Micropropagation of Valeriana Wallichii DC. (Indian Valerian)
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Indian Journal of Biotechnology Vol 14, January 2015, pp 127-130 Micropropagation of Valeriana wallichii DC. multiplying the elite germplasm of V. wallichi. (Indian Valerian) through nodes Although in vitro propagation was reported via callus culture3, suspension cultures5, shoot buds6, encapsulated 7 Sandeep Singh, Vijay K Purohit*, P Prasad and apical and axial shoot buds , but multiple shoot formation A R Nautiyal through nodes of in vitro germinated seedlings of High Altitude Plant Physiology Research Centre, V. wallichii has not been reported so far. Therefore, in the H N B Garhwal University, Srinagar 246 174 (Uttarakhand), India present study, development of in vitro micropropagation Received 2 May 2013; revised 10 October 2013; protocol through multiple shoot formation has been accepted 14 December 2013 reported using nodal explants from the 2-month-old in vitro germinated seedlings of V. wallichii. In vitro propagation method was developed for obtaining large Seeds of V. wallichii were collected from the wild number of plantlets of Valeriana wallichii DC. (Valerianaceae), an indigenous high value medicinal and aromatic plant species of population at Khirsu (1800 m asl), Pauri (Garhwal), th Indian Himalayan region, using nodes of in vitro grown seedlings. Uttarakhand, India in the 4 week (wk) of April. High frequency shoot proliferation was induced in explants cultured Surface disinfection of seeds was performed using a on Murashige and Skoog (MS) medium supplemented with plastic net as seeds were tiny. Seeds were wetted in a different concentrations of 6-benzyladenine (BA), kinetin (Kn), adenine sulphate (AS) and thidiazuron (TDZ). Amongst all the detergent solution (Tween 20, two drop v/v) for 10 tested cytokinins, 3.0 µM BA was found to be the most effective min and washed under running tap water for 15 min. and an average of 12.0 shoots per nodal explant were formed after Subsequently, the seeds were repeatedly rinsed in 15 d of culture. Higher concentration of TDZ (3.0) µM was found distilled water and then treated with solutions least effective among all the cytokinins used for shoot induction. containing a systematic fungicide (Bavistin, 0.1%, Rooting (100 %) was observed in PGR-free Murashige and Skoog medium after 12 d of incubation of microshoots. Well rooted w/v) for 20 min, followed by sodium hypochlorite plantlets (30-d-old) were successfully transplanted and established (chlorine 5%) for 5 min, and finally rinsed with sterile in mixture of soil and sand (3:1) under partially shade conditions. distilled water. Each treatment was followed by repeated washing with sterile distilled water. Keywords: Adenine sulphate, 6-benzyladenine, In vitro shoot proliferation, kinetin, nodal explants, thidiazuron, The disinfected seeds were inoculated in 250 mL Valeriana wallichii Erlenmeyer flasks containing PGR-free MS medium with agar (0.8%, w/v) and sucrose (3.0%, w/v). Seeds Valeriana wallichii DC. (Indian Valerian) syn. germinated in 1 wk after inoculation. After 2 months, V. jatamansi (Family: Valerianaceae) grows wild in the nodal parts excised from seedlings served as explants temperate Himalaya, at an altitude of 1300-3300 m asl. (Fig. 1a). Nodal explants (5-6 per culture flask) were It is a small perennial, pubescent herb of 10-35 cm transferred to MS medium containing sucrose (3.0%, height having thick branching stem and fibrous root w/v), agar (0.8%, w/v) and supplemented with various stock1. The species has high medicinal and aromatic concentrations of either 6-benzyladenine (BA, 1-10 value, and is widely exploited for its aromatic roots and µM), kinetin (Kn, 1-10 µM), adenine sulphate (AS, 1- rhizomes, which contain valepotriates and valerinic 10 µM) or thidiazuron (TDZ, 0.1-5 µM). Explants acids2. For high medicinal and aromatic value, it is cultured on medium without plant growth regulators collected from its natural habitat in indiscriminate (PGRs) served as control. Each treatment consisted of manner, which has led to its large scale depletion in the 24 explants and all treatments were repeated twice. wild3. A significant decrease in population density of Data on percentage of shoot formation, shoot number V. jatamansi has also been reported due to human and shoot length were recorded after 30 d of sub- induced pressures4 and steadily deteriorating culture. For root induction, microshoots (3.0-4.0 cm regeneration. Tissue culture could possibly be useful in height with 4 or 5 leaflets) were excised from the bunch of multiple shoots and transferred to PGR free basal MS medium. After shoot harvest, the remaining *Author for correspondence: Tel: +91-1346-252172/253760; Fax: +91-1346-252070 explants subcultured in the same shoot multiplication E-mail: [email protected] medium continued to produce fresh shoots. 128 INDIAN J BIOTECHNOL, JANUARY 2015 The shoots with well developed roots were taken BA, Kn, AS and TDZ resulted in shoot formation out from the culture medium after 30 d. The roots (Fig. 1b). However, the number of shoots formed were gently washed with water to remove traces of varied with the treatments and PGR concentrations agar gel and the plantlets were treated with Bavistin (Table 1). In terms of per cent shoot induction, (0.1%, w/v) before transferring to small thermocol number of shoots per explant and shoot length, BA plastic cups (6.1 cm diam, 8.5 cm height) containing (3.0 µM) was found to be the most effective and garden soil and sand (3:1, v/v). These cups were produced 12.13±0.16 shoots per explant with covered by transparent polythene bags for avoiding 4.53±0.15 cm average shoot length (Table 1, Fig. 1c). moisture loss and maintaining humidity and kept in Incorporation of other cytokinins, Kn (3.0 µM), AS the shadehouse for acclimatization. After the period (5.0 µM) and TDZ (0.1 µM) also produced multiple of 2 wk, polythene bags were removed and plants shoots but the number of shoots was very less as were watered regularly. Following this, growth compared to BA (3.0 µM, Table 1). It was also performance and per cent survival recorded at 45 d observed that nodal explant responded to 100% of interval upto 90 d. Then acclimatized plants were shoot bud induction in all the tested cytokinins, except shifted to nursery conditions with partial shade TDZ. The low concentration of TDZ (0.1 µM) because of shady nature of the plants. The least- produced maximum number of shoots but, at higher significant difference was calculated following the concentrations, shoot induction decreased (Table 1). published methods of Snedecor and Cochran8. PGR-free MS medium induced high frequency of The culture of excised nodal parts (Fig. 1a) of V. rooting of microshoots (av. height 3.0-4.0 cm with 4 wallichii from in vitro germinated seedlings on MS or 5 leaflets) after 12 d of culture (Fig. 1d & e). In this medium supplemented with various concentration of process, 100% microshoots (10.0±0.47 number of Fig. 1 (a-i)—In vitro propagation of V. wallichii: a. Original explants (nodal part excised from 2-month-old seedlings); b. Shoot bud induction from nodal explants after 7 d of culture; c. Bunch of multiple shoots formed in MS medium supplemented with 3.0 µM BA with rhizogenesis; d. Transfer of microshoots for rooting in PGR free MS medium; e. Root formation in microshoots after 12 d of culture; f. Well rooted microshoots after 30 d of culture and ready for soil transfer; g. Rooted microshoots taken out after 30 d from culture flasks; h. Transfer to plastic cups covered with polythene bags for the process of acclimatization; & i. Well established plants after 90 d following transfer to soil (field). SHORT COMMUNICATION 129 roots/shoot with 5.1±1.20 cm length) were successfully Acclimatization of rooted plantlets was achieved rooted without the formation of basal callus (Figs 1f & by transferring them into plastic cups containing g). Although auxins are essential for root induction in garden soil and sand under shadehouse (75% shade; microshoots but in case of V. wallichi, cytokinins, BA, Figs 1h & i). After 90 d of acclimatization, 100% Kn, AS and TDZ also induced roots in microshoots with survival was recorded with increasing growth of low rooting potential, in comparison to the PGR-free plantlets (Table 2, Fig. 1h). Significant increase in MS medium with 100% rooting potential. growth parameters was observed during 90 d of growth period. Table 1—Effect of MS medium supplemented with different concentrations of plant growth regulators (PGR) on multiple In an earlier study, superiority of BA over other shoot induction in nodal explants of V. wallichii cytokinins in shoot induction was reported in 9 PGR Conc. % shoot Av. no. of Av. length of Andrographis paniculata . Further, efficiency of treatmen (µM) induction shoots/explants shoots shoot multiplication using BA had been reported for (±SE) (cm±SE) several medicinal and aromatic plant species, such 10 11 BA 1.0 100 3.96±0.13 3.31±0.19 as, Ocimum basilicum , Withania somnifera and 12 3.0 100 12.13±0.16 4.53±0.15 Hippophae rehmnoides . In the present study, PGR- free MS medium produced shoots in V. wallichii, 5.0 100 4.26±0.11 4.19±0.19 which suggests that shoot induction is not growth 10.0 100 4.03±0.31 3.52±0.06 regulator dependent but BA plays significant role for Kn 1.0 100 2.93±0.19 3.78±0.18 high degree of shoot formation and multiplication. 3.0 100 4.43±0.35 3.85±0.30 At higher concentrations of cytokinins, however, 5.0 100 4.20±0.26 3.16±0.24 shoot proliferation decreased.