period was found to be superior and abundantly increased the efficiency of hairy root induction (Figure 2).  These variations in the requirement for a definite co-cultivation period may originate in the specificity of the plant tissue, the Agrobacterium strain or the medium used for bacterial culture and co-cultivation. Co-cultivation is an important phase in transfer of gene from bacteria to plant. But co-cultivation period produced successful transformants on depending upon the plant species and time duration. For R1000 strain, transformation efficiency increased in line with co-cultivation duration and stability on 5th day of culture. During the co-cultivation period, many factors influence the efficiency of the genetic transformation process. After Agrobacterium infection, the plant tissues and bacteria are cultivated for a few days. Important events occur during co-cultivation; plant cells and bacteria divide further, and T-DNA is transferred from the bacteria to the plant cells (Kim et al., 2007). The length of the  co-cultivation period  also  influences  transformation efficiency (Tao and Li, 2006). These results confirm that the optimization of co-cultivation period is critical for higher hairy root induction in W. somnifera.

Effect of explants on hairy root induction

Among the various explants tested, only leaf was a greater explant to the infection of both strains. The leaf explant produced higher number of hairy roots by being infected with both the strains R1000 and A4. But the cotyledon explants showed lower number of hairy roots by being infected with both the strains when compared to leaf explants. The leaf explants infected by R1000 and A4 strains produced 28.2 and 24.3 number of hairy roots/explants, respectively and the cotyledon explants infected by R1000 and A4 strains produced 17.6 and 14.5 numbers of hairy roots, respectively (Figure 2). Most of the authors reported that leaf explants only showed the best response for hairy root induction in W. somnifera, but Murthy et al. (2008) obtained lower transformation efficiency in cotyledon explants (3%) whereas in the present study, cotyledon explants also produced hairy roots almost equal to leaf explants. Kang et al. (2006) showed the importance of explant choice by producing more hairy roots of Aralia elata on the root segment than on the petiole explant. It has been reported in several previous studies that the morphological patterns and hairy root production characteristics of infected tissues differed substantially (Ottani et al., 1990). These differences in hairy root production may depend upon differences between species, plant organs, or sites of infection. The choices of explants for hairy root induction after infection with A. rhizogenes constitute the most salient of the integrated factors for a successful hairy root transformation. Plant transformation efficiency differs significantly according to the source of the explant (Alpizar et al., 2006). Therefore, leaf explants possibly influence the rate of hairy root induction than the cotyledon explants by optimizing different factors such as co-cultivation, acetosyringone and bacterial strain in W. somnifera.

Prof. A. Ganapathi is thankful to University Grants Commissions (UGC), Government of India, for the award of UGC-BSR (Basic Scientific Research Fellowship). The first author gratefully acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India, for the award of CSIR-SRF.

The author(s) have not declared any conflict of interest.