Plant Tissue Culture Medium : M S Medium

PREPARATION OF PLANT TISSUE CULTURE MEDIUM

AIM:  To prepare Murashige and Skoog (MS) media for plant tissue culture.

INTRODUCTION: 

Murashige and Skoog medium or (MSO or MS0 (MS-zero)) is a most commonly used plant growth medium in the laboratories for cultivation of plant cell culture. MSO was invented by plant scientists Toshio Murashige and Folke K. Skoog in 1962 during Murashige's search for a new plant growth regulator.

The culture medium is one of the most important components of plant cell and tissue culture method. The successful application of plant culture medium with its right composition in addition to its components is one of the most important pre-requisite of a plant tissue culture experiment. Another important function of the culture medium is to provide the right physical environment for cells and tissues to grow on solid medium. The media also performs a function like soil by providing a physical support matrix. All plant cells require water, nutrients and plant growth regulators.

The requirements of plant cell culture may be subdivided into 3 groups:

1.      Inorganic nutrients

2.      Organic nutrients

3.      Plant growth regulators

Inorganic nutrients: Inorganic nutrients are mineral elements and based on their essential concentration they are classified into 2 groups: macroelements, present in large supplies (mM concentration) and microelements as trace elements.

MACROELEMENTS:

·         Nitrogen: supplied in the form of nitrate ions and ammonium ions (KNO₃ and NH₄NO₃).     Nitrogen is a component of protein, nucleic acid, chlorophyll, some coenzymes and is required in greatest amounts.

·         Sulphur: supplied in the form of sulphate ions. Sulphur is a vital component of amino acids, vitamins, cofactors, iron sulphur protein (electron carrier).

·         Phosphorous: supplied as a phosphate ion (PO₄­). Added as Na/K dihydrogen phosphate. It is an essential component of nucleic acid, phospholipids and in energy rich compounds.

·         Cations: supplied as KNO₃ salt. Potassium functions as osmoregulators, cation-anion balance, and pH stabilization.

MICROELEMENTS:

·         Iron: added as iron sulphate [Fe (III)]. EDTA is usually used in conjunction with iron sulphate. This complex with iron so as to allow slow and continuous release of iron into the medium. Uncomplexed iron usually precipitates out of the medium as ferric oxide. It is required by the plant for chlorophyll synthesis as a constituent of cytochrome and Fe-S protein.

·         Boron: added as boric acid. It is a component of cell wall required for cell division of optical meristem.

·         Cobalt: added as cobalt chloride. It is a component of some vitamins.

·         Copper: added as copper sulphate. It as serves as an enzyme cofactor and function in electron reactions.

·         Iodine: improves growth of roots and callus, added in the form of KI.

·         Manganese – Added in the form of MnSO₄. Manganese is found in metalloproteins.

·         Molybdenum – Added as sodium Molybdate. It is an enzyme cofactor.

·         Zinc – Added as ZnSO₄ required for activity of various enzymes.

Organic Nutrients:

          While green plants are autotrophic, most culture system in early stages is heterotrophic. Plant culture system requires an organic carbon and energy source and complex organic molecule such as vitamins and cofactors for healthy growth.

·         Carbon Source: Sucrose is most commonly used sugar in plant tissue culture media, but glucose, fructose and sorbitol are used in some formulations.

·         Vitamins and Co-Factors: two vitamins, thiamine (B1) and myoinositol( Vit B)  are essential for plant tissue culture. Other vitamins such as biotin, pantothenic acid (niacin), pyridoxine (Vit B6), folic acid, ascorbic acid (Vit C) and tocopheryl acetate (Vit E) are added to the media.

·         Complex Organic Supplements: complex additions such as banana powder and liquid endosperm of coconut are frequently used in plant tissue culture media such as kinetin, benzyl aminopurine and zeatin media to improve growth.

Plant Growth Regulators:

         There are 5 main classes of plant growth regulators.

·         Auxins: they promote both cell division and cell growth. Auxins are synthesized in stems and root spaces and transport along the plant axis. Auxins commonly used in plant tissue culture are 2, 4-dichlorophenoxy acetic acid (2, 4-D), Indole 3-acetic acid (IAA), Indole-3-butyric acid (IBA), 1-napthalene acetic acid (NAA). Auxins together with cytokines are used in plant tissue culture to control differentiation and morphogenesis.

·         Cytokinins: they promote cell division, growth and development. Naturally occurring cytokinins are purine derivatives. They are frequently used in plant tissue culture media such as kinetin, benzyl aminopurine and zeatin.

·         Gibberellins: Major action of gibberillin is stimulation of cell stem elongation and flowering. Gibberillic acids are commonly used in plant tissue cultures (GA, GA4, and GA3).

·         Abscissic Acid: Primarily involved in water-stem responses, seed germination and enhances somatic embryogenesis.

·         Ethylene: is gaseous and is associated with fruit ripening in climatic fruits.

Support Matrices of Gelling Agents:

In some plant cell and tissue culture system a solid or semi solid matrix is needed to support tissue explants while allowing contact with media. The most commonly used support matrices are formed from gelling agents such as agar, agarose, gelatin, gelatin gums.

Agar is the most commonly used gel in plant tissue culture. It is a complex polysaccharide extracted from red algae (Rhodophyceae) and comprises 2 fractions-agar (70%) & agar pectic (30%). Agarose in the jelly fraction and consists of a polymer of D-galactose and 3, 6-anhydrogalactose monosaccharide units. Agropectin is the non gelling fraction and consists of polymer of sulphur.

The required basal medium can be prepared by either dissolving the commercially available basal salt mix on by preparing the mix from individual ingredients. An alternative is to prepare macronutrients & micronutrients stock solution and store these at 4⁰ C. Until used, organic compounds should not be stored in solution for more than 2 weeks. Heat labile compounds such as plant growth regulator, antibiotics are added to the medium after autoclaving.

MATERIALS AND REQUIREMENTS:

·         Distilled water, sucrose, gelling agent, 1M NaOH or 1M HCl to adjust the pH, micronutrients stock, macronutrients stock, organic compound stock.

·         Equipment – Balance, pH meter, autoclave, LAF cabinet.

·         Glassware – conical flask, phyta jars, beakers, pipettes.

PROCEDURE:

·         Add approximately half the volume of water to a conical flask

·         Weigh out the appropriate amount of basal salts (4.4g) or measure out appropriate volume of stock solution .Add the water and 30 g sucrose

·         When all solids have dissolved , adjust the pH 5.7 using 0.1M NaOH or HCl

·         Make up the volume to 1 litre using distilled water

·         Weigh 10 g agar and add to the above medium

·         Melt the agar by heating

·         Divide the medium into tissue culture bottles and close the bottles with screw caps

·         Sterilize the culture bottles by autoclaving at 121⁰ C for 15 min 15 psi

·         After autoclaving, ensure the caps are immediately lightened.

PREPARATION OF HORMONE STOCKS:

·         Weigh out 0.3g of IAA

·         Sterilize the IAA by placing under UV light for 30 mins. Transfer to a sterile volumetric flask.

·         Dissolve the IAA in few drops of ethanol and make up the volume to 100 ml using sterile distilled water.

·         Weigh out 0.1mg of kinetin and sterilize under UV light

·          Transfer to a flask and dissolve in 100 ml of sterile distilled water

·         Add 30 ml of IAA stock and 1.5 ml of kinetin stock to each bottle aseptically. Mix well and allow the contents to solidify and use immediately or store at 4⁰C .

Murashige and Skoog (MS) media composition:

Macroelements	g
NH4NO3	1.65
KNO3	1.90
CaCl2.2H2O	0.44
MgSO4.7H2O	0.37
KH2PO4	0.17
Microelements	mg
FeSO4.7H2O	27.80
Na2EDTA.2H2O	33.60
KI	0.83
H3BO4	6.20
MnSO4.4H2O	22.30
ZnSO4.7H2O	8.60
Na2MoO4.2H2O	0.25
CuSO4.5H2O	0.025
CoCl2.6H2O	0.025
Organic supplements	mg
Myoinositol	100
Nicotinic acid	0.05
Pyridoxine HCl	0.05
Thiamine HCl	0.05
Glycine	0.20
Sucrose	30g
Agar	10g

Dissolve the microelements, macroelements and organics in 800ml of distilled water. Add the required hormone solutions. Adjust the medium pH to 5.7 by adding 1M NaOH or 1M HCl. Add additional distilled water to adjust the final volume to 1litre. Add the agar and heat the medium to dissolve the agar. Dispense the medium into culture tubes or vessels and autoclave for 20minutes at 121°C.


Half (1/2) M S Media :

Some refer to half-MS as half of everything (macro, micro, sucrose, etc.).

Some use half macro and micro and full sucrose. This term has not been standardized in the literature.

Generally Half M S media uses half macro elements only....The micro elements are full strengh...

REFERENCE: 

Biotechnology: Fundamentals and Applications; S.S.Purohit and S.K.Mathur; Agro Botanical Publishers; page no.s 112-117.
http://plant-tc.cfans.umn.edu/listserv/2002/log0205/msg00141.html

Lab protocol for Protease Production and Estimation by Lowry's method

LAB MANUAL

Production And Estimation Of Protease Activity Through Solid State Fermentation

Aim :

To produce and estimate protease produced by Aspergillus niger and Bacillus sp. using wheat bran and dried skimmed milk respectively by solid state fermentation.

Introduction :

Proteases are the most important industrial enzymes and comprise about 25% of commercial

enzymes in the world. It refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze various peptide bonds. Ex : fungal protease, pepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin.

The mycelium of many fungi store sufficient amount of nutrient proteins required for their growth. The enzyme protease becomes active during growth and breakdown of stored proteins into peptides and amino acids which can be used by the fungi for its metabolic and energy requirements. The amount of amino acids released gives the measure of activity of the enzyme protease in the organism.

Proteases are divided into four major groups according to the character of their catalytic active site and conditions of action: serine proteinases, cysteine (thiol) proteinases, aspartic proteinases, and metalloproteinases.

Common protease producing microbes are Aspergillus sp., Aeronomas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp. and Pseudomonas sp.

The present uses of the protease in industrial applications are as detergents, cosmetics, leather processing, solubilising agent, in food industry as softening agent and as well as pharmaceutical  industry to treat digestive ailments.

Principle :

Sterile wheat and skimmed milk are inoculated with A. niger and Bacillus respectively. The fungal cultures are incubated for a week and the enzyme is extracted using phosphate buffer. When protease enzyme acted upon protein, the peptide bonds were cleaved resulting in the liberation of free amino acids. First the proteins are pre-treated with copper ion in alkaline solution and then the amino acids in the treated sample reduce the phosphomolybdate and phosphotungstate acid present in the Folin ciocalteu reagent. The reagent reacts with phenolic and non-phenolic substances and reduce them to heteropolymolybdenum by the copper-catalyzed oxidation of aromatic acids and finally produces blue coloured complex that can be detected spectrophotometrically at 660nm.

.

Requirements:

Bovine Serum albumin, skimmed milk, wheat bran, wheat bran media, phosphate buffer, folin ciocalteu reagent, mortar & pestle, NaOH, CuSO₄, volumetric flask, pipettes and test tubes.

Reagents:

·         Phosphate Buffer: 58.9ml of 0.1M KH₂PO₄ and 61.1ml of 0.1M Na₂HPO₄.

·         Bovine Serum Albumin: 1% Bovine serum albumin (1g of BSA in 100ml of distilled water) used for enzyme activity.

·         Standard Stock: 50mg of BSA was dissolved in 100ml of distilled water to give a concentration of 500mg/ml.

·         Folin ciocalteu reagent: the commercially available reagent is diluted in the ration of 1:2 with water.

Procedure:

Preparation of wheat bran media:

Peptone	1g
CaCl2	100mg
NaNO3	1g
K2HPO4	100mg
MgSO4	100mg

Dissolve the above ingredients in 100ml of distilled water and autoclave. Take 10g each of wheat bran and skimmed milk powder in separate flask and sterilize.The medium is cooled. 10ml of wheat bran media is added aseptically in to the flask containing wheat bran and skimmed milk using sterile pipette.

Inoculation and Incubation:

A small amount of A. niger and Bacillus sp. is inoculated in the flask containing wheat bran and skimmed milk powder respectively. After incubating for a week enzymes are extracted from the medium using phosphate buffer.

Preparation of Standard Graph:

        i.            Different aliquots of standard protein solution (500mg/ml) were pipetted out in to 5 test tubes (100-500mg/ml).

      ii.            Ranging from 0.2ml to 1.0ml with a total volume of 1ml using distilled water. 3ml of alkaline copper reagent was added and the tubes were incubated at room temperature for 15mins.

    iii.            0.5ml of folin ciocalteu reagent was added and the tubes were incubated at room temperature for 30 mins.

    iv.            The absorbance was read at 640nm.

      v.            A standard graph was plotted by taking absorbance on Y- axis and protein concentration on X- axis.

Extraction and estimation of protein:

Extraction:

 For fungi:

1g of fermented substrate is taken and 10ml of phosphate buffer is added homogenised with mortar and pestle. Centrifuge at 3000rpm for 15 minutes. Take the supernatant for enzymatic activity.

For Bacteria:

Directly centrifuge 1ml of culture and homogenised in 10ml of phosphate buffer  and centrifuge at 1000rpm for 10-15 minutes. Take the supernatant.

Estimation

        i.            1g of mycelial mat is homogenised with 10 ml of ice cold phosphate buffer solution.

      ii.            The homogenate is centrifuge at 3000rpm for 10 mins.

    iii.            1ml phosphate buffer is taken in to 3 test tubes with 1 kept as blank.

    iv.            Add 0.5ml of substrate solution (1% BSA) to each of the tubes, allow to stand for 10 minutes at room temperature.

      v.            Add 2ml of TCA

    vi.            Centifuge at 3000 rpm for 10mins.

  vii.            Transfer 1ml of supernatant to fresh tubes.

viii.            Add 2ml of 0.5N NaOH, 0.5ml of CuSO4 and 0.5ml of Folin’s reagent.

    ix.            Incubate at room temperature for 30mins.

      x.            Measure the absorbance at 640nm.

 RESULT:

The protease activity was found for

Fungal Protease_______________

Bacterial Protease ___________

 Observation and Calculation :

Conc. Of BSA (mg/ml)	Vol. of stock (ml)	Vol. of D/W (ml)	Total vol. (ml)	Vol. of alk. Cu reagent (ml)	Incubation @ RT (min)	Vol. of folins ciocalteu reagent (ml)	Incubation @ RT (min)	OD at 640 nm
100	0.2	0.8	1	3	15	0.5	30
200	0.4	0.6
300	0.6	0.4
400	0.8	0.2
500	1.0	0.0
Blank	0.0	1.0
Unknown

Enzyme Activity :

Test tube	Enzyme ext (ml)	PO4 buffer	BSA	Incubation	10% TCA	Centrifugation	1 ml of supernatant + 2 ml (0.5N NaOH) + 0.5 ml Folins reagent	Incubation	OD
Blank	1	1 ml	0	10 min	2 ml	3000 rpm for 10 min		30 min @ RT
Test	1	1 ml	0.5	10 min	2 ml

References :

http://www.bio.davidson.edu/people/jowilliamson/Techniques/Protocolweek5.html

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/6/47641dat.Par.0001.File.tmp/47641dat.pdf

www.nature.com/protocolexchange/protocols/267