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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Results

STOCK
1X
MACRO 10X (g/800 mL)
(1 litre MS) g/L
NH4NO3 1.6500 (1.65 x 10)(800/1000) = 13.2 g
KNO3 1.9000 (1.90 x 10)(800/1000) = 15.2 g
CaCl2.2H2O 0.4400 (0.44 x 10)(800/1000) = 3.52 g
MgSO4.7H2O 0.3700 (0.37 x 10)(800/1000) = 2.96 g
KH2PO4 0.1700 (0.17 x 10)(800/1000) = 1.36 g

1X
MICRO 1000X (g/100 mL)
(1 litre MS) g/L
(0.00083 x 1000)(100/1000)
KI 0.00083
= 0.083 g
(0.00620 x 1000)(100/1000)
H3BO3 0.00620
= 0.62 g
(0.02230 x 1000)(100/1000)
MnSO4.H2O 0.02230
= 2.23 g
(0.00860 x 1000)(100/1000)
ZnSO4.7H2O 0.00860
= 0.86 g
(0.00025 x 1000)(100/1000)
Na2MoO4.2H2O 0.00025
= 0.025 g
(0.000025 x 1000)(100/1000)
CuSO4.5H2O 0.000025
= 0.0025 g
(0.000025 x 1000)(100/1000)
CoCl2.6H2O 0.000025
= 0.0025 g

1X
FERUM 100X (g/500 mL)
(1 litre MS) g/L
(0.03730 x 100)(500/1000)
Na2.EDTA 0.03730
= 1.865 g
(0.02780 x 100)(500/1000)
FeSO4.7H2O 0.02780
= 1.39 g

1X
VITAMIN 1000X (g/100 mL)
(1 litre MS) g/L
(0.0005 x 1000)(100/1000)
Nicotinic acid 0.0005
= 0.05 g
(0.0001 x 1000)(100/1000)
Thiamine HCl 0.0001
= 0.01 g
(0.002 x 1000)(100/1000)
Glycine 0.002
= 0.2 g
(0.0005 x 1000)(100/1000)
Pyridoxine HCl 0.0005
= 0.05 g

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Plant Growth Regulator g/L g/100 mL

Benzylaminopurine
1.000 1 (100/1000) = 0.1 g
(BAP)
Naphthaleneacetic acid
1.000 1 (100/1000) = 0.1 g
(NAA)

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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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)