Influence of Plant Growth Regulators on Indirect Shoot Organogenesis and Secondary Metabolite Production in Aconitum Violaceum Jacq
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Vol. 12(44), pp. 6287-6293, 30 October, 2013 DOI: 10.5897/AJB2013.13390 ISSN 1684-5315 ©2013 Academic Journals African Journal of Biotechnology http://www.academicjournals.org/AJB Full Length Research Paper Influence of plant growth regulators on indirect shoot organogenesis and secondary metabolite production in Aconitum violaceum Jacq. Janhvi Mishra Rawat1,2, Balwant Rawat2,3*, Anup Chandra1 and Subhash Nautiyal1 1Botany Division, Forest Research Institute, Dehradun-248006, India 2Society for the Conservation of Nature, Parnkuti Anantpur, University Road, Rewa-486 002, Madhya Pradesh, India 3GB Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora-263 643, Uttarakhand, India Accepted 17 October, 2013 Influence of plant growth regulators on indirect regeneration and secondary metabolite production in Aconitum violaceum Jacq. was evaluated. Among the different plant growth regulators studied, 2.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.25 µM kinetin (Kn) promoted the highest frequency of callus production for indirect regeneration. 6-Benzyl aninopurine (BAP) was more effective in improving shoot regeneration and secondary metabolite production compared to thidiazuron (TDZ). The highest frequency of regeneration (61.8%) was obtained when calli were transferred to Murashige and Skoog medium supplemented with 1 µM BAP and 0.5 µM α-naphthalene acetic acid (NAA) and was more than two-times higher when compared to the treatments with cytokinin only. Supplementation with low NAA concentrations resulted reduction in in vitro secondary metabolite production in most cases, when compared to treatments with cytokinin only. Moreover, differences in cytokinin concentrations significantly affected secondary metabolite production in some cases. The current findings highlighted the differential effects of auxin-cytokinin interactions on indirect shoot regeneration and the production of secondary metabolites in A. violaceum. Key words: Cytokinins, auxins, plant tissue culture, indirect regeneration, aconitum violaceum, secondary metabolites. INTRODUCTION Aconitum violaceum Jacq. member of family to the medicinal properties of the plant (Anonymous, Ranunculaceae is an important medicinal plant found in 1988). The crude extract of underground parts possess subalpine and alpine areas of Indian Himalayan Region antipyretic and analgesic properties and traditionally been at 3500-4000 m elevations, and shares its position with used in renal pain, rheumatism, high fever and for the threatened plant species of IHR (Chaudhary and Rao, treatment of snake and scorpion bites, contagious 1998; CAMP, 2003). Roots of this plant are the natural infections and inflammation of the intestines (Kirtikar and source of alkaloid aconitine, a neurotoxin which attributes Basu, 1984; Ameri, 1998; Chauhan, 1999). *Corresponding author. E-mail: [email protected]. Tel: +915962241014. Fax: +915962241150. Abbreviations: MS medium, Murashige and Skoog medium; 2,4-D, 2,4-dichlorophenoxyacetic acid; TDZ, thidiazuron; BAP, 6- benzylamino purine; NAA, α-naphthalene acetic acid; Kn, kinetin; IAA, indole-3- acetic acid; PGRs, plant growth regulators; HPLC, high performance liquid chromatography. 6288 Afr. J. Biotechnol. Due to increased demand from pharmaceutical gation following the protocol of Mishra-Rawat et al. (2013). Shoot industries, uncontrolled collection and lack of organized tips and nodal segments of the plants were used as explants for the cultivation, plant is going depleted in natural habitat. establishment of in vitro cultures. Surface sterilization of the explants was done with 0.5% Bavistin (30 min) and 0.1% mercuric Therefore, to protect the natural germplasm and to chloride solution (2 min) followed by four times subsequent washing collaborate with rising demand of the plant material there with sterile water. The sterile explants were cultured on MS is an immense need of biotechnology based interventions (Murashige and Skoog, 1962) basal medium containing 0.8% (w/v) to ascertain secure conservation of elite germplasm. In agar and sucrose (3% w/v) without any plant growth promoters vitro propagation techniques have contributed signify- (Figure 3). The pH of the medium was adjusted to 5.8 before autoclaving. Cultures were maintained at 25 ± 2°C in 16/8 h cantly to the growth of pharmaceutical industry over the light/dark cycle on racks fitted with cool fluorescent tubes (Philips past several decades in a variety of ways including 40 W; 42.0 and 60.0 µmol/m2/s irradiance inside and outside the varietal improvement. The use of in vitro techniques for culture flasks, respectively). Sub-culturing was carried out at three rapid and mass propagation offers possibilities for to four weeks interval for optimal growth. ‘recovery’ of endangered species, thus reducing the risk of extinction (Nadeem et al., 2001). Callus induction, maintenance and plant regeneration Regeneration from secondary meristems is useful for both these approaches because it facilitates the produc- Three types of explants (leaf, shoot tip and nodal segment, taken tion of nonchimeric plants. The indirect regeneration of from the same mother plant) were inoculated on the MS medium adventitious shoots is an alternative method to somatic supplemented with 0.5 to 10.0 µM 2,4-dichlorophenoxyacetic acid embryogenesis in obtaining whole plant regeneration of (2,4-D) and 0.25 to 2.0 µM kinetin (Kn), 3% (w/v) sucrose and 0.8% explants. Shoot organogenesis and somatic embryoge- agar for callus induction. Callus cultures were maintained through subculturing after every 30 days. Consequently, calli subcultured nesis from various explants have been reported earlier within three generations were used to induce adventitious shoots. (De Wit et al., 1990; Kintzios et al., 1999; Vergne et al., Adventitious shoot induction medium was MS medium supple- 2010). One of the major factors affecting the success of mented with various concentration of 6-benzyl aninopurine (BAP), in vitro plant propagation is the choice of plant growth thidiazuron (TDZ) and α-naphthalene acetic acid (NAA) (0.5 to 5.0 regulators (PGRs). As a result, PGRs (especially µM BAP, 0.5 to 5.0 µM TDZ and 0.25 to 2.0 µM NAA: single or in combination) and ultimately plants were regenerated. Regenerated cytokinins and auxins) are often added to culture media shoots were cultured up to eight weeks and further used for for the purpose of controlling different physiological secondary metabolite analysis. Culture conditions were same as responses in vitro, leading to the production of tissues described above for shoot induction. (such as callus), organs (such as shoots and roots) or whole plants. In spite of extensive hype in worldwide research Experimental design and statistical analysis interests in medicinally important plants and pharmaceu- Each experiment was repeated three times and each repeat had tically active compounds there from, no conventional seven replicates. Data were analyzed in a factorial based on scientific attempts have so far been reported to conserve completely randomized design (CRD). Data were analyzed using A. violaceum. The reports on in vitro multiplication and statistical programs MSTAT-C and SPSS. Statistically significant conservation of genetic diversity of genus Aconitum averages were compared using Duncan’s Multiple Range tests. provides a realistic insight of its medicinal importance Graphs were plotted with the Excel program. Differences were regarded as significant at P ≤0.05. (Cervelli, 1987; Shiping et al., 1988; Giri et al., 1993; Watad et al., 1995; Giri et al., 1997; Mitka et al., 2007; Hatwal et al 2011). Thus, due to uncontrolled exploitation, Active ingredient analysis the risk of loss of natural genetic resource of A. violaceum and biodiversity conservation aspects has Ten gram (FW) of plant material of each treatment was used for analysis. Plants were washed to remove media particle and dried at drawn the attention in current research. Keeping above room temperature (25°C) for 20 days. The air dried material were points in mind, the present study was aimed to powdered and made into a composite mixture before chemical investigate the role of plant growth regulators in analysis. Extraction of active ingredients was done following the improving/optimizing indirect shoot regeneration in A. method of Hikino et al. (1983). The powdered samples (1.0 g) were violaceum. The influence of an auxin-cytokinin interaction extracted (25 ml x 3; 30 min each) with ammoniacal ether (ether on secondary metabolite production in vitro was also containing 5% v/v, ammonia solution); the residue was then extracted with methanol (25 ml) for 16 h followed by two more studied. extractions for 3 h each. MATERIALS AND METHODS Column chromatography Plant material and culture conditions Samples were further purified on neutral alumina (Sisco Research Laboratories Pvt Ltd., Mumbai) columns (8 x 2 cm; length and Plants of A. violaceum Jacq. were collected from Hemkund (30° diameter) eluted with 50 ml of ethyle acetate and methanol (7.3, 41’55” N to 79° 47’ E 4100 m amsl, District Chamoli, Uttarakhand) v/v). The eluates were dried in vacuo (30°C) in a rotary film of Garhwal Himalaya during the month of September. Plants were evaporator, dissolved in high performance liquid chromatography brought to the laboratory and used immediately for in vitro propaga- (HPLC) grade methanol (1.0 ml) for further analysis by HPLC. Rawat et al. 6289 Frequency of callus induction callus of Frequency Figure 1. Effects of different