Mafatlal M. KHER1*, Deepak SONER1, Neha SRIVASTAVA1, Murugan NATARAJ1**, Jaime A. TEIXEIRA Da SILVA2*** 1B.R
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Journal of Horticultural Research 2016, vol. 24(1): 21-28 DOI: 10.1515/johr-2016-0003 _______________________________________________________________________________________________________ MICROPROPAGATION OF CLERODENDRUM PHLOMIDIS L.F. Mafatlal M. KHER1*, Deepak SONER1, Neha SRIVASTAVA1, Murugan NATARAJ1**, Jaime A. TEIXEIRA da SILVA2*** 1B.R. Doshi School of Biosciences, Sardar Patel University, Sardar Patel Maidan, Vadtal Road, Satellite Campus, Post Box No. 39, Vallabh Vidyanagar-388120, India 2P.O. Box 7, Miki-cho post office 3011-2, Ikenobe, Kagawa-ken 761-0799, Japan Received: March 2016; Accepted: May 2016 ABSTRACT Clerodendrum phlomidis L. f. is an important medicinal plant of the Lamiaceae family, particularly its roots, which are used for various therapeutic purposes in a pulverized form. The objective of this study was to develop a standard protocol for axillary shoot proliferation and rooting of C. phlomidis for its prop- agation and conservation. Nodal explants were inoculated on Murashige and Skoog (MS) medium that was supplemented with one of six cytokinins: 6-benzyladenine, kinetin, thidiazuron, N6-(2-isopentenyl) adenine (2iP), trans-zeatin (Zea) and meta-topolin. Callus induction, which was prolific at all concentrations, formed at the base of nodal explants and hindered shoot multiplication and elongation. To avoid or reduce callus formation with the objective of increasing shoot formation, the same six cytokinins were combined with 4 µM 2,3,5-tri-iodobenzoic acid (TIBA) alone or in combination with 270 µM adenine sulphate (AdS). Nodal explants that were cultured on the medium supplemented with 9.12 µM Zea, 4 µM TIBA and 270 µM AdS produced significantly more and longer shoots than on medium without TIBA and AdS. Half-strength MS medium supplemented with 8.05 µM α-naphthaleneacetic acid was the best medium for root formation. Most (75%) in vitro rooted plantlets were successfully acclimatized under natural conditions. Key words: auxin inhibitor; axillary shoot multiplication; callus; nodal explants; 2,3,5-tri-iodobenzoic acid INTRODUCTION antiviral, antihypertensive, hypolipidemic and anti- oxidant activities of various plant parts (Shrivastava Clerodendrum phlomidis L. f. (syn. C. multi- & Patel 2007). The hexane and ethyl acetate extracts florum (Burm. f.) Kuntze; C. phlomidis f. rubrum of roots show anti-tubercular activity against Myco- (Roxb. ex Voigt) Moldenke; C. phlomidis var. bacterium tuberculosis H37Rv (Yadav & Gupta rubrum Roxb. ex Voigt; Volkameria multiflora 2014) – attributed to a phenylethanoid glycoside – Burm. f.) is a member of the Lamiaceae family (The while the methanolic extract of its leaves has anti- Plant List 2016). This shrub is distributed in India, diarrhoeal properties (Rani et al. 1999). The aque- Sri Lanka and Southeast Asia (Raja & Mishra 2010) ous extract of the root bark displays anti-inflamma- and is commonly known in India as Clerodendrum tory activity (Parekar et al. 2012). Yadav and Gupta or wind killer in English and Arni in Hindi. (2014) attributed this activity to three compounds in C. phlomidis is an important medicinal plant used the methanolic extract of roots, including lupeol. extensively in Ayurveda, Unani, Siddha and folk- The chloroform extract of leaves show ovicidal and lore medicines for the treatment of various types of oviposition activities against Earias vittella (Muthu ailments (Raja & Mishra 2010) and is well known et al. 2013) because of the presence of the flavonoid for the anti-inflammatory, anti-rheumatic and anti- pectolinaringenin and larvicidal activity against asthma activities of its root bark; antimicrobial aciv- early fourth-instar larvae of the filarial vector Culex ity of its leaves; as well as antidiabetic, antimalarial, quinquefasciatus and dengue vector Aedes aegypti *Corresponding authors: e-mail: *[email protected], **[email protected], ***[email protected] 22 M.M. Kher et al. ____________________________________________________________________________________________________________________ (Muthu et al. 2012), which is due to the presence of proliferation from nodal explants and the successful pectolinaringenin. The medicinal and biological acclimatization of regenerated plants in soil. properties of C. phlomidis are thus broad and firmly established. MATERIALS AND METHODS Owing to these properties, C. phlomidis is a highly traded medicinal plant from tropical forests Plant material and surface sterilization with an estimated consumption or trade of about Nodal cuttings of C. phlomidis were collected 300 metric tons/year and is sold under the trade from the same 10-year-old mother plant growing in name of Arnimul (leaf and root), with a price rang- the Anand Agricultural University Medicinal Plant ing from 0.32 to 0.5 US$/kg (Raja & Mishra 2010). Garden, Anand, Gujarat. They were washed in run- Owing to its therapeutic importance, the plant is in- ning tap water for 10 min. Explants (nodal cuttings discriminately collected without any regulation, with four nodes) were surface sterilized in sterile with a risk to natural populations. Therefore, there distilled water (SDW) containing a few drops of is interest in developing a suitable in vitro method 10% (v/v) Tween-20 followed by a 0.1% (w/v) for the mass propagation of C. phlomidis to reduce HgCl2 solution for 3 min followed by five rinses in pressure on natural populations. Micropropagation SDW. Sterilized nodal explants were used for in by axillary shoot multiplication offers a simple but vitro culture studies as described next. reliable, rapid and reproducible method for clonal Composition of culture media and culture condi- propagation and germplasm conservation of threat- tions ened medicinally important plants. To date, several The culture medium consisted of MS salts, vit- in vitro culture protocols have been developed for amins and 3% (w/v) sucrose. The pH of all media other members of the Clerodendrum genus (Nataraj was adjusted to 5.8 with 0.1 N NaOH or HCl before et al. 2016), but only three reports on the microprop- adding 0.8% (w/v) agar-agar (bacteriological grade; agation of C. phlomidis exist (Devika & Kovilpillai Merck, Ahmedabad, India) and autoclaved at 2012; Raaman et al. 2011; Soner et al. 2015). 121 °C for 15 min. Cultures were maintained at 25 Devika and Kovilpillai (2012) and Raaman et al. ± 2 °C under a 16-h photoperiod at a photosynthetic (2011) used nodes and leaves as initial explants, photon flux density of 35 µmol·m-2·s-1 provided by while Soner et al. (2015) used leaves, internode and cool white fluorescent tubes (36W TL-D Super 80; petiole explants to induce somatic embryogenesis of Philips, Anand, India). All in vitro culture condi- C. phlomidis. Raaman et al. (2011) induced shoots tions were based on in-house optimization trials. through an intermediate callus phase in the presence Borosilicate glassware (Borosil, Bharuch, India) of 6-benzyladenine (BA) and α-naphthaleneacetic was used for all cultures within this study. acid (NAA) in Murashige and Skoog (1962) (MS) Induction of axillary shoots on media containing basal medium. Devika and Kovilpillai (2012) and different cytokinins Raaman et al. (2011) reported shoots from callus, Surface sterilized nodes, each with two vege- but such callus can lead to somaclonal variation tative buds, were inoculated in round glass bottles (Bairu et al. 2011). Although this is generally an un- (400 ml capacity) containing Murashige and Skoog desirable outcome for clonal propagation, it can be (1962) – MS medium supplemented with 2.22, 4.44, beneficial in medicinal plants by obtaining plants 6.66, 8.88 and 11.1 µM of 6-benzyladenine (BA); with altered levels of secondary metabolites. Infor- 2.33, 4.65, 6.98, 9.30 and 11.63 µM of 6-furfuryla- mation about explant age, surface sterilization and minopurine (kinetin, Kn); 2.46, 4.92, 7.38, 9.84 and culture conditions are not described by Devika and 12.3 µM of N6-(2-isopentenyl) adenine (2iP); 2.07, Kovilpillai (2012), making their protocol difficult to 4.14, 6.21, 8.28 and 10.35 µM of 6-(3-hydroxyben- repeat, while somatic embryogenesis require further zylamino) purine meta-topolin, mT); 2.28, 4.56, improvement (Soner et al. 2015). The present study 6.84, 9.12 and 11.4 µM of 6-(4-hydroxy-3- describes a method for the in vitro propagation and methylut-2-enylamino)purine (trans-zeatin, Zea), multiplication of C. phlomidis through axillary bud all purchased from Himedia (Mumbai, India) and Micropropagation of Clerodendrum phlomidis L.F. 23 ____________________________________________________________________________________________________________________ 2.27, 4.54, 6.81, 9.08, 11.35 µM of 1-phenyl-3- Duncan’s multiple range test (DMRT) at p = 0.05 (1,2,3-thiadiazol-5-yl)urea) (thidiazuron, TDZ), pur- using Statistical Package for Social Science (SPSS) chased from Sigma (Bangalore, India) for axillary ver. 19 for Windows (SPSS Inc., Chicago, IL, shoot multiplication. After two weeks of culture, USA). Results were expressed as means ± standard prolific callus formed at the cut ends of nodal ex- deviation (SD). plants, which inhibited effective shoot formation and multiplication (Fig. 1A). RESULTS AND DISCUSSION Influence of TIBA and AdS on axillary shoot multiplication Axillary shoot growth and multiplication To prevent an excessive amount of callus for- Nodal segments are preferred explants for mi- mation, nodal explants were cultured in round glass cropropagation because of the presence of pre-ex- bottles (400 ml capacity) containing MS medium isting meristems, which can develop easily into supplemented with 4 µM of 2,3,5-tri-iodobenzoic shoots while maintaining clonal fidelity. In this acid (TIBA) (Himedia), a polar auxin inhibitor, and study, nodal explants of C. phlomidis were inocu- 8.88 µM of BA, 9.3 µM of Kn, 9.84 µM of 2iP, lated on MS basal medium supplemented with dif- 8.28 µM of mT, 9.12 µM of Zea or 9.08 µM of TDZ ferent concentrations of a single cytokinin (BA, Kn, alone or in combination with 270 µM of adenine TDZ, 2iP, Zea and mT), while cultures without plant sulphate (AdS) (Himedia), which was determined to growth regulators (PGRs) served as the control.