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).
