NSave Nature to Survive

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1 ISSN: NSave Nature to Survive : Special issue, Vol. VI: : 2014 AN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES STANDARDIZATION OF PROTOCOL FOR IN VITRO MULTIPLICATION OF ORCHIDS Abhinash Moirangthem et al., KEYWORDS In vitro NAA BAP PLBs Proceedings of National Conference on Harmony with Nature in Context of Environmental Issues and Challenges of the 21 st Century (HORMONY ) November 28-0, 2014, Udaipur, organized by Department of Environmental Sciences, Faculty of Earth Sciences M. L. Shukhadia University, Udaipur (Rajasthan) in association with National Environmentalists Association, India 221

2 NSave Nature to Survive QUARTERLY ABHINASH MOIRANGTHEM 1 *, N. INDRAKUMAR SINGH 2 AND S. S. PATIL 1 Department of Floriculture, Medicinal and Aromatic Plants, U.B.K.V., Pundibari, Coochbehar , West Bengal, INDIA 2 Scientist, Biotech Park, Imphal , Manipur, INDIA Department of Horticulture, University of Agricultural Sciences, Dharwad , INDIA maddynash12@gmail.com ABSTRACT The investigation was carried out at College of Agriculture, University of Agricultural Sciences, Dharwad during the year to find out the effect of different treatment combinations for in vitro multiplication of PLB in terms of number of shoots per explants, length of shoot, rooting percentage, number of roots, etc. Eight treatments and their effects on multiplication and preservation of PLBs were measured based on different parameters. Results revealed that all treatments for all the parameters and among the treatments for proliferation, T 5 with BAP at 2.0 mg/l exhibit the maximum number of shoots proliferation, shoot length, fresh weight and dry weight. In case of root proliferation T 4 with NAA at 1.5 mg/l exhibited the highest rooting percentage, number of roots and length of roots. T 4 with 1/ 2 M.S. + BAP 5 mg/l proved to be the most efficient treatment for in vitro preservation (IVP) in terms of number of PLBs developed, number of plantlets per culture respectively. The maximum PLBs development was found to be highest at 150 days of IVP, thereby reducing trend in PLBs development was visible in subsequent days of preservation. Significant results of IVP were observed in both treatments. Highest cost benefit ratio for shoot proliferation and root proliferation were observed in T 5 (1:8.10) and T 4 (1:8.29) respectively. Therefore, it is concluded that T 4 treatment may be recommended for in vitro multiplication, preservation and regeneration in future use. INTRODUCTION Cymbidium is one the most important genera of the family orchidaceae, the largest flowering plants among monocotyledons. Nearly, 100 are estimated to be naturally available in India, of which, 600 species are available in the north-eastern region of India (Rao, 1979). Cymbidium alone has 70 species of various terrestrial, lithophytic or epiphytic plants. It grows well under temperate condition in the north-eastern Himalayan region. as the climate of this region offers a congenial growing condition for commercial purpose on a profitable basis. This reduced the cost of production which otherwise needed to be incurred on provision of optimum environment and also ensures better flower quality for cut flowers. States like Sikkim and Darjeeling district of West Bengal have already ventured out successfully in Cymbidium cut flower production and are front runners in production and export in India. Other than its cut flower value, the Cymbidium elegance is used in local medicine for the treatment of nervous disorders, in colouring as dye and the pseudo bulb of some Cymbidium species are used as food by some tribes of Manipur. So far very limited work has been carried out with regards to in vitro preservation and regeneration of protocorm like bodies through modified culture medium method, the concept being new, contributes to slow growth system of in vitro preservation. This technique would be beneficial in providing ready to serve propagating materials at any time of the season and could be successfully adopted in germplasm conservation. Since the PLBs were obtained from shoot tip, a true to type planting materials can be generated as and when required. Saradvathi, et al, (2009) established a standardized protocol for rapid, reproducible protocol for regeneration of Talinum cuneifolium through mature nodal explant culture. Callus induction was also reported in coconut milk supplemented media and in combination of different Phytohormones like 2, 4-D + IAA, 2, 4-D + IBA, IAA + KN, 2, 4-D + KN. Best result was observed in 2, 4-D in.0 ppm ppm (Kumari and Pandey, 2010). Physiological state of the explant is one of the factors for morphogenic response to the culture medium (Mahato et al., 2009). Keeping the ever increasing demand for cut flowers from innumerable hybrids and inexhaustible varieties and simultaneously the ever changing taste of the people, the need to maintain the stock was realized for future use. This is most efficiently possible through in vitro preservation wherein supervision, maintenance and space requirement is comparatively less as compared to ex situ preservation besides having many advantages. Therefore, the present study on In vitro multiplication and preservation of protocorm like bodies of Cymbidium rivulux Cooks Bridge through modified culture medium was undertaken with an objective to standardize the protocol for in vitro multiplication of orchids. MATERIALS AND METHODS *Corresponding author The present experiment on was conducted in the Tissue Culture Laboratory of the Department of Horticulture, College of Agriculture, University of Agricultural Sciences, Dharwad during the year The details of material used and 222

3 STANDARDIZATION OF PROTOCOL methods followed are presented below. Nutrient media The nutrient media used in the present study was the Murashige and Skoog media (Table 1). For preparing the nutrient media, different stock solutions were prepared along with the stocks of growth regulators- Stock A: Macro salts; Stock B: Micro salts; Stock E: Vitamins. a) Kinetin-1000 ppm - For 1000 ppm stock solution, 25 mg of kinetin was weighed and the weighed kinetin was transferred into a beaker. Kinetin was then dissolved by adding of few ml of 0.1 N or 1 N HCl. The final volume of the solution was made up to 25 ml by adding distilled water. Then labeled and stored in a refrigerator. b) 6-Napthalene acetic acid-0.1 ppm (stock solution) - For 0.1 ppm stock solution, 10 mg of NAA was weighed and dissolved by adding ethyl alcohol drop by drop till NAA completely dissolves. Then, added few drops of 1 N sodium hydroxide which increases the solubility of NAA. The final volume was made up to 100 ml by adding distilled water there on, stored in a stock solution bottle, labeled and placed in freezer/ refrigerator. c) Abscisic acid (ABA) ppm stock solution - ABA, 600 mg was weighed and it was dissolved by adding few drops of ethyl alcohol. The final volume was made upto 50 ml. The amber bottle containing stock solution was labeled and stored under refrigerated condition; 1 ml/l of solution gave 6 ppm. d) 6-benzyl aminopurine ppm of BAP stock solution, 500 mg of BAP was measured and poured into a 100 ml glass beaker and dissolved in 1 N hydrochloric acid. The final volume was made upto 50 ml by adding distilled water. It was labeled and stored under refrigerated condition, 1 ml/l of solution gave 5 ppm. While preparing individual medium, using stocks, required amount of stock solutions were added along with sucrose and by adding distilled water the volume was made up. The ph was checked and adjusted to 5.6 to 5.8 with the help of 0.1 N HCl or 0.1 N NaOH. The volume was finally made up and the required amount of agar was added into the medium. Agar in the medium was completely melted by gentle heating upto 90 o C ml of medium was poured into 25x150 mm pre-sterilized glass culture bottles. These bottles were then autoclaved. Additive materials a) Calcium chloride, Activated charcoal-1 g/l, Agar-agar- 0.8%, ready made Murashige and Skoog medium -1 g/l and MS plant salt mixture. RESULTS AND DISCUSSION In vitro multiplication of protocorm like bodies (PLBs) The effect of different treatments on in vitro multiplication of PLBs based on the proliferation of shoot and initiation of root exhibited by the in vitro cultures is given here under with appropriate headings. Number of shoots per explants Effect of different concentrations of BAP with the number of shoots per explants (Table 2) yielded maximum shoot number (5.) at 2 mgl -1 from T 5 followed by T 4 at 1.5 mgl -1 and T at 1.0 mgl -1 with 4.50 and 4.0, respectively. Lowest number of shoot was obtained from T 8 at.5 mgl -1 (1.90). Length of shoots per explants Significant differences were observed among the treatments as depicted in the Table 2 and plate 5 for average length of shoot per explants. Longest shoot was exhibited by T 5 (4.52 cm) at 2 mgl -1 followed by T 4 and T with 4.45 cm at 1.5 mgl-1 and 4.08 cm at 1.0 mgl -1, respectively. Minimum shoot length was recorded in T 1 with 2.68 cm. The type and concentration of growth regulators are an initial consideration for micropropagation of orchid species (Genkov and Ivanova, 1995). Addition of cytokinin to the medium increase the number of shoots which demonstrates the significance of exogenous cytokinin to enhance the multiple shoots. BAP influences shoot proliferation by stimulating quick cell divisions to induce large number of multiple shoots (Yakimova et al., 2000; Roy and Banerjee, 2002). Yakimova et al. (2000) demonstrated that the culture plants producing large number of shoots exhibit minimum shoot length because all the nutrients are utilized for the formation of lateral shoots. From the available data of shoot proliferation there were significant differences among the treatments with respect to shoot number. Maximum number of shoot was observed in the treatment T 5 (MS + BAP 2.0 mg/l) and T 4 (MS + BAP 1.5 mg/l). This might be due to the presence of BAP which is a growth promoting hormone. Results were also supported by the findings of Roy and Banerjee (2002) who reported BAP enhances the shoot multiplication more actively. BAP provided increase in number of axillary buds. BAP showed the best elongated shoots in T 5 followed by T 4. As such, the results coincide with the findings of Sirchi et al. (2008) that BAP Table 1: Composition of murashige and skoog media (1962) Components Murashige and skoog media (mg) Macro-elements NH 4 NO 1650 KNO 1900 CaCl 2.2 H 2 O 440 MgSO 4.7 H 2 O 70 KH 2 PO Micro-elements KI 0.8 H BO 6.2 MnSO 4.4 H 2 O 22. ZnSO 4.7 H 2 O 8.6 Na 2 MoO 4.2 H 2 O 0.25 CuSO 4.5 H 2 O CaCl 2.6 H 2 O Na 2.6H 2 O FeSO 4.7 H 2 O 27.8 Vitamins and organics to be added Myo-Inositol 100 Nicotinic acid 0.5 Pyridoxine HCl 0.5 Thiamine HCL 0.1 Glycine 2.0 Sucrose 0 g Agar 8 g ph

4 ABHINASH MOIRANGTHEM et al., actively stimulates the nutrient mobilization from source to sink areas due to which it act as a triggering element to increase the length of micro shoots. Higher concentration of BAP than the specific limit of concentration caused a reduction in their number and their length of shoots. Roy and Banerjee (2002) reported that application of exogenous cytokinins at optimal levels have inhibitory impacts on shoot length and number. Similar findings were also reported by Saradvathi, et al. (2009). Fresh weight of the culture plant per treatment (mg) Fresh weight of the culture plant was recorded after fulfilling the stipulated conditions of the culture as mentioned in Chapter III. Significant differences were observed among the treatments in terms of fresh weight (Table 2). T 5 recorded the maximum fresh weight of mg followed by T 4 and T with mg and 62.5 mg respectively. Lowest response was associated with T 1 with mg. Dry weight of the shoots (mg) Observation on dry weight is furnished in Table 2. Significant differences were observed among the treatments with respect to dry weight. Highest dry weight was obtained from T 5 with mg followed by T 4 and T with mg and 5.22 mg. Lowest dry weight was associated with T 8 (1.99 mg). Fresh and dry weight, due to biomass accumulation of cytokinin, increased number of leaves, shoots and shoot length by stimulating cell division and elongation through mobilization (Peres et al., 2001). In the present study fresh and dry weights of proliferated shoots gave highest weight in treatment T 5 (MS + BAP 2.0 mg/l) and T (MS + BAP 1.5 mg/l). 4 According to Vulysteker et al. (1997), fresh weight is dependent on the shoot number and it might decrease due to reduced number of shoot. George et al., (2008) demonstrated that supra-optimal concentrations of BAP illustrated the deleterious effects and reduced the fresh as well as dry weights of proliferated shoots. Higher concentrations of BAP are responsible for elevating the ethylene production causing senescence in plant tissues, and it subsequently gives smaller fresh weights. Rooting percentage on MS with various concentrations of NAA Significant differences were observed among the treatments as depicted in the Table for rooting percentage of culture. Highest rooting percentage was observed in T 4 with 85.0 % at 1.5 mgl -1 followed by T and T 5 with 75.0% at 1.0 mgl -1 and 67.0% at 2 mgl -1, respectively. Lowest rooting percentage was observed in T 8 with 44.7 % at.5 mgl -1. Auxins are considered as the efficient plant growth regulators which accelerate the processes of root induction and development by differentiation of vascular bundles. These auxins may not have direct effect on the development of shoots, but may be effective mostly through induction of roots (Husen and Pal, 2007). In the present study, highest rooting percentage was recorded in T 4 (MS + NAA 1.5 mg/l) and T (MS + NAA 1 mg/l). This might be due to the presence of NAA as a growth promoting hormone. The results were supported by findings of Henrique et al. (2006) that NAA promote rooting in the plants through changes in the biological systems of the plants. Number of roots per plant Table and plate 6 with respect to average number of roots per plant showed significant differences. T 4 showed higher potential with 4.0 at 1.5 mgl -1 followed by T and T 5 with.5 Table 2: Effect of different concentration of BAP on average number of shoots, length (cm) and fresh as well as dry weight (mg) of orchid Cymbidium rivulux Treatment Number of shoots per explants Shoot length (cm) Fresh weight of shoots (mg) Dry weight shoots (mg) T 1 - MS T 2 - MS + BAP 0.5 mg/l T - MS + BAP 1.0 mg/l T 4 - MS + BAP 1.5 mg/l T 5 - MS + BAP 2.0 mg/l T 6 - MS + BAP 2.5 mg/l T 7 - MS + BAP.0 mg/l T 8 - MS + BAP.5 mg/l Mean SEm ± CD (0.01) CV Table : Effect of different concentration of NAA on average rooting percentage root number and length (cm) of Cymbidium rivulux Treatment (mg/l) Rooting percentage Number of roots Length of roots (cm) T 1 - MS T 2 - MS + NAA 0.5 mg/l T - MS + NAA 1.0 mg/l T 4 - MS + NAA 1.5 mg/l T 5 - MS + NAA 2.0 mg/l T 6 - MS + NAA 2.5 mg/l T 7 - MS + NAA.0 mg/l T 8 - MS + NAA.5 mg/l Mean SEm ± CD (0.01) CV

5 STANDARDIZATION OF PROTOCOL at 1 mgl -1 and.25 at 2 mgl -1, respectively. Lowest number of roots per plant was produced by T 8 with 1.65 at.5 mgl -1. Length of the root (cm) Significant differences were observed among the treatments. As depicted in the Table for average length of the root per plant, longest root was exhibited by T 4 with.25 cm at 1.5 mgl -1 followed by T and T 5 with.10 cm at 1 mgl -1 and.07 cm at 2 mgl -1. Lowest length of root was obtained from T 8 with 1.65 cm at.5 mgl -1. Auxin application to micro shoots is said to intensify the number of adventitious roots by increasing the level of endogenous contents of enzymes. They are considered to have an increased effect on cell division, elongation and differentiation (Husen and Pal, 2007). Han et al. (2009) revealed that auxins induces the sprouting of shoot buds which then stimulates the growth substances present in the roots for their growth and elongation. In the present study, highest root number and root length was observed in T 4 (MS + NAA 1.5 mg/l) and T (MS + NAA 1 mg/l) this might be due, to the presence of NAA as it promotes the number and length of roots by enhancing cell division in the root primordial. The findings were in conformity with the reports of Rout (2006). Higher concentrations of NAA were found to have a reduced number and length of roots than the optimum level. Ozel et al. (2006) reported that higher levels of NAA applied to plants, inhibits the formation of shoot buds and this might further stop the production of roots as the auxin in the root primordial is shifted from the shoot apex. All the treatments tested for in vitro multiplication of PLBs yielded significant results with respect to the parameters considered for investigation. Among the treatments for shoot proliferation, T 5 with BAP at 2.0 mg/l exhibited the maximum number of shoots proliferation, shoot length, fresh weight and dry weight. In case of root proliferation T 4 with NAA at 1.5 mg/ l exhibited the highest rooting percentage, number of roots and length of roots. REFERENCES Genkov, T. and Ivanova, I Effect of cytokinin active phenylurea derivatives on shoot multiplication, peroxidase and superoxide dismutase activities of in vitro cultured carnation. Bulgaria Journal of Plant Physiology. 21: 7-8. George, E. F., Hall, M. A. and Deklark, G. J Plant propagation by tissue culture. Springer. 1: Han, H., Zhang, S. and Sun, X A review on the molecular mechanism of plant rooting modulated by auxin. African J. Biotechnol. 8: Henrique, A., Campinhos, E. N., Ono, E. O. and Pinho, S. Z. D Effect of plant growth regulators in the rooting of pinus cuttings. Brazil Arch. Biol Tech. 49: Husen, A. and Pal, M Metabolic changes during adventitious root primordium development in Tectona grandis Linn. F. (teak) cuttings as affected by age of donor plants and auxin (IBA and NAA) treatment. New Forests. : Kumari, S. and Pandey, R. K In vitro germination and callus induction from seeds of Carthamus tinctorius L. The Bioscan. 5(2): Mahato, S., Mehta, A. and Pandey, R. K In vitro regeneration and callus formation from different parts of seedling of Plantago ovate Forsk. The Bioscan. 4(1) : Ozel, C. A., Khawar, K. H., Mirici, S. Arslan, O. and Sebahattin, O Induction of ex vitro adventitious roots on softwood cuttings of centaurea tchihatcheffii Fisch. et. Mey using indol--butyric acid and á-naphthalene acid. Int. J. Agric. Biol. 1 : Peres, L. E. P., Majerowicz, N. and Kerbauy, E. G. B Dry matter partitioning differences between shoots and roots in two contrasting genotypes of orchids and their relationships with endogenous levels of auxins, cytokinins and abscicic acid. R. Bras. Fisiol. Veg. 1: Rao, A. S Orchid of India. National Book Trust, New Delhi, India. Rout, G. R Effect of auxins on adventitious root development from single node cuttings of Camellia sinensis (L.) kuntze and associated biochemical changes. Plant Growth Regul. 48: Roy, J. and Banerjee, N Rhizome and shoot development during in vitro propagation of Geodorum densiflorum (Lam.) Schltr. Sci. Hort. 94: Saradvathi, J., Reddy, K. and Savithramma, N High frequency in vitro shoots Multiplication of Talinum cuneifolium (Vahl) Willd. The BioScan. 4(): Sirchi, M. H. T., Kadir, M. A., Rashid, P. A. A., Rafat, A. and Javadi, M. B Plant regeneration as affected by plant growth regulators (PGR) in mangosteen (Garcinia mangostana L.). African J. Biotechnol. 7: Vulysteker, C., Leleu, O. and Rambour, S Influence of BAP and NAA on the expression of nitrate reductase in excised chicory roots. J. Exp. Bot. 48: Yakimova, E., Toteva, V. K., Groshkoff, I. and Ivanova, G Effect of BAP and CPPU on protease and á-amylase activity of in vitro cultured explants of Rosa hybrida L. Bulgarian J. Plant Physiol. 26:

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