Plant Biotechnology
LABORATORY EXERCISE Preparation of MS Medium from Stock Solutions Introduction Murashige and Skoog medium (MS) is the most suitable and most commonly used basic tissue culture medium for plant regeneration from tissues and callus. It was developed by Toshio Murashige and Folke K. Skoog in 1968; based primarily on the mineral analysis of tobacco tissue. This is a “high salt” medium due to its content of K and N salts. Kiss et al. (1995) indicated shoot proliferation of pineapple Ananas comosus was achieved on MS medium with low levels of some inorganics and vitamin supplements. The shoots after transfers to MS medium containing NAA or IBA gave good rooting. They also reported the in vitro axillary bud formation on the etiolated shoots and their rooting on hormone-free MS medium. Litchi, one of the important subtropical fruit crop of Southeast Asia, has been successfully regenerated in vitro using cotyledon, hypocotyls, and root segments on MS medium supplemented with 0.05 mg/L NAA, 0.05 mg/L 2-Ip, 0.2 mg/L ABA, and 45 g/l sucrose (Chandra (Chandra and Padaria, 1 999). Objectives 1. To improve how to prepare Murashige and Skoog (MS) culture medium. 2. To improve how to calculate quantity required from the stock solutions for a given concentration concentration and volume of MS medium. Materials and Methods Beakers (1000, 500, and 100 ml); graduated cylinders (1000, 500, and 100 ml); conical flasks (100 ml); reagent bottles/Schott bottles (1000, 500, and 100 ml); micropipettes (1000 µl); distilled water; magnetic stirrer; spatulas; chemical balance; weighing boat; tissue; pH meter; aluminium foil; autoclave machine; and chemicals needed: stock solutions of macronutrient; micronutrient; iron source; vitamins; plant growth regulators such as BAP and NAA; sucrose; myo-inositol; agar; and NaOH and HCl to adjust pH. Methodology 1. Five hundred millilitres of MS medium was made. The required volume of each stock solution were calculated calculated and obtained into 500 ml beaker on a magnetic stirrer. 2. 15 g sucrose and 0.05 g myo-inositol were added, and then stirred until fully dissolved. 3. The volume was adjusted to about 500 millilitres with distilled water. 4. 2 ml (2000 µl) of NAA was added (to get 4 mg/L NAA) then pH was adjusted to 5.75 ± 0.05 with NaOH and HCl. 5. 3.1 g of agar was added and stirred for complete mixing. 6. The medium was heated up in microwave oven to dissolve the agar. 7. Medium was transferred into 500 ml Schott bottle. Bottle was labelled before autoclaving. 8. The culture medium medium was autoclaved for 15-20 min at 121°C. 9. After autoclaved, medium was distributed approximately 20 ml to each sterile disposable petri dishes in the laminar air flow and medium are allowed to solidify followed by exposing to UV light for 20 minutes and stored for use.
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Plant Biotechnology
Results Calculation for preparation preparation of 500 ml MS medium from stock solutions Formula - M 1V1 = M2V2 M1 = Concentration of the stock V1 = Volume will be taken from the stock (x) M2 = Concentration of MS medium V2 = Volume of medium = 500 mL Component
Macronutrient
Micronutrient
Ferum/iron source
Vitamin
NAA
Calculation
M1V1 = M2V2 (10) (x) (x) = (1) (500 mL) x = 500/10 = 50 mL from stock M1V1 = M2V2 (1000) (x) = (1) (500 mL) x = 500/1000 = 0.5 mL from stock M1V1 = M2V2 (100) (x) = (1) (500 mL) x = 500/100 = 5 mL from stock M1V1 = M2V2 (1000) (x) = (1) (500 mL) x = 500/1000 = 0.5 mL from stock M1V1 = M2V2 (1000) (x) = (4) (500mL) x = 2000/1000 = 2 mL from stock
Amount taken for 500 ml MS medium
50 mL
0.5 mL
5 mL
0.5 mL
2 mL
Discussion Comparing MS medium with other culture medium MS medium - one of the most successful media - was formulated formulated by analyzing the inorganic components components in tobacco plants and then adding them to medium in amounts similar to those found in the plants. It has comparatively high salt levels compared to White’s medium (White, 1963) but lower than B5 medium (Gamborg et al., 1968). The type of tissue culture medium selected depends on the species to be cultured. According to Andreu and Marin insititia (2005), WPM was the medium that promoted a better establishment of the Prunus insititia cultures, but MS supported higher multiplication rates. In contrast, MS was better than WPM for both explant establishment and multiplication of chokecherry Prunus virginiana (Zhang et al ., ., 2000), and mature wild cherry (Hammatt and Grant, 1997) Carbon sources In tissue culture, plants lose their ability to synthesize their own food, thus they depend on external factors to make energy. Sucrose as a carbon source is known to be the best. This is because it is cheap and easily available. Added to that, sucrose can be autoclaved along with
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Plant Biotechnology
the media, which is preferred over filter sterilization. Sucrose hydrolyses during autoclaving into more efficiently utilized energy source, such as fructose and glucose. Sucrose is essential for various metabolic activities. It is needed for differentiation of xylem and phloem in cultured cells. Sucrose is cheaper than glucose and has a more positive water potential. However sucrose hydrolysis during autoclaving is dependant on pH factors, and it does not occur at pH settings of 6.0 (George et al., 2007). Sucrose is less effective for monocot plants (Bhojwani and Razdan, 1996). Sucrose can also pose as a limiting factor to growth in certain cultures. The presence of sucrose in the media specifically inhibits chlorophyll formation and photosynthesis, thus it endures a less feasible autotrophic condition. Sucrose may not promote organogenesis as good as glucose. Alternative types of carbon source available; respectively in decreasing order are glucose, maltose and raffinose. Fructose is less effective and mannose and lactose were the least suitable. Other types of carbon source are galactose, cellobiose, melibiose, and trehalose, but these carbon source show inferior results as compared to sucrose. Agar Agar is the most commonly used gelling agent. When agar is mixed with liquid, it forms a gel that melts at about 100°C and solidifies at about 45°C. It is highly stable, clear, non-toxic and resistance to metabolism during culture (Henderson and Kinnersley, 1988; Sahay, 1999). All agars contain impurities, such as inorganic salts, organic compounds, phenolics, and long chain fatty acids: amounts and types vary depending on the manufacturer. Interactions were found between agar performance performance and plant species and regeneration processes. Agar quality could affect, in principle, all developmental processes, where the regeneration of adventitious adventitious shoots and roots being the most sensitive (Scholten and Pierik, 1998).
The concentration of agar may be critical to plant response in culture. Stoltz (1971) described the effects of concentrations of agar on the growth of mature Iris embryos. Somatic embryos of Picea (Tremblay and Tremblay, 1991), shoot apical meristems of Picea (Romberger and Tabor, 1971), protoplasts of red cabbage (Koda et al., 1988), and anther cultures of tobacco (Kohlenbach and Wernicke, 1978) have been shown to be sensitive to agar quality. The agar concentrations concentrations commonly used in plant culture culture media range between 0.5% and 1%. Scholten and Pierik (1998) reported that no relationship could be found between the price and quality of the agars. The performance of agars can be improved by washing agars before use (Shillito et al., 1983) When greater purity is needed, agarose may be used. Because of the additional purification (Johansson, 1988), agarose is considerably more expensive than agar (Kao, 1981). Medium solidified with Gelrite has the advantage of being clear, which agar-solidified medium is not. Consequently contamination is more easily detected at an early stage (Anonymous, 2006a). Gelrite requires more stirring than agar when being added to media. Phytagel is an agar substitute produced from a bacterial substrate but results in clumping. Therefore, to
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Plant Biotechnology
prevent clumping, phytagel should be added to rapidly stirring culture medium at room temperature temperature (Anonymous, 2006b) The selection of a gelling agent for specific plants is generally empirical. For unknown reasons, tissues of some species grow more vigorously on one gelling agent than on another. Podwyszynska (1994), who done a research on species belonging to the Araceae and Liliaceae families - e.g. Homalomena sp. and Cordyline sp.; described that macroelements availability and uptake depends on the gelling agent and varies between species. However, gelling agent did not influence the multiplication rate of any species significantly significantly (Podwyszynska and Olszewski, 1995). Myo-inositol Myo-inositol, is the only one of the nine thereotical stereoisomers of inositol has significant biological importance. It has been classed as the member of Vitamin-B complex and is required for the growth of cells in tissue culture. Myo-inositol has been classified as plant “vitamin”. Several research states that presence of myo-inositol in culture media initiates the cell division process. For example, Kaul and Sabharwal (1975) states that formation of shoot buds on the callus of Haworthia spp. was shown to be dependant on the availability of myo-inositol. Adjusting pH Adjusting of pH is necessary for the addition of agar. This is necessary for the agar to set properly. At pH below 5.5 the agar will not gelled properly while at above 6.0, the agar will become too firm (Debergh, 1983). Also the required pH is necessary to support different phases of growth and differentiation. pH was adjusted by adding NaOH or HCL. Conclusion From this experiment, we become aware and understood on how to improve preparation of MS medium and the significance of each component in MS medium. References Andreu, P. and Marin, J.A. 2005. In Vitro Culture Establishment and Multiplication of the Prunus Rootstock ‘Adesoto 101’ (P. insititia L.) as Affected by the Type of Propagation of the Donor Plant and by the Culture Medium Composition. Scientia Horticulturae. 106 (2): 258-267.
Anonymous. 2006a. PhytoTechnology Lab Catalogue . PhytoTechnology Laboratories Inc, Shawnee Mission, Kansas, USA. Anonymous. 2006b. Plant Culture Catalogue . Sigma Aldrich Chemical, St. Louis, Missouri, USA. Bhojwani, S.S. and Razdan, M.K. 1996. Plant Tissue Culture: Theory and Practice. Elsavier, Netherlands. Chandra, R. and Padaria, J.C. 1999. Litchi Shoot Bud Culture for Micropropagation. Journal of Applied Horticulture. 1(1): 38-40.
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Debergh, P.C. 1983. Effect of Agar Brand and Concentration on the Tissue Culture Media. Physiologia Plantarum. 59(4): 270-276. Dekhuijizen, H.M. 1971. Sterilization of Cytokinins. In Bragt, J.V., Mossel, D.A.A., Pierik, R.L.M. and Veldtra, H. (eds.), Effects of Sterilization on Components in Nutrient Media. pp 129-139. Kniphorst Scientific, Wageningen, Netherlands. Netherlands. Gamborg, O. L., Miller, R. A., and Ojima, K. 1968. Nutrient Requirements of Suspension Cultures of Soybean Root Cell . Experimental Cell Research . 50 (4): 151-158. Gray, D.J. and Trigiano, R.N. 2000. Plant Tisue Culture: Concepts and Laboratory Exercises . CRC Press, New York, USA. George, E.F., Hall, M.A, and De Klerk, G.J. 2007. Chapter 4. In Thorpe, T.A. (ed.), Plant Propogation by Tissue Culture Third Edition. pp 115-174. Springer, New York, USA. Hammatt, N. and Grant, N.J. 1997. Micropropagation of Mature British Wild Cherry. Plant Cell, Tissue, and Organ Culture . 47 (5): 103-110. Henderson, W.E. and Kinnersley, A.M. 1988. Corn Starch as an Alternative Gelling Agent for Plant Tissue Culture. Plant Cell, Tissue, and Organ Culture . 15 (1): 17-22. Johansson, L.B. 1988. Increased Induction of Embryogenesis and Regeneration in Anther Cultures of Solanum tuberosum. Potato Research. 31: 145-149. Kao, K.N. 1981. Plant Formation from Barley Anthers Cultured with Ficoll Media. Z. Pflanzenphysiol . 103 (2): 437 –443. Kaul, K. and Sabharwal, P.S. 1975. Morphogenetic Studies On Haworthia: Effects of Inositol On Growth And Differentiation. Differentiation. American Journal of Botany . 62: 655-659. Kiss, E.J., Kiss, G., Gyulai, K., and Heszky, L.E. 1995. A Novel Method for Rapid Micropropagation Micropropagation of Pineapple. Horticultural Science. 30(1): 127-129. 127 -129. Kumar, A. 1936. Recent Advances In Plant Biotechnology and Its Applications . I.K. International International Publishing House, New Delhi, India. Koda, T., Ichi, T., Yamagishi, H., Yoshikawa, H. 1988. Effects of Phytohormones and Gelling Agents on Plant Regeneration from Protoplasts of Red Cabbage. Agricultural Biology and Chemistry . 52 (9): 2337-2340. Kohlenbach, H.W. and Wernicke, W. 1978. Investigations on the Inhibitory Effect of Agar Pflanzenphysiol . 86 (5): 463and the Function of Active Carbon in Anther Culture. Z. Pflanzenphysiol 472.
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Podwyszynska, M. 1994. Effect of Growth Regulators on Rooting and Acclimatization of Miniature Rose Cultured In Vitro . PhD Thesis. Research Institute of Pomology and Floriculture, Floriculture, Poland. Podwyszynska, M. and Olszewski, T. 1995. Influence of Gelling Agents on Shoot Multiplication and the Uptake of Macroelements by In Vitro Culture of Rose, Cordyline and Homalomena. Scientia Horticulturae. 64 (2): 77-84. Razdan, M.K. 2003. Introduction to Plant Tissue Culture. Science Publishers, New York, USA. Romberger, J.A. and Tabor, C.A. 1971. The Picea abies Shoot Apical Meristem in Culture; Agar and Autoclaving Effects. American Journal of Botany . 58 (2): 131-140. Sahay, S. 1999. The Use of Psyllium (Isubgol) as an Alternative Gelling Agent for Microbial Culture Media. World Journal of Microbiology and Biotechnology . 15 (2): 733-735. Scholten, H.J. and Pierik, R.L.M. 1998. Agar as a Gelling Agent: Differential Biological Effect In Vitro. Scientia Horticulturae. 77 (2) 109-116. Shillito, R.D., Paszkowski, J., and Potrykus, I. 1983. Agarose Plating and a Bead Type Technique Enable and Stimulate Development of Protoplast-derived Colonies in a Number of Plant Species. Plant Cell Report . 2(5): 244-247. Stoltz, L.P. 1971. Agar Restriction of the Growth of Excised Mature Iris Embryos. Journal of American Society of Horticultural Science . 96 (5): 618-684. Tremblay, L. and Tremblay, F.M. 1991. Effects of Gelling Agents, Ammonium Nitrate, and Light on the Development of Picea mariana (Mill) BSP. (black spruce) and Picea rubens Sarg. (red spruce) Somatic Embryos. Plant Science. 77(2): 233-242. White, P. R. 1963. A Handbook of PIant Tissue CuIture . Jacques Cottell, Pennsylvania, USA. Zhang, Z., Dai, W.H., Cheng, Z.M., and Walla, J.A. 2000. A Shoot Tip Culture Micropropagation Micropropagation System for Chokecherry. Journal of Environmental Horticulture . 18 (2): 234-237.
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