In vitro propagation of Wedelia trilobata (L.) using Phormidium subincrustatum extracts: A novel approach

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1 AsPac J. Mol. Biol. Biotechnol Vol. 20 (4), 2012 Vol. 20 (4) : In vitro Propagation of Wedelia trilobata 1 In vitro propagation of Wedelia trilobata (L.) using Phormidium subincrustatum extracts: A novel approach M. Keerthiga 1 *, S. P. Anand 1, V. Nandagopalan 1, A. Doss1 and S. R. Senthilkumar 2 1 PG & Research Department of Botany, National College (Autonomous), Tiruchirappalli , Tamil Nadu, South India 2 Department of Botany, St. Joseph s College (Autonomous), Tiruchirappalli , Tamil Nadu, South India Received 8 August 2012/ Accepted 24 December 2012 Abstract. Most micropropagation involves the proliferation of callus, shoot and root tissue using MS medium supplemented with commercial growth hormones. While such media have arguably been too successful in terms of multiplication yields, it has become increasingly important to improve productivity and reduce the time taken to multiply commercially important material. The present study reveals the potential effect of extracellular products (EP) and biomass water extracts (BWE) of Phormidium subincrustatum on regeneration of Wedelia trilobata. The growth parameters of plantlets (11cm shoot length and 12 leaves per shoot) were proliferated from the nodal explants when cultured on basal MS media supplemented with 10% cyanobacterial extracts as in the positive control. Initiation of callus growth was observed on the cut surfaces of the leaf sections within days of culture with MS medium with BWE, compared to the control. Tremendous increase in shoot length and callus volume over a short period indicates that MS media with added cyanobacterial extracellular product can be used as a better alternative to other chemically synthesized growth regulators in MS media for callus and shoot induction. Keywords: Callus induction, Phormidium subincrustatum, Plant growth regulators, Shoot induction, Wedelia trilobata INTRODUCTION Efficient plant regeneration is a vital objective of many studies involving plant tissue culture. Most of the growth regulators used are chemically synthesised, but plant hormones have central role in the control of in vitro organogenesis. In particular, auxins and some cytokinins play a crucial role in gene expression during plant development in vitro. For the improvement of economically and medicinally important plants, tissue culture is one of the biotechnological tools that play an important role in the genetic modification of plants. Success of any tissue culture depends upon the standardization of medium used. The use of cyanobacterial culture could overcome one of the barriers to effective micropropagation: the involvement of where costly synthetic chemicals (Banerjee et al., 2008). Cyanobacteria produce a variety of bioactive compounds including growth phyto-regulators, which could be used in the in vitro production of vegetables, fruits, fungi and ornamental flowers (Zaccaro et al., 2006). Among the growth regulators, gibberellins, auxins, cytokinins, ethylene, abscisic acid and jasmonic acid have been detected in cyanobacteria (Stirk et al., 2002; Gupta et al., 1973; Ordog et al., 2005). There is accumulating evidence that cyanobacteria produce plant hormone-like activity, and it has recently been demonstrated that a number of cyanobacteria produce, accumulate and liberate various plant regulators. Wedelia trilobata (L.), (Singapore daisy) is a flowering plant of the group Asteraceae. A tropical perennial medicinal herb, with deeply lobed, fleshy leaves, growing up to 10 inch tall, spreading like a mat, it produces a dense cover, and blossoms profusely with orange and yellow flowers. It is an interesting source of potentially bioactive molecules such as iridoid compounds, flavonoids, diterpenoid derivatives, and phytosteriods with antioxidant, anti inflammatory, anti microbial, hepatoprotective, analgesic, antihistamine, anti-implantation, anti-asthmatic and anti-cancer activity (Meena et al., 2011, Govindappa et al., 2011, Chethan et al., 2012). The aim of this present work was to improve the plant tissue culture medium by the addition of intra- and extracellular substances from Phormidium subincrustatum (cyanobacterial) extracts as an organic supplementation of MS media, and observe the effects on the in vitro propagation of Wedelia trilobata and to compare these effects with those produced by synthetic phyto-regulators. * Author for correspondence: M. Keerthiga, PG & Research Department of Botany, National College (Autonomous), Tiruchirappalli , Tamil Nadu, South India.

2 148 AsPac J. Mol. Biol. Biotechnol. Vol. 20 (4), 2012 In vitro Propagation of Wedelia trilobata MATERIALS AND METHODS Cyanobacteria and Growth Conditions. The cyanobacterium, Phormidium subincrustatum was supplied by Department of Botany, University of Madras, Chennai, India, and cultured in 250 ml Erlenmeyer flasks in BG11 medium (Rippka et al., 1979). The culture was grown and maintained under light intensity of 30 μ Em-2S-1 at 25±10 C. The culture chamber was fitted with an automatic timer (Sangamo Weston Ltd., S F model) set to alternating 12 hours light and 12 hours dark cycles. The culture was harvested at the stationary growth phase by centrifugation. Measurement of Cyanobacterial Growth. Growth was determined by measuring chlorophyll a for every fifth day up to 30 days, using the method of McKinney (1941). Uniform suspension of the culture was obtained by homogenizing using a syringe, and a sample was taken for the estimation of chlorophyll a concentration. After centrifugation at 6,000 rpm for 10 minutes in a refrigerated centrifuge, the pellet was suspended in 5 ml of 80% acetone and left in the dark at 4ºC for overnight extraction. The suspension was again centrifuged and the optical density of the supernatant was read at 663 nm in a Beckman DU-40 spectrophotometer with 80% acetone as a blank. Biomass water extracts (BWE) and extracellular products (EP). After 30 days Phormidium subincrustatum biomass in the medium was separated from the culture medium by centrifugation at 10,000 rpm at 5 C. The fresh biomass was homogenized using acid wash sand (1g biomass to 3g acid wash sand) and extracted with sterile distilled water (1g fresh biomass to 5ml distilled water). After centrifugation, the BWE supernatant was obtained, and the ph adjusted to 5.8. The cultured medium contained the extracellular products (EP). The BWE and EP were sterilized by ultrafiltration (0.22 μ). Preparation of Plant Material. Leaf and nodal segments were collected as explants from 2-4 week-old Wedelia trilobata plants. The explants were washed thoroughly in running tap water for 20 minutes, before being dipped in 70% ethanol for 60 seconds and rinsed with sterile distilled water, surface sterilized using 0.1% mercuric chloride solution then again washed well with sterile distilled water. The explants were blotted using sterile Whatmann No. 1 filter paper. Culture media. Different combinations growth regulators were used for in vitro cultures of Wedelia trilobata in culture media (Table 1). The commercial growth regulators BAP and NAA were added to a final concentration of either 0.5 or 2mgL -1, and the media contained 3% sucrose and 0.7% agar. The ph was adjusted to 5.8, before the media was sterilized in tubes (14 x 2.5cm). Treatment I : MS (Control) Treatment II : 8 ml MS + 2 ml EP Treatment III : 8 ml MS + 1 ml EP+ 1ml NAA+ 1 ml BAP Treatment IV : 8 ml MS + 2 ml BWE Treatment V : 8 ml MS +1ml BWE +1ml BAP +1ml NAA Treatment VI : 8 ml MS + 2 ml distilled water mg/l NAA mg/l BAP. Table 1. Culture media composition for six treatments, used for in vitro cultures of Wedelia trilobata. In vitro culture. Explants (leaves and nodes) were inoculated aseptically onto culture medium of various compositions. After inoculation, the culture flasks were maintained at 25±2 ºC and 16 hour photoperiod. The number of regenerated calli and shoots after 45 days of culture were recorded. RESULTS Growth Curve. Isolated axenized cultures of Phormidium subincrustatum were harvesed at mid log phase, homogenized to a uniform suspension and inoculated into 100 ml of BG 11 medium. The cultures were grown at 25±1 ºC under a light intensity of 45 µmol photon x-2 seq-¹ and L/D cycle of 12 hrs each for 30 days. Phormidium subincrustatum cultures (Figure 1a) were harvested at 5 day intervals and growth was measured in terms of chlorophyll a presence. Chlorophyll a content was µgml-1 on the 25th day and then declined (Figure 1b). The biomass water extracts (BWE) and extracellular products (EP) were extracted from 30 day cultures of Phormidium subincrustatum. Secondary metabolite production was very high in the stationary phase, though they are not involved in the growth of microorganisms.

3 AsPac J. Mol. Biol. Biotechnol. Vol. 20 (4), 2012 In vitro Propagation of Wedelia trilobata 149 Figure 1a. Phormidium subincrustatum in culture. Figure 2a. Effect of various medium compositions on shoot induction. Figure 1b. Estimation of chlorophyll a content in culture medium over time. Figure 2b. Effect of various medium compositions on callus induction. Plant Tissue Culture. Leaf and nodal regions were used as primary explants. Initiation of shoot growth was observed on the cut surface of the leaf sections after days of culture only on MS medium with added BWE (treatment IV) and to a lesser extent on MS medium with added EP (treatment II) (Figure 2a). The highest rate of calli development in comparison to the control treatment was also observed in treatments IV and II which were grown on MS with added NAA and BAP (Figure 2b). Morphological observation showed that callus growth was smooth, compact and yellow in colour (Figure 3). Plant growth regulators derived from biomass water extracts (intracellular) of Phormidium subincrustatum showed positive effects on micropropagation; elongated shoot induction (11 cm) and a stimulation of callus development were recorded in MS medium with added BWE (treatment IV). MS with extracellular product added (treatment II) showed elongated shoot and callus development but comparatively less than treatment IV (MS+BWE). The number of leaves was more or less same with the addition of both extracellular and intracellular products (treatment IV and II); an acceleration of differentiation was seen with the addition of EP and BWE to the growth medium. A healthy strong Wedelia trilobata plantlet (11cm shoot length) proliferated from nodal explants (1 cm length) when cultured onto basal MS medium supplemented with BWE (Figure 4). Plantlets grown on MS with added NAA and BAP were 2.5 cm in length. The effects of various media additives on in vitro regeneration of Wedelia trilobata are summarised in Table 2. Normally in any tissue culture media the organic growth regulators are used in the chemical form. In our study, however, MS with added BWE (treatment IV) and MS with added EP (treatment II) led to the growth of an elongated shoot with many leaves and the regeneration capacity was also better than in explants grown on MS with added NAA and BAP (treatment VI) or other treatments. Plant tissue culture medium can thus be improved by the addition of

4 150 AsPac J. Mol. Biol. Biotechnol. Vol. 20 (4), 2012 In vitro Propagation of Wedelia trilobata Figure 3. In vitro callus induction in MS medium with or without the addition of cyanobacterial extract. a) MS without additions (control), b) MS+EP, c) MS+BWE. Figure 4. In vitro shoot induction in MS medium with or without the addition of cyanobacterial extract. a) MS without additions (control), b) MS+EP, c) MS+BWE. compounds extracted from cyanobacteria. The addition of a minimum fresh weight of Wedelia trilobata to the in vitro culture medium proves that BWE and EP are natural additives which may be better alternatives to NAA and BAP for shoot and callus induction. DISCUSSION The techniques of in vitro cell and tissue cultures were originally developed to allow the study of plant nutrition and morphogenesis. Recently the use of regenerants derived from these cultures has become one of the most fundamental approaches to practical plant biotechnology. Most micropropagation methods involve the proliferation of calli using MS medium containing only chemically defined complex nutritive compounds (Hall, 1999). Micropropagation by conventional techniques is typically a lengthy, labour intensive but economical means of clonal propagation. Among the various combinations of growth regulators tested in previous studies, MS medium containing BAP and NAA was found to be the best for induction of shoot and callus growth from leaf and nodal explants in the studies of Sedlak and Paprstein (2008) in sweet cherry plant, Pant et al., (2007) in Daucus carota and Anand, et al., (2011) in Premna latifolia. Any improvement on this conventional media would be welcome if it enabled growers to regenerate plants at a faster rate. Use of cyanobacteria in this process might just be such an option. Tremendous increase in root length and volume over a short period was achived by the addition of Plectonema sp. extracellular product to the MS during the screening of rice genotypes for water stress reactions (Manickavelu et al., 2006). The efficient induction of adventitious shoot and root growth using cyanobacterial extracts has been demonstrated by Meenakshi Banerjee et al. (2010) in Bacopa monnieri and Sarika shrivastava et al. (2009) in Jatropha curcas. Recently in vitro regeneration of Stevia rebaudiana was reported using cyanobacteria as media and as an additive to MS media (Meenakshi Banerjee, 2005 and Tiwari Seema et al., 2011). Here, we studied the use of extracts of a cyanobacteria species, Phormidium subincrustatum, for its effect on shoot induction and callusing of Wedelia trilobata. In vitro callusing of Wedelia trilobata has previously been studied and shown to be a slow process (Tripti Seema et al., 2011). But, in this work, the plant tissue culture medium was modified using the compounds of cyanobacteria, which resemble natural plant hormones such as gibberellins, auxins and cytokinins (Ordog et al., 2005 and Manickavelu et al., 2006), and the difference in growth of the plant was easily visible.

5 AsPac J. Mol. Biol. Biotechnol. Vol. 20 (4), 2012 In vitro Propagation of Wedelia trilobata 151 Media composition Shoot induction Callus induction No. of nodal explants Percentage No. of leaf explants Percentage MS (control) 4.33 ± MS+EP 14 ± MS+EP+NAA+BAP 9.33 ± MS+BWE ± MS+BWE+NAA+BAP ± MS+NAA+BAP 8 ± Table 2. Effect of Various Media Composition on In vitro Regeneration of Wedelia trilobata CONCLUSION In the present study, leaf and nodal regions of Wedelia trilobata plants were used to form primary explants. Initiation of calli was observed on the cut surface of the leaf sections within days of culture only when biomass water extracts (BWE) of Phormidium subincrustatum were added to the culture medium. Earlier regeneration is the primary goal of efforts to improve in vitro studies and the number of days taken for callus and shoot induction was seen to be reduced by this change to the culture medium. The present paper deals with the use of MS media with cyanobacterial extract as a liquid culture media for the induction and development of shoots to ensure high yield of biomass of the medicinal herb Wedelia trilobata. This protocol is very cost effective and totally novel. REFERENCES Anand, S.P., Nandagopalan, V. and Jeyachandran, R In vitro plant regeneration of Premna latifolia from nodal explants. Asian Journal of Microbiology, Biotechnology and Environmental Science 13(4): Banerjee, M. and Shrivastava, S An improved protocol for in vitro multiplication of Bacopa monnieri (L.). World Journal of Microbiology and Biotechnology 24: Banerjee, M In vitro callusing in Stevia rebaodiana Bertoni using cyanobacterial media. International Journal of Biology 3(3): Banerjee, M. and Modi, P Micropropagation of Bacopa monnieri using cyanobacterial liquid medium. Plant Tissue Culture & Biotechnology 20(2): Chethan, J., Sampath Kumara, K.K., Niranjana, S.R. and Prakash, H.S Evaluation of antioxidant and antibacterial activities of methanolic flower extract of Wedelia trilobata (L.) Hitch. African Journal of Biotechnology 11(41): Govindappa, M., Naga Sravya, S., Poojashri, M.N., Sadananda, T.S. and Chandrappa, C.P Antimicrobial, antioxidant and in vitro anti-inflammatory activity of ethanol extract and active phytochemical screening of Wedelia trilobata (L.) Hitch. Journal of Pharmacognosy and Phytotherapy 3(3): Gupta, A.B. and Agarwal, P.R Extraction, isolation and bioassay of a gibberellin like substances from Phormidium foveolarum. Annals of Botany 37(152): Hall, T BioEdit computer program, version Retrieved from bioedit.html. MacKinney, J Absorbtion of light by chlorophyll solution. Journal of Biological Chemistry 140: Manickavelu, A., Nadarajan, N., Ganesh, S.K., Ramalingam, R., Raguraman, S. and Gnanamalar. R.P Organogenesis induction in rice callus by cyanobacterial extracellular product. African Journal of Biotechnology 5(5):

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