In Vitro Culture Media

In Vitro Culture Media

The purpose of an in vitro culture medium is to provide optimum conditions for the growth of the explants.  These conditions are determined largely by physical factors, such as the composition of nutrients and growth regulators, pH, solidness of  the medium, and temperature.  A universal medium cannot be developed.  Each variety of a genus, and also explants from different parts of the same plant have different requirements for optimum growth.

In the last three or four decades several in vitro culture media have been developed for specific uses of purposes.  These basic media are usually named after the research worker who developed them.  A complete medium consist of basic medium and varying amounts of organic nutrients, growth regulators, and often agar, according to the specific purpose of the medium.

1.      Basic medium

A basic medium is composed of a number of elements which are added in salts in the form of ions.  Those elements required in large amounts (macroelemnets) are ; Nitrogen, phosphorus, sulphur, calcium, potassium, magnesium.  Those elements required in smaller amounts (microelements) are; iron, manganese, copper, zinc, boron, and molybdenum.

Some of these ions are added in more than one salt.  In the MS medium, the K –ion is contributed to by KNO3 and KH2PO4 and the NO3-ion by KNO3 and NH4NO3.  It is advisable to add iron as ferri sodium ethylenedianine tetraacetate (NaFe EDTA), or dinatrium ethylenedianine tetra acetate (Na2EDTA) in combination with FeSO4.   The presence of this compound in the medium also ensures that iron is available over a wide pH range.

2.      Organic Nutrients

To obtain optimum growth of the tissue, it is essential to supplement the medium with one or more vitamins and amino acids, because most cells in a culture plant only synthesize these in suboptimal quantities.  Thiamine (vitamin B1), added as thiamine HCL, has been proved to be essential; other vitamins, especially pyridoxine (vitamin B6), nicotinic acid (vitamin B3), calcium panthotenate (vitamin B5) and myo-Inositol have been shown to improve the growth of cultured plant material.

Numerous complex nutrient mixtures of undefined composition, such as, casein hydrolysate, coconut milk, malt extract, and yeast extract have also been used to promote the growth of plant tissues.  However, the use of these extract should be avoided, because of difficulties in reproducing results.  The growth promoting constituents in these extract vary considerably.  It is preferable to replace these substances with a single amino acid.

Tissues which lose their green pigment in culture, depend on an external source of carbon; the most commonly used is sucrose, at concentration of 2 to 5 %.

3.      Hormones and growth Regulators

Hormones are organic substances synthesized in plants and affect physiological processes, such as, growth, flowering, and senescence.  Growth regulators are artificially produced organic substances, which have the same characteristics as hormones.  The most important hormones and growth regulators in vitro culture are auxins and cytokinins.

Auxins are used to induce cell division and root differentiation.  In general, at a low concentration (1 mg/l) root differentiation occurs, and at a higher concentration ( 10 mg/l) callusing occurs.  The auxins most commonly used are the hormone indole 3 acetic acid (IAA), and the growth regulators indole 3 butyric acid (IBA), naphtalane acetic acid (NAA) and dicholorophenoxyacetic acid (2,4D).   IAA is thermo- and photo-unstable.  2,4D is effective in inducing growth of callus, but also in inducing mutations.

Cytokinins are used in invitro culture to stimulate growth and differentiation.  They induce cell division, especially when an auxin is also added to the medium.  In high concentrations (1-10 mg/l) they can induce growth of adventitious root formation is normally inhibited.

Those most commonly used in invitro culture are the hormone zeatin (Z) and the growth regulators benzyl aminopurine (BA), furfuryl aminopurine (kinetin), 2 isopentenyl adenine (2iP) and isopentenyl adenosine (IPA).

 

4.      Agar

In order to overcome the problem of lack of oxygen when the explants is submerged, the liquid medium is gelled with a concentration of 0,5 to 1 % agar, which is a polysaccharide obatained from seawees.  Silidified media are widely used because they are easy to maintain and handle, and can be used for many purposed.

5.      pH

The pH of the medium is usually adjusted to between 5 and 6 before sterilization.  In general, a higher pH (6) gives a fairly solid medium, and a lower pH (5) does not allow the agar to gel.  The ph is adjusted by using a HCl or KOH solution.

6.      Selection of a Medium

Several methods to formulate a medium for specific explants have been described.  Bhojwani (1983) advise starting with a well-kown medium, such as, MS or B5, and making minor qualitative and quantitative adjustments on the basis of a series of experiments in which inorganic and organic constituens are treated separately.  Attention should be paid to the nitrogen source, because the addition of reduced nitrogen in the form of NH4Cl or NH4NO3 as well as KNO3 may be necessary.  In these experiments care should be taken not to raise the total salt concentration excessively.

7.      Medium Preparation

For routine purposes, commercially available dry powdered medium, which contains the necessary compounds, can be used, and also fully automatic medium preparation units are available.  For experimental work, where it is necessary to change the constituent of the medium, a popular method is to prepare a series of concentrated solutions of some compounds in the basal medium.  Various combinations of “stock solution” will result in media of different composition.  It should be remembered not to combine salts which precipitate in one solution.   Stock solution should be made in large quantities in order to avoid small variations which occur when made up in small quantities.  However, stock solution can be stored in small quantities and thawed before use.   Stock solution containing growth regulators can only be kept in a refrigerator for one to two months.   Other components, such as amino acids, should be added to the medium directly after weighing the necessary amount.

8.      Medium Sterilization

To destroy contaminants present in a medium, the containers need to be closed with a suitable bacteria-proof cap and autoclaved, that is, sterilized under pressure.  This is done at 0,7 kg/Cm² over-pressure at a temperature of 115º C for 20 menits from the time the medium inside reaches the required temperature and pressure.  Ussually, for experiments, volume of 5 to 10 ml in test tubes or culture tubes are autoclaved.  It is essential  that all the solution reaches the required temperature in the required time.  Therefore, for larger quantities a much longer period of autoclaving at a higher temperature is required.  Preferably, the media should be autoclaved in small volumes, so that irregulaties in larger containers do not affect autoclaving.  Care must be taken in cooling the solution, because rapid loss of pressure exceeding the rate of reduction in temperature will make the liquid boil vigorously.

Some growth regulators and amino acids are thermolabile, and therefore should be filter sterilized and added to the medium after it has been autoclaved.  Ussually, glassware is sterilized together with the medium, otherwise it can be sterilized in an oven at temperature of 150ºC for a period of three hours.  Plastic containers made of polypropylene, polymethylpentane, polyallomer, tefzel ETFE, tubing can also be autoclaved.

9.      Physical Conditions

Physical conditions in the culture room must be controlled carefully while explants are growing in culture tubes.  This should be done with the aid of automatic time switches and independently controlled temperature cannot be given because methods of culturing and the purpose for culture vary.  (disadur dari; Introductory course on in vitro culture, J.Beelen, Department of Tropical Crop Science, Wageningen).