Plant Biotechnology

LABORATORY EXERCISE Preparation of Stock Solutions for Plant Tissue Culture Introduction Murashige and Skoog (MS) medium was formulated in 1962 and the usage of this media is considerably high compared to other types of plant media. This is because MS medium was found to be the most suitable medium used for plant regeneration from tissues and calluses. MS medium was invented by plant scientists Toshio Murashige and Folke K. Skoog during Murashige's search for a new plant growth regulator. Initially, the media was produced based on mineral analysis of tobacco. Since the MS media contains high amount of nitrogen fixing agents, it was found out that the MS medium was equally compatible for other plant species as well. To have a complete form of MS media, the major nutrients required were the macronutrient, micronutrient, ferum, vitamins and in most cases, presence of plant growth regulators or hormones gives off a better result of root and shoots formation. And in high presence of these plant growth regulators, callus can be induced as well. Preparation of stock solutions for MS medium is a vital step in media preparation. Several nutrients must be prepared and stored in order to have a good working medium. Therefore, to have a working solution, the stock must be prepared beforehand. Objectives 1. To improve the preparation of stock solutions for MS culture medium. 2. To improve and calculate quantities of chemicals needed for given concentrations and stock volumes. Materials and Methods Beakers (1000, 500, and 100 ml); graduated cylinder (1000, 500, and 100 ml); conical flask (1000. 500, and 100 ml); reagent bottles (1000, 500, and 100 ml); distilled water; magnetic stirrer; spatulas; chemical balance; weighing boat; tissue; label and pen; and chemicals for macronutrient, micronutrient, iron source, vitamin, etc (according to table) 1. Each group was assigned and stock solutions were prepared. 2. Appropriate amount of each components were weight. Different spatula and weighing boat were used for different types of chemicals. 3. All chemicals for each stock solution were weight and dissolved in specified amount of distilled water using magnetic stirrer. 4. Stock solutions were labelled and date of preparation was indicated. 5. All stock solutions were stored in a chiller at 4°C.

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Plant Biotechnology

Results STOCK MACRO NH4NO3 KNO3 CaCl2.2H2O MgSO4.7H2O KH2PO4 MICRO KI H3BO3 MnSO4.H2O ZnSO4.7H2O Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O 1X (1 litre MS) g/L 1.6500 1.9000 0.4400 0.3700 0.1700 1X (1 litre MS) g/L 0.00083 0.00620 0.02230 0.00860 0.00025 0.000025 0.000025 1X (1 litre MS) g/L 0.03730 0.02780 1X (1 litre MS) g/L 0.0005 0.0001 0.002 0.0005 10X (g/800 mL) (1.65 x 10)(800/1000) = 13.2 g (1.90 x 10)(800/1000) = 15.2 g (0.44 x 10)(800/1000) = 3.52 g (0.37 x 10)(800/1000) = 2.96 g (0.17 x 10)(800/1000) = 1.36 g 1000X (g/100 mL) (0.00083 x 1000)(100/1000) = 0.083 g (0.00620 x 1000)(100/1000) = 0.62 g (0.02230 x 1000)(100/1000) = 2.23 g (0.00860 x 1000)(100/1000) = 0.86 g (0.00025 x 1000)(100/1000) = 0.025 g (0.000025 x 1000)(100/1000) = 0.0025 g (0.000025 x 1000)(100/1000) = 0.0025 g 100X (g/500 mL) (0.03730 x 100)(500/1000) = 1.865 g (0.02780 x 100)(500/1000) = 1.39 g 1000X (g/100 mL) (0.0005 x 1000)(100/1000) = 0.05 g (0.0001 x 1000)(100/1000) = 0.01 g (0.002 x 1000)(100/1000) = 0.2 g (0.0005 x 1000)(100/1000) = 0.05 g

FERUM Na2.EDTA FeSO4.7H2O

VITAMIN Nicotinic acid Thiamine HCl Glycine Pyridoxine HCl

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Plant Biotechnology

Plant Growth Regulator Benzylaminopurine (BAP) Naphthaleneacetic acid (NAA)

g/L 1.000 1.000

g/100 mL 1 (100/1000) = 0.1 g 1 (100/1000) = 0.1 g

Discussion Macronutrient Macronutrients are normally required in millimolar (mM) quantities in most plant media. The macronutrient normally contains high amount of nitrogen fixing agents in the form of ammonium (NH4+) and nitrate ions (NO3-) ions. Apart from nitrogenic substances, potassium, magnesium, calcium, sulphur and phosphorus are also added as macronutrient in the form of diluted salts. Also, macronutrients serve as components for structural and protoplasmic tissue. Micronutrient Micronutrients are needed in a very small amount. The low requirement of micronutrients can be accounted for participation of these elements in enzymatic reactions and as constituents of growth hormones. The micronutrient is the mixture of boron, cobalt, manganese, molybdenum, copper and zinc. Excessive amount added to the medium can cause suffocation and premature death of the explants used. Ferum Ferum is required in metabolic functions such as nitrogen fixation, photosynthesis and electron carrier during respiration’s electron transfer process. It is usually present in the form of FeSO4.7H2O and Na2EDTA. In some cases, ferum is prepared together with micronutrient. Ferum oxidises in the presence of sunlight. Furthermore, high concentrations will cause precipitation to occur. Therefore, preparing ferum and storing the stock solution in dark environment can prolong the shelf life of ferum stock solution. Vitamin Vitamin is essential as it is involved in carbohydrate metabolism and the biosynthesis of some amino acids. Normally thiamine is deemed as the most important vitamin and it is introduced as thiamine hydrochloride. Other vitamins like pyridoxine, nicotinic acid are added as well. Certain plant species requires special requirements of vitamin like biotin, riboflavin, folic acid and more. Vitamins are associated with metabolic activity of the plant. Therefore, to have good yield, sufficient amount of vitamin should be added to the MS medium. Plant growth regulators Plant growth regulator (PGR), functions in initiating the root and shoot development of explants and embryos. They also stimulate cell division and expansion. Certain parts of plants have plant growth regulators readily available in the explants. In cases of PGR absence, PGR supplemented through medium enables growth of the

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Plant Biotechnology

explants. There are two major classes of PGRs; cytokinin and auxin. Cytokinin (e.g. BAP) is used to generate shoots while auxins (e.g. NAA) are used to induce roots. In considerably high concentrations, certain explants prefer callus formation. Therefore, PGRs can be seen as the growth inducer in plants. Why preparing stock solution? The amount nutrients used are considerably very low. Weighing out the salts for each time media being prepared is a tedious job as the quantity required is very small. Therefore, accuracy will not be established. Preparing the stock solution requires vast quantity and this ensures accuracy at the same time. It is also time saving. Conclusion From this experiment, we become understood in calculating quantities of chemicals needed for given concentrations and stock volumes; and improving our skills in preparation of stock solutions for MS medium. References Fageria, N.K. 1992. Maximizing Crop Yields. Marcell Dekker, New York, USA. Fageria, N.K., Baligar, V.C., and Jones, A.C. 1997. Growth and Mineral Nutrition of Field Crops. Marcell Dekker, New York, USA. Mengel, K. and Kirkby, E.A. 1978. Principles of Plant Nutrition. Kluwer Academic Publishers, Dordrecth, Netherlands. Trigiano, R.N. and Gray, D.J. 2000. Plant Tissue Culture Concepts and Laboratory Exercises. CRC Press, Boca Raton, Florida, USA. http://en.wikipedia.org/wiki/Murashige_and_Skoog_medium (141009)

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