Micropropagation of Stemona tuberosa Lour. An endangered and rare medicinal plant in Eastern Ghats of India

Transcription

1 Indian Journal of Biotechnology Vol 12, July 2013, pp Micropropagation of Stemona tuberosa Lour. An endangered and rare medicinal plant in Eastern Ghats of India K Sri Rama Murthy 1 *, M Chandrasekhara Reddy 1, R Kondamudi 1 and T Pullaiah 2 1 Department of Botany and Biotechnology, School of Conservation Biology and Plant Biotechnology Montessori Mahila Kalasala, Vijayawada , India 2 Department of Botany, Sri Krishnadevaraya University, Anantapur , India Received 17 January 2012; revised 25 June 2012; accepted 10 August 2012 An in vitro technique of multiple plantlet regeneration was developed for conservation of endangered wild medicinal plant Stemona tuberosa Lour.. Murashige and Skoog (MS) medium supplemented with 7.0 mg/l Kn (kinetin) was found to be the optimum for axillary bud proliferation and multiple shoot induction. Excision and culture of nodal segments from the in vitro-shoots on medium containing 7.0 mg/l Kn and 4.0 mg/l TDZ (thidiazuron) showed maximum number of shoot multiplication with 7.10±0.37 shoots/node. Rapid shoot growth with simultaneous tuberous root formation was also observed in the same concentration. Shoots developed were rooted best on ½ strength MS with 1.0 mg/l IAA (indole acetic acid). Plantlets established in pots exhibited 85% survival. Plantlets successfully established in field exhibited morphological characters identical to mother plants. Keywords: Micropropagation, multiple shoots, Stemona tuberosa, tuberous roots Introduction Stemona tuberosa Lour. is a medicinally important twining herb, which belongs to the family Stemonaceae 1. The genus Stemona is characterized by rhizome with tuberous roots, which contain stemona alkaloids. The plant has officially been listed as antitussive medicinal herb in Chinese pharmacopoeia due to the presence of alkaloids, neostenine and neotuberostemonine 2. The crude extract obtained from the species of Stemona has been used in the treatment of respiratory and skin diseases, and as an antihelmintics 3. Nayar and Sastry 4 have reported that existence of Stemona species has become restricted to remote pockets in the Himalayas and the Western Ghats in Peninsular India, the two biodiversity hotspots, and designated as rare. The genus Stemona has now been added to the list of Indian endangered plants 4. Since in situ conservation effort has had the limited impact on halting the decline of S. tuberosa population, it is necessary to establish ex situ conservational methods like micropropagation as a supplementary measures to increase its population. Even though all the species of Stemona are *Author for correspondence: drksrmurthy@yahoo.com medicinally important, studies on micropropagation were conducted only on a few species, such as, S. japonica 5, S. tuberosa S1 clone 6 and S. curtisii 7. Therefore, the present study was undertaken to develop a protocol for in vitro propagation S. tuberosa in order to prevent the extinction of this endangered species. Materials and Methods S. tuberosa plant material were collected during August and September, 2009 from the wild population of Akashaganga, Tirumala hills, Chittoor district of Eastern Ghats, India. The voucher specimen was deposited in the herbarium of the Department of Biotechnology, Montessori Mahila Kalasala, Vijayawada, Andhra Pradesh, India. The availability of seeds in the natural habitat was poor, therefore vegetative plant was collected. The healthy, mature and juvenile twigs were washed uder running tap water for 2 to 3 times to remove the debris and dust particles, followed by a fungicide and bactericide (0.3% each) treatment for 10 min. The twigs were rinsed in an agitated solution of 1% (v/v). Tween 20 for 10 min, followed by rinsing with distilled water. Then the surface sterilization was carried out with 0.1% HgCl 2 (w/v) for 3 to 4 min, followed by 5 rinses with sterilized double distilled water. After

2 MURTHY et al.: MICROPROPAGATION OF S. TUBEROSA LOUR. 421 sterilization, the nodal segments were excised and used as explant. A single explant was inoculated into each culture tube. The explants were cultured onto MS medium 8 solidified with agar 0.8% (w/v) (HiMedia, Mumbai) and supplemented with different growth regulators, such as, benylaminopurine (BAP), kinetin (Kn) and thidiazuron (TDZ), at different concentrations either alone or in combinations. The medium was autoclaved at 121 C and 15 lbs pressure for 20 min after adjustment of ph to 5.7±2 with 1 N NaOH and 1 N HCl. All the in vitro cultures were maintained at 24±2 C and illuminated for 16 h with fluorescent light (18-24 µ mol m -2 s -1 ), followed by 8 h dark period. About 60-80% relative humidity was maintained within the 250 ml bottles and mm 2 culture tubes covered with the aluminum foil. In vitro regenerated shoots were inoculated for rooting on ½ strength MS medium supplemented with IAA (indole acetic acid), IBA (indol butyric acid) and NAA (naphathalene actic acid). The rooted shoots were washed with distilled water to remove the traces of the medium. The in vitro rooted plantlets were transferred to plastic cups containing autoclaved vermiculite and sand in 3:1 ratio. These plastic cups were covered initially with polythene bags to maintain humidity and placed in a mist chamber. After every alternative day, ½ strength MS medium salt solution was supplied to the plantlets. After 45 to 50 d of growth, the complete plants were established, acclimatized and thrived in greenhouse conditions. Each experiment was repeated at least thrice and for each experiment ten replicates were developed. Data were recorded periodically for shoot multiplication and rooting. Data were subjected to analysis of variance (ANOVA) and comparison among mean of experiments were made by Tukeys HSD test using statistical software Graphpad instat. The values with P 0.05 were considered to be statistically significant. Results and Discussion Among different cytokinins tested in the present study, Kn (7.0 mg/l) showed 90% response with maximum number (6.70±0.36) and maximum length (8.27±0.39 cm) of multiple shoots, followed by TDZ (4.0 mg/l) and BAP (2.0 mg/l) (Table 1; Figs 1A-C). Plant growth regulators* BAP Kn TDZ Table 1 Effect of cytokinins on in vitro shoot multiplication in S. tuberosa % shoot response No. of shoots** Length of the shoots** ± 0.36 b 6.48 ± 0.23 b ± 0.38 a 7.07 ± 0.23 a ±0.30 b 6.04 ± 0.24 b ± 0.43 b 5.32 ± 0.25 b ± 0.39 b 4.49 ± 0.31 c ± 0.40 c 4.16 ± 0.23 c ± 0.39 c 4.81 ± 0.17 bc ±0.63b c 5.32 ± 0.21 b ± 0.69 b 6.19 ± 0.28 b ± 0.51 b 6.93 ± 0.35 a ± 0.58 a 7.75 ± 0.34 a ± 0.36 a 8.27 ± 0.39 a ± 0.27 a 7.21 ± 0.33 a ± 0.45 b 6.67 ± 0.28 a ±0.35 c 4.34 ± 0.23 c ± 0.33 c 3.71 ± 0.17 c ± 0.36 b 4.38 ± 0.30 c ± 0.37 a 5.54 ± 0.36 b ± 0.37 a 6.43 ± 0.31 b ±0.34 b 4.89 ± 0.35 b

3 422 INDIAN J BIOTECHNOL, JULY 2013 In case of Kn, the number of shoots and shoot length increased with increasing concentration up to 7.0 mg/l; however, further increase in concentration (8-10 mg/l) decreased both the number and length of shoots. Among all the shoots proliferated, shoots formed on Kn containing medium showed better characteristics like healthy stem with dark green leaves, more similar to that of the in vivo grown plants, in comparison to shoots formed on the TDZ and BAP. These results are in consonance with the finding reported in Portulaca oleracea 9 and Ceropegia juncea 10. However, Kn supplemented medium showed very poor response in case of Aegle marmelos 11. In contrary to the present findings, multiple shoot induction was better in BAP and TDZ supplemented medium in case of S. tuberosa S1 clone 6, S. curtisii 7 and Ceropegia hirsuta 12. Further, the response of nodal explants with combination of Kn and TDZ was found comparatively better than individual cytokinins tested. The combination of TDZ 4.0 mg/l+kn 7.0 mg/l showed highest shoot regeneration response (95%) with maximum number of shoots (7.10±0.37) and maximum shoot length (8.35±0.28cm) (Table 2; Figs 1D-E). Multiple shoots developed with a combination of TDZ and Kn grew faster as compared with the shoots formed in the single cytokinin containing medium. The efficient role of TDZ and Kn in induction and elongation of multiple shoots was reported in Givotia rottleriformis 13. The cultures initiated on 0.1 mg/l TDZ produced the maximum number (7.8 shoots/explants) of shoots with an average shoot length of 3.2 cm on secondary medium containing of 0.3 mg/l Kn. Furthermore, synergistic effect of auxin in combination with cytokinins has Fig. 1 (A-G) In vitro micropropagation of S. tuberosa: A. Axillary bud proliferation on MS+BAP 2.0 mg/l after 3 wk of inoculation; B. Multiplication and elongation of shoots on MS+Kn 7.0 mg/l after subculture; C. Induction of multiple shoots on MS+TDZ 4.0 mg/l after 5 wk; D. Simultaneous multiple shoot and tuberous development on MS+Kn 7.0 mg/l+tdz 4.0 mg/l after another 6 wk; E. In vitro plantlet with well developed tuberous roots; F. In vitro rooted plantlets on ½ strength MS+IAA 1.0 mg/l; & G. Acclimatized plantlet ready to transfer into field conditions after 2 months. Table 2 Effect of cytokinin combinations on in vitro multiple shoot and tuberous root formation in S. tuberosa Cytokinin* Multiple shoot** TDZ Kn % response No. of shoots Length of shoots Tuberous root % response No. tuberous roots Length of tubers ± 0.41 c 4.24 ± 0.49 c ± 0.37 c 5.15 ± 0.39 c ± 0.34 c 1.95 ± 0.65 c ± 0.29 bc 7.47 ± 0.41 b ± 0.36 b 3.98 ± 0.30 b ± 0.37 a 8.35 ± 0.28 a ± 0.36 a 4.38 ± 0.25 a ± 0.43 b 6.13 ± 0.46 b ± 0.22 c 1.31 ± 0.68 c

4 MURTHY et al.: MICROPROPAGATION OF S. TUBEROSA LOUR. 423 been reported in Caralluma sarkariae 14 and only cytokinin combinations in Wattakaka volubilis 15. Normally cytokinins inhibit the root induction and enhance the shoot proliferation and elongation during in vitro propagation of plants. However, in the present study, the combination of Kn and TDZ also stimulated simultaneous tuberous root formation along with the formation of multiple shoots. The maximum number (3.00±0.36) and size (4.38±0.25 cm) of tuberous roots were observed on medium containing 3% sucrose+tdz 4.0 mg/l + Kn 7.0 mg/l (Table 2). Auxins play a vital role in root induction. In the present study, differences were noticed in the nature of roots induced depending on the auxins used in the medium. When individual shoots with 3 to 4 nodes were inoculated on MS medium containing different auxins, initially swelling of the basal part was observed and then slender root formation started. Among the three auxins tested, the best root induction was observed in IAA followed by NAA and IBA (Table 3). The maximum (75%) of root regeneration was observed at IAA 1.0 mg/l with the maximum root number (5.60±0.40) and root length (6.19±0.56 cm) (Fig. 1F). Similar results were also found in S. curtisii 7, whereas auxins had no positive effect on root induction in S. tuberosa S1 clone 6. The benefit of any micropropagation system can, however, be fully realized only by the successful transfer of plantlets from tissue culture to the ambient conditions found ex vitro 16. In the present study, healthy plantlets with well developed roots were removed from culture tubes keeping the roots intact and then gently rinsed with distilled water to remove the traces of agarified medium. The regenerates were planted in plastic cups containing sterilized vermicompost and sand in 3:1 ratio and covered with plastic bags to maintain relative humidity. The plantlets were irrigated for every 2 d with ½ strength MS liquid medium. After 1 month, the polythene bags were holed here and there to reduce the relative humidity, and finally removed to adjust the ambient conditions. Then the hardened plants were transferred to earthen pots. Vermin compost plays a vital role in primary stage of hardening because it is rich in nutrients and has a good water holding capacity to maintain moist condition. Successful hardening by using vermiculite was reported in several other species like Elettaria cardamomum 17 and Citrus jambhiri 18. The plantlets of S. tuberosa were successfully established with 85% survival rate in field conditions (Fig. 1G). Table 3 Effect of auxins on in vitro root induction in S. tuberosa Auxins* IAA IBA NAA % response No. of roots** Length of roots** ± 0.44 c 4.38 ± 0.34 b ± 0.60 c 5.67 ± 0.34 a ± 0.40 a 6.19 ± 0.56 a ± 0.34 b 4.73 ± 0.44 b ± 0.34 c 2.49 ± 0.25 c ± 0.47 c 3.73 ± 0.37 b ± 0.42 b 5.31 ± 0.30 a ± 0.51 b 4.14 ± 0.53 b ± 0.31 c 2.27 ± 0.24 c ± 0.47 c 1.79 ± 0.26 c ± 0.26 c 3.35 ± 0.27 b ± 0.49 c 3.77 ± 0.31 b ± 0.41 a 4.92 ± 0.29 a ± 0.43 b 2.90 ± 0.37 b ± 0.45 c 2.17 ± 0.25 c

5 424 INDIAN J BIOTECHNOL, JULY 2013 Acknowledgement KSM expresses gratitude to the University Grants Commission, South Eastern Regional Office (SERO), Hyderabad for the financial assistance provided to the present study. References 1 Reddy K N & Reddy C S, First red list of medicinal plants of Andhra Pradesh, India Conservation assessment and management planning, Ethnobot Leaflets, 12 (2008) Pilli R A, Rosso G B & de Oliveira M D C F, The Stemona alkaloids, Alkaloids Chem Biol, 62 (2005) Chung H S, Hon P M, Lin G, But P P & Dong H, Antitussive activity of Stemona alkaloids from Stemona tuberose, Planta Med, 69 (2003) Nayar M P & Sastry A R K, Red data book of Indian plants, vol 1 (Botanical Survey of India, Calcutta) 1987, Yang Z-D, Huang S-X & Lu T-L, Tissue culture and rapid propagation of medicinal plant Stemona japonica, Zhongcaoyao, 33 (2002) Montri N, Wawrosch C H & Kopp B, In vitro propagation of Stemona tuberosa Lour., an antitussive medicinal herb, Acta Hortic, 812 (2009) Montri N, Wawrosch C H & Kopp B, Micropropagation of Stemona curtisii Hook. f., a Thai medicinal plant, Acta Hortic, 725 (2006) Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962) Sharma M M, Singh A, Verma R N, Ali D Z & Batra A, Influence of PGRs for the in vitro plant regeneration and flowering in Portulaca oleracea L.: A medicinal and ornamental plant, Int J Bot, 7 (2011) Nikam T D & Savanth R S, Multiple shoot regeneration and alkaloid ceropegin accumulation in callus culture of Ceropegia juncea Roxb., Physiol Mol Biol Plant, 15 (2009) Nayak P, Behera P R & Manikkannan T, High frequency plantlet regeneration from cotyledonory node cultures of Aegle marmelos (L.) Corr., In Vitro Cell Dev Biol (Plant), 43 (2007) Nikam T, Savanth R S & Parage R S, Micropropagation of Ceropegia hirsuta Wt. & Arn. A starchy tuberous asclepiad, Indian J Biotechnol, 7 (2008) Samuel K, Debashish D, Madhumita B, Padmaja G, Prasad S R et al, In vitro germination and micropropagation of Givotia rottleriformis Griff., In Vitro Cell Deve Biol (Plant), 5 (2009) Sreelatha V R, Sandhya Rani S, Reddy P V K, Naveen M, Ugraiah A et al, In vitro propagation of Caralluma sarkariae Lavranos & Frandsen An endemic and endangered medicinal plant, Indian J Biotechnol, 9 (2009) Arulanandam L J P, Kumar S G & Sowmim M, Micropropagation and conservation of rare medicinal plant Wattakaka volubilis. (Linn.) Stapf., Indian J Biotechnol, 10 (2011) Hazarika B N, Acclimatization of tissue cultured plants, Curr Sci, 85 (2003) Manohari C, Backiyarani S, Jebasingh T, Somanath A & Usha R, Efficient plant regeneration in small Cardamom (Elettaria cardamomum Maton.) through somatic embryogenesis, Indian J Biotechnol, 7 (2008) Saini H K, Gill M S & Gill M I S. Direct shoot organogenesis and plant regeneration in rough lemon (Citrus jambhiri Lush.), Indian J Biotechnol, 9 (2010)