In vitro propagation of Spilanthes acmella (L.) Murray using semisolid and liquid medium

Indian Journal of Biotechnology Vol 14, January 2015, pp 112-116 In vitro propagation of Spilanthes acmella (L.) Murray using semisolid and liquid medium Veenu Joshi, Kishan Lal Tiwari and Shailesh Kumar Jadhav* School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur 492 010, India Received 25 June 2013; revised 30 August 2013; accepted 20 September 2013 Spilanthes acmella (L.) Murray (Family: Asteraceae) is a multifunctional medicinal plant, which is a major source of alkamides including spilanthol reported to exhibit various pharmacological activities. Therefore, an efficient in vitro propagation protocol is requisite for conserving and meeting the ever increasing industrial demands of this plant. The present study investigates the effect of agar concentration and liquid medium on in vitro proliferation of S. acmella. Nodal segments of a healthy plant were surface sterilized and inoculated on MS medium supplemented with different concentrations of BA (benzylaminopurine). Maximum bud break was observed in MS containing 0.5 mg/l BA. Maximum multiple (6.20±0.45) with good mean shoot length (8.01±0.87 cm) was observed in liquid MS with 1 mg/l Kn (kinetin) in comparison with the semi solid medium. The regenerated showed rooting without addition of any auxins but rooted best in MS medium containing 1 mg/l IBA and successfully hardened to ex vitro conditions using cocopeat irrigated with ½ MS, followed by transfer to soil with 100% survival. Keywords: Agar concentration, antimalarial, liquid medium, propagation, Spilanthes acmella, spilanthol Introduction Spilanthes acmella (L.) Murray (Family: Asteraceae), commonly known as Toothache Plant, is a multipurpose herb with considerable traditional value. It grows throughout the tropical and subtropical regions of the world. In India, it is reported from the regions of Chhattisgarh, Jharkhand and Rajasthan. The plant grows well in damp, marshy areas near sea or sewage discharge areas, and generally found as a weed of road-sides and cultivations 1. The plant is a rich source of alkamide spilanthol, which is supposed to be responsible for most of its medicinal properties. S. acmella has been reported to possess larvicidal activity against Aedes aegypti, Anopheles and Culex mosquitoes and thus can be developed into a potent antimalarial agent. It is traditionally reported for its uses as spice, antiseptic, anti-bacterial, anti-fungal, and as remedy for toothache, stomatitis, throat complaints and tuberculosis 2-5. It has experimentally proven to be *Author for correspondence: Tel:+91-771-2263022 shailesh_07@sify.com Abbreviations: MS, Murashige and Skoog (1962) medium; BA, Benzylaminopurine; IBA, Indole-3-butyric acid; Kn, Kinetin; NAA, α-naphthalene acetic acid; HgCl 2, mercuric chloride. diuretic, insecticidal, vasorelaxant, antioxidant, antinociception, anti-inflammatory, antimicrobial, immunomodulatory, antipyretic, analgesic and aphrodisiac. The conventional propagation of Spilanthes is hampered due to its poor vegetative propagation and low rate of seed germination 6, which is posing a hurdle in coping up with the ever increasing industrial demands. In this respect, in vitro culture is considered as a promising technology to play a major role in mass multiplication, secondary metabolite production and sustainable use of valuable herbs. Although several workers have studied the in vitro regeneration of S. acmella through hypocotyl 7, leaf 8, axillary bud 9 and nodal explants 10,11, but shoot proliferation using liquid medium has not yet been reported. Use of liquid medium is effective in improving the shoot growth, reducing the culture transfer frequencies and thus overall cost of producing plants for the commercial purposes 12. Thus the present paper reports the effect of agar concentration and liquid medium on in vitro proliferation of S. acmella. Materials and Methods Source and Explant Treatment Nodal explants were collected from a healthy mother plant growing in the Pt. Ravishankar Shukla

JOSHI et al: IN VITRO PROPAGATION OF S. ACMELLA 113 University Campus, Raipur (Chhattisgarh), India. The explants were thoroughly washed in running tap water for 5-10 min and disinfected by immersion in ethanol 70% (v/v) for 30 sec, followed by rinsing three times with double distilled water. The effect of surface sterilization using (1% & 4%) sodium hypochlorite and (0.01% & 0.1%) HgCl 2 for different exposure time periods (5, 10 &15 min) was tried. Surface sterilized explants were then rinsed 4 to 5 times with autoclaved double distilled water and further trimmed before inoculating onto nutrient medium. Surface sterilization and trimming operations were performed in a laminar air flow cabinet. Media Preparation and Culture Conditions The formulation of MS 13 basal medium supplemented with plant growth regulators was used throughout the study. The liquid MS medium was gelled with 0.8, 0.5 and 0.2% agar and sterilized by autoclaving at 121 C and 15 Psi for 20 min. All the inoculated cultures were incubated in the culture room maintained at 25±2 C under 16/8 h light/dark cycle with the light intensity of 3000 lux. Culture Establishment Sterilized nodal explants were inoculated singly in culture tubes containing 15 ml of semisolid MS medium supplemented with 0-4 mg/l BA. The explants were observed on daily basis for recording time taken in bud break and per cent bud break. The initial data of per cent response was recorded after 28 d of culture. Shoot Multiplication When the axillary attained a length of 3-4 cm, they were excised and inoculated onto semisolid media supplemented with various combinations and concentrations of BA, Kn and NAA to determine best medium for multiplication and growth. Further, in vitro explants were inoculated on best obtained multiplication media gelled with 0.5, 0.2 and 0% agar. About 20 explants were cultured per treatment and the data were recorded after 30 d of culture. Rooting, Hardening and Acclimatization In vitro grown of 4-6 cm height were excised and transferred to rooting (MS) medium supplemented with 0.5-4.0 mg/l IBA. Plantlets obtained after in vitro rooting were carefully removed from medium, washed thoroughly with sterile water to remove agar, followed by transfer to sterilized cocopeat irrigated with ½, full and without MS medium. Subsequently, they were maintained in the culture room for in vitro hardening. After a period of 3 wk, plants were transferred to greenhouse for ex vitro hardening, followed by transplanting into pots with soil for acclimatization to the natural conditions. Results and Discussion Explant Response Sodium hypochlorite treatment of explants was found ineffective, giving high percentage of contamination. Also, a higher exposure time towards 0.1% HgCl 2 proved lethal for the explants. However, treatment with 0.1% HgCl 2 for 5 min proved quite effective in providing 80% of the aseptic cultures (complete data not shown). In about 5-6 d of inoculation, swelling at the base followed by bud break was observed on the initiation medium. The maximum percentage (70%) of bud break was recorded in 0.5 mg/l BA along with maximum mean shoot length (2.89±0.33) and leaf number (7.09±0.59) (Table 1; Fig 1a). Increase in concentration of BA resulted in the increase in callusing and decrease in mean shoot length and mean node number. A similar trend was observed in Eucalyptus ficifolia 14 and Cleistanthus colinus 15. It has been reported that higher concentration of BA induces the formation of callus tissue that causes the chromosomal instability of the regenerated plants 16. Shoot Proliferation In the present study, the maximum shoot multiplication (3.05±0.23 mean ) was observed on MS medium supplemented with the combination of BA (1 mg/l) and Kn (2 mg/l) but with poor mean shoot length (2.25±0.12) (Table 2). Further increase in the concentration of BA or Kn showed decline in Table 1 Effect of different concentration of cytokinin BA on culture initiation in S. acmella (mg/l) % bud break Day of bud break Length of (cm±se) leaves MS 50 6 th d 1.20±0.10 3.62±0.27 5.77±0.30 MS+0.5 BA 70 5 th d 1.46±0.13 2.89±0.33 7.09±0.59 MS+1.0 BA 65 6 th d 1.60±0.19 2.41±0.31 6.20±0.58 MS+2.0 BA 62 5 th d 1.86±0.19 1.75±0.26 4.70±0.49 MS+4.0 BA 65 5 th d 2.06±0.18 1.55±0.23 5.27±0.55

114 INDIAN J BIOTECHNOL, JANUARY 2015 Fig. 1 (a-h) In vitro culture of S. acmella: a. Initiation of culture in 0.5 mg/l BA supplemented semisolid MS medium; b. Multiplication in 1.0 mg/l Kn supplemented semisolid MS medium; c. Multiplication in 1.0 mg/l Kn supplemented MS medium gelled with 0.5% agar; d. Multiplication in 1.0 mg/l Kn supplemented MS medium gelled with 0.2% agar; e. Multiplication in 1.0 mg/l Kn supplemented liquid MS medium; f. Rooting in 1.0 mg/l IBA; g. In vitro hardening of plantlets; & h. Ex vitro hardened plants. [Bar = 1.0 cm and it represents the length of the plant] shoot length as well as leaf number. Similar results were also reported in case of Psoralea corylifolia 17, Clerodendrum incisum 18 and Decalepis arayalpathra 19 and for such effects, hyperhydricity could have possibly be responsible 20. A similar number of mean (3.00±0.23) with good mean length (4.59±0.49) and leaf number (5.18±0.32) was recorded with 1 mg/l Kn without any auxin (Table 2; Fig. 1b). This indicates that these explants contain sufficient level of endogenous auxins or capable of its de novo syntheses, which can induce shoot formation even in a medium containing cytokinin alone 18. Addition of 0.1 mg/l NAA did not show any significant change in mean number of with the combinations (Table 2). Shoot proliferation was found to be higher in liquid MS medium in comparison to semisolid medium. Comparison of proliferation of on the semisolid and liquid media is shown in Table 3. The results revealed that shoot numbers and their length increased with the decrease in the concentration of agar (0.5% < 0.2% < liquid media without agar). The maximum mean shoot number (6.20±0.45) and shoot length (8.01±0.87) was observed in liquid MS media supplemented with 1 mg/l Kn, which was twice the mean shoot number observed in semi-solid medium with the same hormone concentration (Table 3; Figs 1c-e). This clearly shows that liquid medium not Table 2 Effects of different cytokinins and their combinations on shoot multiplication in S. acmella (mg/l) Length of (cm ±SE) leaves MS 1.75±0.13 6.45±0.55 8.86±0.62 MS+0.5 BA 2.31±0.10 6.32±0.61 16.6±0.54 MS+1.0 BA 2.70±0.15 2.56±0.25 10.31±0.44 MS+2.0 BA 2.31±0.21 1.54±0.23 6.41±0.96 MS+4.0 BA 2.42±0.24 2.13±0.21 8.41±0.69 MS+0.5 Kn 2.55±0.16 2.76±0.51 5.91±0.34 MS+1.0 Kn 3.00±0.23 4.59±0.49 5.18±0.32 MS+2.0 Kn 2.57±0.19 2.16±0.28 6.13±0.54 MS+4.0 Kn 1.61±0.15 2.62±0.26 6.85±0.51 MS+1.0 BA+0.5 Kn 2.85±0.24 1.89±0.19 5.15±0.43 MS+0.5 BA+0.5 Kn 2.80±0.29 3.09±0.40 7.03±0.57 MS+0.5 BA+1.0 Kn 2.33±0.12 3.23±0.27 6.65±0.34 MS+1.0 BA+2.0 Kn 3.05±0.23 2.25±0.12 3.34±0.20 MS+0.5 BA+4.0 Kn 1.78±0.11 1.90±0.15 6.68±0.36 MS+1.0 BA+1.0 Kn 2.27±0.16 3.58±0.26 6.92±0.32 MS+4.0 BA+4.0 Kn 0.57±0.21 0.67±0.06 2.25±0.28 MS+0.5 BA+0.1 NAA 2.20±0.32 3.20±0.07 5.48±0.21 MS+1.0 Kn+0.1 NAA 2.81±0.14 3.26±0.21 5.92±0.13 MS+2.0 Kn+1.0 BA+ 0.1 NAA 2.67±0.22 3.01±0.6 4.49±0.43 MS+1.0 Kn+0.5 NAA 2.00±0.00 2.16±0.44 4.46±0.43 only improves the shoot proliferation but also increases the shoot growth. Such response is possibly due to better contact between plant tissue and medium with subsequent better diffusion of medium constituents 21. Similar results were also observed for shoot proliferation of Aloe vera in liquid medium 22. Advantages of liquid media for enhancing shoot propagation have also been reported for Allium sativus 23. Rooting and Acclimatization In cases, multiplication medium also regenerated roots on axillary shoot explants. These observations are in accordance with the previous studies on Acmella radicans 6 and S. acmella 9. However, good quality roots were produced on MS+1 mg/l IBA with 100% response (Table 4; Fig 1f). Well-rooted plants were successfully hardened in vitro and ex vitro in cocopeat irrigated with ½ MS (Figs 1g & h), acclimatized and showed 100% survival after 4 wk of transfer to the soil (complete data not shown).

JOSHI et al: IN VITRO PROPAGATION OF S. ACMELLA 115 MS medium with different conc. of agar Table 3 Effects of semi-solid and liquid MS media on shoot multiplication in S. acmella Length of (cm±se) leaves 0.5 mg/l BA+0.5% agar 3.60 ab ±0.64 4.61 a ±0.94 7.40 a ±1.30 1.0 mg/l Kn+0.5% agar 3.54 ab ±0.13 5.23 ab ±0.21 5.84 ab ±0.15 0.5 mg/l BA+0.2% agar 3.60 ab ±0.47 5.68 ab ±1.24 8.12 b ±1.36 1.0 mg/l Kn+0.2% agar 4.03 bc ±0.33 4.76 a ±0.21 10.21 cd ±0.64 Liquid MS 3.14 a ±0.15 6.32 b ±0.67 10.00 c ±0.87 0.5 mg/l BA+Liquid MS 4.53 c ±0.77 6.34 b ±0.64 11.84 d ±1.35 1.0 mg/l Kn+Liquid MS 6.20 d ±0.45 8.01 c ±0.87 11.80 d ±0.98 1.0 mg/l BA+2.0 mg/l Kn+Liquid MS 6.10 d ±0.65 6.20 b ±1.20 10.34 cd ±1.10 Means followed by different letters differ significantly at 5% as analysed by Duncan Multiple Range Test using SPSS Table 4 Effect of different IBA concentrations on rooting of in S. acmella % rooting MS+0.5 mg/l IBA 66.34 MS+1.0 mg/l IBA 100 MS+2.0 mg/l IBA 80.00 MS+4.0 mg/l IBA 76.40 Conclusion The present investigation suggests one line protocol for clonal propagation of S. acmella with MS+0.5 mg/l BA for initiation of culture, MS liquid+1.0 mg/l Kn for good proliferation of and 1.0 mg/l IBA for rooting, followed by in vitro and ex vitro hardening by transferring to cocopeat irrigated with ½MS and subsequent establishment in soil. The in vitro propagation protocol developed in the present study with higher response of shoot growth and multiplication on liquid culture medium can be useful for large-scale multiplication of S. acmella. Thus it can effectively be utilized for producing large quantity of plants. Acknowledgement The authors are grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi for providing financial assistance to carry out the work. References 1 Tiwari K L, Jadhav S K & Joshi V, An updated review on medicinal herb genus Spilanthes, J Chin Integr Med, 9 (2011) 1170-1178. 2 Di Stasi L C, Hiruma C A, Guimarães E M & Santos C M, Medicinal plants popularly used in Brazilian Amazon, Fitoterapia, 65 (1994) 529-540. 3 Akah P A & Ekekwe R K, Ethnopharmacology of some Asteraceae family used in Nigerian traditional medicine, Fitoterapia, 66 (1995) 351-355. 4 Storey C & Salem J I, Lay use of Amazonian plants for the treatment of tuberculosis, Acta Amazon, 27 (1997) 175-182. 5 Ramsewak R S, Erickson A J & Nair M G, Bioactive N-isobutylamides from the flower buds of Spilanthes acmella, Phytochemistry, 51 (1999) 729-732. 6 Rios-Chavez P, Ramirez-Chavez E, Armenta-Salinas C & Molina-Torres J, Acmella radicans var. radicans: In vitro culture establishment and alkamide content, In Vitro Cell Dev Biol-Plant, 39 (2003) 37-41. 7 Saritha K V, Prakash E, Ramamurthy N & Naidu C V, Micropropagation of Spilanthes acmella Murr., Biol Plant, 45 (2002) 581-584. 8 Pandey V & Agrawal V, Efficient micropropagation protocol of Spilanthes acmella L. possessing strong antimalarial activity, In Vitro Cell Dev Biol-Plant, 45 (2009) 491-499. 9 Yadav K & Singh N, Micropropagation of Spilanthes acmella Murr. An important medicinal plant, Nat Sci, 8 (2010) 5-11. 10 Yadav K & Singh N, Rapid plant regeneration from nodal explants of Spilanthes acmella (L.) Murr. An endangered medicinal plant, An Univ Oradea-Fascicula Biol, 19 (2012) 35-39. 11 Yadav K & Singh N, In vitro flowering of regenerated from cultured nodal explants of Spilanthes acmella Murr. An ornamental cum medicinal herb, An Univ Oradea- Fascicula Biol, 18 (2011) 66-70. 12 Mehrotra S, Goel M K, Kukreja A K & Mishra B N, Efficiency of liquid culture system over conventional micropropagation: A progress towards commercialization, Afr J Biotechnol, 6 (2007) 1484-1492. 13 Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol Plant, 15 (1962) 473-497. 14 De Fossard R A, Bennett M T, Gorst J R & Bourne R A, Tissue culture propagation of Eucalyptus ficifolia, F. Muell., Int Plant Propagators Soc Comb Proc, 28 (1978) 427-435. 15 Quraishi A, Koche V & Mishra S K, In vitro micropropagation from nodal segments of Cleistanthus collinus, Plant Cell Tissue Organ Cult, 45 (1996) 87-91. 16 Čellárová E & Kimáková K, Morphoregulatory effects of plant growth regulators on Hypericum perforatum L. seedlings, Acta Biotechnol, 19 (1999) 163-169.

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