Use of various concentrations of growth hormones for callogenesis of anti cancerous plant Viola odorata L.

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1 Use of various concentrations of growth hormones for callogenesis of anti cancerous plant Viola odorata L. Krupali V. Haralkar, Sanjay R. Biradar Tissue Culture Research Centre Dept. of Botany, Shri Chhatrapati Shivaji College, Omerga , Dist. Osmanabad, (MS). INDIA sanjaybiradar2006@rediffmail.com Abstract The present investigation aimed at developing in-vitro propagation protocol, which can be used for conservation of Viola odorata L. a medicinal herb growing in Kashmir and western Himalayas. Viola odorata L. is generally used in the treatment of various diseases especially in the treatment of cancer and whooping cough [Ghani UK et.al. (1997)]. The leaf and petiole used as a explants for inoculation on MS medium [Murashige and Skoog (1962)] contains various concentrations and combinations of growth hormones for callus induction like Kin, BA, NAA, IAA, and 2, 4-D and better results occurred in concentration of BA and 2,4-D ( ) for callus induction in 12 and root formation occurred in concentration of BA+2,4-D ( ) in 110. Keywords- Viola odorata L, callus induction, anti cancerous plant, callogenesis INTRODUCTION Viola odorata L. belongs to family Violaceae, order Violales, subclass Dilleniide, class Magnoliatae. The Genus Viola comprises almost 500 species, spread all over the world. The spreading area of this species is very large: Europe, Minor Asia, Syria, Caucasus, and North America. Sweet violet (Viola odorata L.) is one of the first perfumed flowers in the spring. [Ravarut et.al. ( 1967), Savulescu T et.al.( 1955)]. In India, Viola odorata L. in Jammu and Kashmir is mostly found in regions like Kud, Patnitop, Reasi, Kishtwar, Mahore and Panchari. Viola odorata L. is generally used in the treatment of various diseases especially in the treatment of cancer and whooping cough [Ghani UK et.al. (1997)]. It contains salicylic acid, which is used to make aspirin and thus is effective in the treatment of headaches, migraine and insomnia. The whole plant plays role in anti-inflammatory, antidyslipidemic and antihypertensive activity [Siddiqi HS et.al. (2012)]. It is taken internally in the treatment of bronchitis, respiratory problems, coughs, asthma, and cancer of the breast, lungs or digestive tract. Externally, it is used to treat mouth and throat infections. It is with this backdrop that the genuine plants of V. odorata need to be conserved and plant tissue culture techniques provide the most efficient and reliable method for multiplication and conservation of such plant [Zahoor A et. al. (2013)]. In vitro propagation methods deliver powerful methods for the mass multiplication and germplasm conservation of economically important species [Snyman et al., (2000)]. Previously [Babber & Kulbhushan (1991)] in an experiment got callus from root hypocotyls and cotyledonary segments of Viola tricolor but unsuccessful to get shoot formation. In a report [Sato et al., (1995)] managed regeneration of plantlets from petiole callus of wild Viola. In yet another report [Wijowska et al., (1999)] achieved callus independent endosperm and root In vitro by culturing unfertilized ovules of Viola odorata L. There are various reports about the breeding, inheritance, cultivation and regeneration of Viola odorata L., however to date there have 1 ISBN:

2 been no reports on the successful In vitro callogenesis and organogenesis of Viola odorata L. using leaf explant [Rupinder & Ramash, (1998)]. MATERIALS AND METHODS Collection of plant material The plant Viola odorata L. was collected from Sanjeevini vatika of Department of Horticulture, UAS, GKVK, University of Agriculture Science Bangalore - 65, India. Explants preparation The explants used in the present study were (Leaves and apical buds) as these preformed buds are the potential source for the large scale clonal propagation of the plants. The green plant parts ware used as explants (leaf, petiole). The excised leaf or petiole was washed in tap water with laboline for 15 minutes then explants were sterilized by 70% alcohol then treated with 0.1% HgCl2 for 1 min followed by several time washing with DDW under aseptic condition. Media Preparation For inoculation of explants the MS media [Murashige & Skoog (1962)] was prepared supplemented with cytokinins and auxins like NAA, IAA, Kin, BA and 2,4-D. ph of the medium was adjusted at 5.8 and prior to autoclaving, added 0.6% agar agar for solidification (Himedia, Mumbai), then autoclaving at 15psi for 30mins of MS media were carried out. Table 1. Different combinations of hormones with media code used for callus induction of Viola odorata L. Sr. Media code Growth hormone Concentration (mg/l) No. 1 A BA+2,4-D B BA+2,4-D C BA+2,4-D D BA+2,4-D E BA+2,4-D F BA+2,4-D G BA+2,4-D H BA+2,4-D I BA+2,4-D J BA+2,4-D K IAA+Kin L IAA+Kin M IAA+Kin+BA N IAA+Kin+BA O Kin+NAA+IAA Inoculation After complete preparation of MS media the leaves and petiole were cut into small pieces of 1 cm in size rectangular or oval in shape with the help of sterilized scissor were inoculated on basal medium with the help of forceps in the laminar air flow chamber. 2 ISBN:

3 Culture Conditions The cultures were incubated under at 25 ± 2ºC & light (16 hours light and 8 hours dark). Details regarding quantity of callus color, type and number of to callus formation were observed and results were recorded. RESULTS AND DISCUSSION Callus induction was observed on 10 combinations (A,B,C,D,E,F,G,H,I,J) out of the total 15 combinations used for callus culture remaining 5 media code (K,L,M,N,O) didn t get the results. The colour, size, texture and number of were recorded. Best and efficient callus induction was occurred on H media code, which was after 12 of incubation having BA + 2,4-D ( mg/L) and the callus was observed to be green Crystalline (fig E). Table: Data of callus induction on MS media with various concentrations of growth hormones. Sr.No. Media code Growth hormones Concentration (mg/lit) Colour Texture Size Days 1. A BA+2,4-D Yellow Compact small B BA+2,4-D Brown Compact Small C BA+2,4-D White Granulated Small D BA+2,4-D Whitish Compact Large E BA+2,4-D Pale Granulated small 70 yellow 6. F BA+2,4-D Dark Brown Crystalline small G BA+2,4-D Brown Compact Medium H BA+2,4-D Green Granulated Medium I BA+2,4-D Dark Crystalline Large Brown 10. J BA+2,4-D Brown Crystalline Large 30 On D media code having BA+2, 4-D (3+0.25mg/L) induced large size callus occurred with compact and whitish in colour (fig F). F media code having BA+2, 4-D (2.5+2 mg/l) induced small sized and dark brown crystalline colour callus was form after 30 (fig D). 3 ISBN:

4 Fig:A Fig:B Fig:C Fig:D Fig:E Fig:F Fig:G 4 ISBN:

5 Fig:H Fig:I Figs A-I Photo plates showing different stages of callus from leaf and petiole as a explants. A. Flower bearing plant Viola. odorata L. B. Uprooted plant. C, H and I. Basal callus formation with root hairs from petiole. D, E and F. Induction of callus from leaf. And G. Basal callus formation from petiole. C media code having BA+2,4-D ( ) induced small sized callus, granulated and white in colour after 90 and root formation occurred after 110 (Fig.I). REFERENCES 1. Babber, S and S. Kulbhushan Study of anatomy of vitrified structure in Viola tricolor L. Ann. Biol. (Ludhiana), 7(1): Ghani UK, Saeed A, Alam MT. Indusyunic Medicine. Department of Pharmacognosy, University of Karachi,1997; pp Murashige,T.and F.skoog (1962). A revised medium for rapid growth and bioassay with tobacco tissue cultures. physiol. Plant. 15: Ravarut M., Anghel GH., Buia AL., Niarady A., Morlova Irina Botanică. Edit. Did. Pedag., Bucureşti, 459 pp. 5. Rupinder, K. and K. Ramesh Effect of nitrogen, phosphorus and plant spacing on seed yield in pansy (Viola tricolor) cv Swiss Giant. J. Ornamental Hort. New Series., 11: Sato, T., O. Kwon, H. Miyake, T. Tangiguchi and E. Maeda Regeneration of plantlets from petiole callus of wild Viola (Viola patrinii DC.). Plant Cell Rep., 14: Savulescu T., Beldie AL., Buia AL., Grintescu GH., Grintescu I., Gusuleac M., Nyarady A., Nyarady E.I., Ravarut M., Şerbanscue I., Ţopa E., Zahariadi C Flora R.P.R, vol. III, Edit. Acad. R.P.R., pp.) 8. Siddiqi HS, Mehmood MH, Rehman NU, Gilani AH. Studies on the antihypertensive and antidyslipidemic activities of Viola odorata leaves extract. Lipids in Health and Disease, 2012; 11: Snyman, S.J., M.P. Watt, F.C. Huckett and B.I. Botha Direct somatic Embryogenesis for rapid, cost effective production of transgenic Sugercane ( Saccharum ssp. Hybrids). Pro. South Africa. Suger Technol. Assoc., 74: Wijowska, M., E. Kuta and L. Przywara In vitro culture of unfertilized ovules of Viola odorata L. Acta Biol Cracov Ser. Bot., 17: Zahoor. A. Kaloo, Rukhsana Akhtar, Zafrul-Haq and B. A. Wafai. (2013) Effect of growth regulators on the in vitro multiplication of Viola odorata. Int. Jour.of medicinal plant research.issn X Vol.2 (4), pp ISBN: