INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 6, No 2, Copyright by the authors - Licensee IPA- Under Creative Commons license 3.

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 6, No 2, 2015 Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article ISSN In-vitro micropropagation and comparative study on antimicrobial activity of micropropagated and naturally Vidwans H 1, Moghe R 2, Upadhyay A 3 1- PhD student Ankur seeds pvt ltd, Nagpur, 2- Technical Officer, Ankur Seeds Pvt. Ltd., Nagpur 3- Head of Department, Hislop College, Nagpur. harshada.vidwans2@gmail.com doi: /ijes.6034 ABSTRACT Syzygium cumini is an important medicinal plant. A micropropagation protocol was developed from nodal explants of eight months old plantlets of S.cumini. Shooting was induced on MS medium supplemented with combinations of BAP and IBA. The shoots were rooted in half strength MS medium supplemented with IBA and sucrose. Acclimatization was successful and plantlets established in the field showed good survival response. Antimicrobial activity was carried out against coliforms (directly isolated) and mixed culture (mixture of pure cultures of coliform group of bacteria) with respect to in-vivo and in-vitro leaf extracts of S.cumini. Keywords: antimicrobial activity, micropropagation, agar well diffusion, coliform 1. Introduction Plants are important part of our life. Plants face a variety of antagonists and have evolved multiple defense mechanisms by which they are able to survive with various kinds of biotic and abiotic stress (Ballhorn et al., 2009). Many plant tissues contain a variety of compounds called secondary plant compounds (metabolites) grouped as glucosides, saponins, tannins, alkaloids, essential oils, organic acids and others (Fraenkel 1959). The present study was focused on medicinal plant S.cumini. Its bark is used as astringent in dysentery, seeds are antidiabetic, fruits are used to treat cough, diabetes, dysentery, inflammation (Swami et al., 2012). Khorasani (2010) showed the antioxidant and antimicrobial activities of A.officinalis differ between in vitro and in vivo grown plants. Antimicrobial activity was obtained from in vitro plants against B. cereus. A compound produced in an in vivo plant could be produced at the same or different levels or not produced at all (Verpoorte et al 2002). The variety of compounds produced in in-vivo and in vitro plants can show different bioactivity potentials (Grzegorczyk et al., 2007). Therefore it was decided to compare activities of naturally growing plant and regenerated plant. This study was carried out to develop a protocol for micropropagation of S.cumini from nodal explants and to compare antimicrobial activities of naturally growing and micropropagated plants. Antimicrobial activity was observed against coliforms, as coliforms are considered as indicator organism of fecal contamination of water (WHO 2011). Along with isolated coliforms, antimicrobial activity will be observed against mixed culture of different bacteria from coliform group. Leaf extracts of the above plants can be used to treat bacteriologically contaminated water, thus, it will provide a non-chemical form of disinfectant. Received on July 2015 Published on September

2 2. Material and methods 2.1 Plant material Plants of Syzygium cumini (Jamun) were collected and established in the green house and used as source of plant material. Explants collected form source plant were trimmed to around 2.5 cm. They were thoroughly washed in running tap water to remove dirt, followed by washing with detergent (1 drop of lyzol) for 5 minutes, again washed with double distilled water. Explants were washed with 0.5 % HgCl2 (Syzygium cumini) and again washed with distilled water. These were spread on sterilized petridishes on sterile bloating paper inside laminar air flow hood. Explants were trimmed to about 1.5 cm. 2.2 Tissue culture Nodal segments when inoculated in MS medium containing BAP and IBA showed enhanced shoot prolification. Shoots after initial proliferation on medium, were sub-cultured on fresh medium after 30 days. Incorporation of BAP and IBA into MS medium, supported multiplication of shoots. The surface sterilized explants were cultured on MS medium supplemented with BAP ( mg/l), IBA ( mg/l) either alone or in combination. The media were adjusted to ph 5.8 before gelling with agar (6 g L -1 ). The cultures were incubated at 23±2 C and 16 h photoperiod. For rooting, in vitro shoots (4-5 cm long) were transferred to rooting medium (1/2 MS with 3% sucrose and 0.7% agar) containing various concentrations of IBA ( mg/l). Well developed and rooted plants thoroughly washed in running tap water and planted in soil rite, a commercially available sterile potting mix in net pots and hardened in a shade house under 90-95% relative humidity (RH) for 8-10 days. They were gradually transferred to plastic pots or poly bags. A survival rate of 90-95% was achieved during the hardening. After days, the hardened plants were transferred to field. All experiments were repeated thrice with triplicates. ANOVA was carried out and all values are calculated in mean ± standard deviation. 2.3 Microorganism used A) Mixed culture: Coliform group of organisms namely Klebsiella pneuminiae MTCC 8911, Enterobacter aerogenes 2823, citrobacter frundei 8128, Pseudomonas aeruginosa 7198, Shigella flaxuneri 1457, Proteus vulgaris 744, E.coli MTCC were from procured from MTCC, IMTECH, Chandigarh. Above organisms were revived and transferred to NB, individually. 0.5 ml culture of each microorganism was mixed in a sterile test tube aseptically. B)Coliforms: Coliforms were isolated from well water by membrane filtration technique on EMB agar. Colony with green metallic sheen was picked from plate and was streaked to get pure culture. Isolated colony with green metallic sheen was transferred to NB was incubated at 37 0 C for 18hrs. 2.4 Preparation of leaf extracts The leaves were collected, cut into pieces and then extracted with ethanol in mortar pastel. Each of the test samples was thoroughly homogenized methanol on a rotatory shaker at rpm for 72 hrs, then passed through a muslin cloth and centrifuged at 5000 rpm for 10 min. The supernatant was collected and used for antimicrobial screening. 302

3 3. Well diffusion assay Mueller Hinton agar (MHA) was distributed in sterilized petridishes. Antimicrobial activity was performed by agar well diffusion assay (Perex et al., 1990). An 24hrs old inoculum (10 4 CFU/ml) of each test bacterial isolate was inoculated on dried surface of Mueller -Hinton agar by streaking with sterile cotton - tipped swab to achieve a confluent growth. The inoculated plates were allowed to dry after which wells were punched on the agar using a sterile standard 4 mm cork borer. Extract was introduced into the wells that have been labeled accordingly. Plates were kept in incubator and then examined for the development of clear inhibitory zone. 4. Results and discussion Best response was observed in medium containing BAP 1 mg/l and IBA 0.5 mg/l, average no. of shoots 3.98 ± 0.50 and best shoot length 5.0 ± 0.25 cm (Table 1). According to ANOVA, a significant effect of plant growth regulators over the in vitro phase on measured parameters was observed (P < 0.05). Table 1: Effect of different concentrations of plant growth regulator on in vitro multiplication (2nd subculture) from nodal explant of S.cumini. MS+BAP(mg/L) IBA (mg/l) No of shoots Height of % response per explants shoot ± ± ± ± ± ± NR - - MEAN ± SD Full and half strength of MS Medium without any PGR failed to induce roots in regenerated shoots. Auxin such as IBA induced routing in the medium containing ½ MS medium salts +1% glucose. The best rooting response was observed in the medium containing 0.3mg/l IBA, where roots measuring 2.23 ± 0.27 cm were formed. Figure 1: Micropropagation of S.cumini: A) initiation B) multiplication C) elongation D) in-vitro rooting E) primary hardening 303

4 According to ANOVA, a significant effect of plant growth regulators over the in vitro phase on measured parameters was observed (P < 0.05). Figure 2: Developed plant of S.cumini Syzygium cumini is a medicinally important plant and possess antimicrobial activity against Enterococcus sp, Vibrio sp, etc (Vidwans et al., 2014). Antimicrobial activity was performed with leaf extracts of naturally growing and tissue culture (in-vitro) plants. Experiment was done in triplicate. Both the extracts inhibited the growth of coliforms, but in-vitro plant extracts exhibited higher activity as compared to naturally growing plant extracts. In-vitro leaf extract gave inhibition zone 17 ± 0.50 mm. (Figure 2) Table 2: Evaluation of Antimicrobial activity of S.cumini Sr.No Type of Leaf Extract Zone (mm) SD (coliforms) Zone(mm)+- SD (mixed culture) 1 In-vivo (ethanol) 12 ± ± In-vitro (ethanol) 17± ± 0.33 MEAN ± SD standard deviation (A) In-vivo extract against coliforms (B) In-vitro extracts against mixed culture 304

5 5. Conclusion (C) In-vitro extract against coliforms Figure 3: Antimicrobial Zone of S.cumini Micropropagation protocol was developed and comparison of in-vivo and in-vitro plants was done with respect to phytochemical and antimicrobial properties. In S.cumini, maximum number of shoots was produced in MS medium supplemented with BAP 1 mg/l and IBA 0.5 mg/l. Addition of IBA to1/2 MS medium initiated rooting. Antimicrobial activity of in-vitro extracts of S.cumini showed greater activity as compared to naturally growing plants. Also the in-vitro extracts was able to inhibit mixed culture, but comparatively the antimicrobial activity was greater in case of coliforms. Thus, extracts can be used to treat coliforms in drinking water, further work is needed. 6. References 1. Ballhorn D, Kautz S, Heil M, Hegeman A, (2009), Cyanogenesis of wild lima bean (Phaseolus lunatus L.) is an efficient direct defence in nature, Plant Signaling and Behavior, 4(8), pp Fraenkel G, (1959), The raison d Etre of secondary plant substances. Science, 129, pp Grzegorczyk I, Matkowski A, Wysokinska H (2007). Antioxidant activity of extracts from in vitro cultures of Salvia officinalis L. Food Chemistry, 104(2), pp Khorasani A, Sani W, Philip K, Taha R, Rafat A, (2010), Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur. 5. Perex C, Paul M, Bazerque P Antibiotic assay by agar well diffusion method. Experimental Medicine and Biology, 15, pp Swami S, Singh N, Thakor J, Patil M, Haldankar P, (2012), Jamun (Syzygium cumini (L.)): A Review of Its Food and Medicinal Uses, Food and Nutrition Sciences, 3, pp Verpoorte R, Contin A, Memelink J (2002). Biotechnology for the production of plant secondary metabolites, Phytochemical. Review, 1(1), pp

6 8. Vidwans H, Gaikwad G, Deshpande S, Ramteke D, Wate S, (2014), Selection of Diversified Floral Species from Various Regions in India and their Antimicrobial Activities with respect to Coliform, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Research Journal, 48 (3), pp WHO, (2011), Library Cataloguing-in-Publication Data Guidelines for drinking-water quality 4 Th ed. 1.Potable water - standards. 2. Water - standards. 3. Water quality - standards. 4. Guidelines. I. World Health Organization. ISBN (NLM classification: WA 675) World Health Organization. 306