Post on 25-Jan-2023
PLANT TISSUE CULTURE MEDIA: COMPONENTS AND
PREPARATION
Afolayan Adedotun O. (Mrs)
Scientific Officer 1, National Centre for Genetic Resources and Biotechnology,
Moor Plantation, Apata Ibadan – Federal Ministry of Science & Technology.
PMB 5382, Moor Plantation Ibadan
Culture media
Culture media can be described as a substrate on which in-vitro
propagated explants are placed for proper nourishment and
growth. It is to these plantlets as soil is to naturally raised
plants in their field. Appropriate media selection and
preparation is a vital factor that must be put into
consideration for a successful tissue culture.
The growth of a tissue in vitro may vary in its nutritional
requirements depending on the species. Even tissues from
different parts of a plant may have different requirements for
satisfactory growth. Therefore, no single medium can be
suggested as being entirely satisfactory for all types of plant
tissues and organs; rather, media selection for healthy growth
of any plant tissue is done after carrying out a series of
experimental work under trial and error. However, when starting
with a new system, it is essential to work out a medium that
would fulfill the specific requirements of that tissue.
Over the years, several recipes have been developed due to the
different needs that have arisen.
NUTRIENT MEDIA COMPOSITION
Generally, nutrient media is made up of the following:
- Water
- Inorganic nutrients
- Carbon sources and Osmotica
- Vitamins
- Growth regulators
- Organic supplements
WATER
Essential to the preparation of any medium is the presence of
water as the solvent in which other added components are
dissolved. The water that is often employed in the preparation
of media is de-ionized or de-mineralized water. Often, double
distilled water may be used but not generally acceptable. It is
also important to use freshly de-ionized water. Prolonged
storage should be discouraged as it could pose a problem to the
media.
INORGANIC NUTRIENTS
Apart from carbon, hydrogen and oxygen, cultured plant tissue
also requires in large amounts certain macronutrient elements,
which includes nitrogen, phosphorus, potassium, magnesium and
sulphur.
A concentration of 1 – 3mM calcium, sulfate and magnesium is
usually adequate. Nitrogen is added in the form of either
nitrate or ammonium in a concentration ranging from 2 –20mM.
Also, plant cells need certain micronutrients in minute
quantities. They are usually small in amount and their stock
solution is always prepared in advance. These micronutrient
elements include iron, manganese, zinc, carbon, copper,
molybdenum and chlorine. The stock solution of iron is always
prepared separately because of the problem of iron solubility.
Some media may contain traces of cobalt and iodine in addition
to the known micronutrients. Agar may also be a source of
numerous elements (Dodd & Robinson, 1990).
CARBON SOURCES AND OSMOTICA
Usually, the choice and concentration of carbohydrate source to
be used will depend mainly on the plant tissue to be cultured
and the purpose of the research. Green cells in culture are
generally not photosynthetically active and require a carbon
source. Sucrose or glucose at 2-3%w/v is commonly used in cell
culture. Other carbohydrate source such as fructose and starch
can also be used in protoplast culture while much higher levels
may be used for embryo or anther culture. All cultured cells
utilize sucrose and glucose equally well. The concentration is
between 20 to 30g/l. In the medium, added sucrose is rapidly
converted to glucose and fructose. The hexitols e.g. myo-
inositol, have been found to be very important in tissue
culture. Myo-inositol is an interesting hexitol involved in
cyclitol` biosynthesis, storage of polyhydric compounds as
reserve germination of seed, sugar transport, mineral
nutrition, carbohydrate metabolism, membrane structure, and
cell wall formation. (Cyclitols are any hydroxylated
cycloalkanes containing at least three hydroxyl groups attached
to different carbon atoms. They are cyclic polyols and are
formed in a plant as a response to salt or water stress. Some
are parts of hydrolysable tannins). The cyclitol myo-inositol
(100mg/l) can be added to some culture medium.
Also, cultured plant tissues require some osmotica like sugars,
mannitol or sorbitol which is often used as external osmoticum
(Dodd & Robinson, 1990). Sugar will undergo carmelization if
autoclave for too long (peer, 1971) and will react with amino
compounds (mailard reaction). Carmelization occurs when sugars
are heated, degrade and form melanoidens which are brown, high
molecule weight compounds, which can inhibit cell growth.
VITAMINS
Vitamins are needed in small amounts due to their catalytic
function in enzyme systems. These include Thiamine (B1),
Nicotinic Acid (B3), Pyridoxine (B6), Aminobenzoic Acid,
Vitamin B12, Folic Acid, Biotin and Ascorbic Acid (used as
antioxidant to stop tissue browning). Thiamine was thought to
be essential vitamins for nearly all plant cultures and is used
at low concentration of cytokinins whereas nicotinic acid and
pyridoxine may stimulate growth (Gamborg et al, 1976). Vitamins
stocks are best stored in a freezer and can be made up such
that 5mls aliquots are used per liter of medium prepared. The
vitamins stock is prepared by dissolving in water. Most
vitamins are soluble in water.
GROWTH REGULATORS
Auxin and cytokinin are often used in most tissue cultures.
Gibberellins also may be used in media composition but the
addition of either of these depends on the aim of the
experiment.
The common auxins used in tissues culture are Indole-3-Acetic
Acid (IAA), Naphthalene Acetic Acid (NAA), and 2, 4-
Dichlorophenoxy acetic acid and Indole-3-Butyric Acid. Auxins
are class of compound that stimulates shoots cell elongation,
however IBA is effectively used as rooting agent.
The cytokinins commonly used in tissue culture are kinetin
(synthetic compound), benzyladeline (synthetic compound) and
zeatin (naturally occurring compound). Cytokinins promote cell
division in plant tissues under certain bioassay condition and
also regulates growth and development e.g. kinetin which also
is used for the induction of callus at the concentration of
0.1mg/l.
The ratio of auxin-cytokinin supplements is important in the
regulation of cell division, cell elongation, cell
differentiation and organ formation. Although, it is not always
necessary to include both in every culture at a particular
ratio because some tissue has inherent residual high
concentration of auxin already.
Gibberellins are used in apical meristems culture.
Preparation:
Auxin stocks are usually prepared by weighing out 10mg auxin
into a beaker, adding some few drops of 1N NaOH or KOH until
crystals are dissolved completely. Then distilled water is
added which bring it up to 100ml volume in a volumetric flask.
IAA stock is usually prepared fresh because IAA is degraded by
light within several hours to a few days. Auxins are
thermostable; however IAA is destroyed by low pH, light, oxygen
and peroxides [Pothumus, [1971]. Hence, NAA and 2, 4-D are more
stable forms of auxins.
Cytokinin [kinetin, BA, Zeatin] promotes cell division, shoot
proliferation and shoot morphogenesis. Cytokinnis stocks are
prepared in a similar way to auxin stocks except that HCl is
used to dissolve crystals along with a few drops of water.
Gentle heating is usually required to completely dissolve
crystals. Double distilled water is then added quickly to avoid
the crystals falling out of solution. After which the final
volume is made 100ml volumetric flask. Cytokinnis stocks can be
stored for several months in the refrigerator. Some cytokinnis
[kinetin and zeatin] are thermostable in that no breakdown down
products were detected after 1hour at 120oC [Dekhnyzen, 1971],
while BA is stable for 20mins at 100oC.
Gibberellin is not frequently used in plant cell culture as GA3
can inhibit callus growth. Stock solution of GA3 can be
prepared by dissolving 10mg in water and adjusting the pH to
5.7. GA3 is not thermostable and 20mins at 114oC reduces its
activities by more than 90% (Van braft and Pierk, 1971).
Abscisic acid is a plant hormone involved in leaf and fruit
abscission as dormancy. It is useful in embryo culture. ABA is
heat stable but light sensitive. Stock solution can also be
prepared.
ORGANIC SUPPLEMENTS
Peptons, yeast extract and malt extract are often used. Fruit
juices such as orange juice and tomatoes juice are also
important supplements.
Other added components include:
1. Charcoal
Activated charcoal is widely used in tissue culture to absorb
many organic and inorganic molecules from the cultured medium.
It may help to remove contaminants from agar and secondary
products secreted by cultured tissue. It has also been reported
that activated charcoal may stimulate embryogenesis and may
also have inhibitory effects on growth and morphogenesis in
vitro (Constantin et al, 1977; Kohlenbach and Wernicke, 1978).
2. Amino acids or amides
The names of amino acids or amides that produce positive
results include L-aspartic acid, L-asparagine, L-glutamic acid,
L-glutamine and L-arginine. Amino acids Amino acids and amides
can be very important in morphogenesis. All ‘L’ forms are the
natural form detected by the plant. L-tyrosine can play a role
in shoot initiation, L-arginicine in rooting L-asparagines
sometimes significantly enhance somatic embryogenesis.
3. Medium matrix
Matrixes are substances on which cultures are placed to grow.
It is true that not all plant tissues will grow on a liquid
medium. Addition of a solidifying agent such as Defco Bacta
agar (0.6 – 1% w/v); starch copolymer which can be used as an
agar substitute (Cooke, 1977); sucrose polymer can also serve
as a support matrix and filter paper platform were introduced
by Heller (1965). Glass fibers filters can be used as support
and pretreatment is absolutely necessary before use.
4. Antibiotics
Due to excessive contamination problems with certain plant
explants, fungicides and bactericides have been incorporated
into the culture medium. {Thurston et al 1979}. Although these
additions have not been very useful as they can be toxic to the
explants or the contaminant will re-appear as soon as the
fungicides or bactericides are removed.
SELECTION OF A MEDIUM
The choice of a particular medium depends on the species of
plant, tissues or organ to be cultured and the purpose of the
experiments. Successfully cultured published protocol or
methodology of a particular tissue can be adopted.
Murashige and Skoog (MS) medium has been proven to be very
effective in the production of callus from dicotyledonous
plants due to the presence of high concentration of nitrate,
potassium and ammonium ions. Also, MS vitamin, myo-inositol
(100mg/l) and sucrose 2-3% w/v can be added to MS medium to
improve callus production. The inorganic salt formulations can
vary (Gamborg et al 1976); however, Murashige and Skoog (MS)
(1962) formulation is the most widely used and will be the
major salt formulation used in this training.
The B5 medium developed by Gamborg’s group (Gamborg et al, 1968)
is also effective in callus production especially when 2-4-D
(0.2 – 2mg/l) is added. This is effective for many plant
tissues.
Also, the addition of cytokinin especially kinetin (0.5 –
2mg/l) in combination with IAA (30mg/l) may be helpful in
callus production. Coconut milk can also be added to other
combinations.
In addition to the proper ratio of hormone, shoot formation may
require a medium that is high in phosphate or low in ammonium
nitrate (Miller and Murashige, 1976).
PRECAUTORY MEASURES
1. In all media preparation, always use the glass-
distilled water and never tap water.
2. A portion of water (300-500ml for 1liter vol.) must
always be in your beaker prior to adding the stock
solution otherwise concentrated stocks will co-react
and precipitate out.
3. Adjust the medium pH using 0.5N HCl or NaOH using a
pasture pipette while keeping the medium up final
vol.
4. Never pour excess stocks back into the original stock
solution container.
5. Never put excess sucrose or agar back into the
original container always cleans up spills around
balance and work areas.
6. When melting agar in motion using a magnetic stir bar
on the hot plate or by hand using an autoclave tape.
This is kept in motion to prevent the flask. The agar
must be completely dissolved before dispensing into
culture tubes. The agar is said to be dissolved when
you have a clear solution without any granules the
media can now be dispensed in measured amount into
the culture tubes using a syringe or an automatic
media dispensing equipment. The agar can also be
melted in an autoclave in foil-capped Erlenmeyer
flask for 15min at 121OC. When cool to touch, it is
dispensed aseptically under flow hood into sterile
container.
STEPS BY STEP MEDIA PREPARATION TECHNIQUES
Browse and get the protocol right. Write down the step by step preparation techniques that will be followed
Distill tap water
De-ionize the distilled water
Glassware preparation (washing, oven drying and autoclaving)
Rinse all glassware with deionized water
Arrange all required reagents on the workbench to ensure their availability
Clean all apparatus to be used (measuring balance, pH meter etc)
Measure out the solid reagents
Place the beaker on magnetic stirrer, and put the deionized water that is equivalent to about 10% of the final media volume
Add all liquid stock solutions one after the other {macronutrient stock, micronutrient stock, vitamin stock)
Add weighed solid reagents (sucrose or table sugar, myo-inositol, EDTA+IRON mixture)
Add hormones
Homogenize by mixing the components well
Check and regulate the pH to the desired value
Make up to desired volume using de-ionized water
Add agar (if solid or semi-solid media)
Melt the agar @ 100oC and dispense into smaller culture vessels
Autoclave to ensure sterility
Store appropriately
NATURAL PLANT COMPONENT AS COMPARATIVE DETERMINANT OF CULTURE COMPOSITION
Plant hormones
These are otherwise referred to as phyto-hormones and are
specialized chemical substances produced by plants. Also, they
are the main internal factors controlling growth and
development. They regulate most of the life cycle events in
plants, such as germination, cell division and extension,
flowering, fruit ripening, seed and bud dormancy, and death.
Hormones are produced in one part of a plant and transported to
others, where they are effective in very small amounts.
Depending on the target tissue, a given hormone may have
different effects.
Five plant hormones have long been identified: auxin,
cytokinin, gibberellin, abscisic acid, and ethylene. Recent
discoveries of other plant hormones include brassinosteroids,
salicylates, and jasmonates
Auxin, one of the most important plant hormones, is
produced by growing stem tips and transported to other
areas where it may either promote growth or inhibit it. In
stems, for example, auxin promotes cell elongation and the
differentiation of vascular tissue, whereas in roots it
inhibits growth in the main system but promotes the
formation of adventitious roots. It also retards the
abscission (dropping off) of flowers, fruits, and leaves.
Auxins are primarily responsible for protein synthesis and
promote the growth of the plant's length. The most common
auxin, indoleacetic acid (IAA), is usually formed near the
growing top shoots and flows downward, causing newly
formed leaves to grow longer. Auxins stimulate growth
toward light and root growth.
In plants, auxins are used as herbicides, to induce fruit
development without pollination, and to induce root
formation in cuttings.
The effect of auxin on plant cells is important in
controlling plant functions called tropisms. A tropism is
a plant’s response to external stimuli that causes a
change in the direction of the plant’s growth, such as
bending, turning, or curving. When an indoor plant is
placed in a sunlit window, the plant appears to bend or
grow toward the sun. This response to the stimulus of
light is called phototropism. It is believed that light
destroys auxin where it strikes the stem, causing an
imbalance in which the side of the stem that receives less
light has more auxin. Because more auxin is present, the
cells on the darker side are able to elongate more than
the cells on the lighted side, causing the plant to bend
toward the light.
Geotropism is the response of plants to gravity. If a
growing plant is placed on its side, the stem will tend to
bend upward and the roots will tend to bend downward. As
with phototropism, this is caused by an imbalance in the
distribution of auxin. When the plant is horizontal, the
force of gravity causes the auxin to move to the underside
of the stem. Because of the increased amount of auxin, the
cells on the underside of the stem elongate more than the
cells on the upper part, causing the stem to turn upward.
In the roots, gravity also causes auxin to move to the
underside. However, in roots, the increased auxin inhibits
the elongation of cells. Thus the cells on the upper side
elongate more and the roots turn downwards.
Indole-acetic acid, the most common auxin, is usually
formed near the new stems at the top of a plant and flows
downward to stimulate the elongation of newly formed
leaves. Scientists have developed chemical compounds,
called growth substances, based on naturally occurring
auxins. These synthetic growth substances, in the form of
sprays or powders, are used to slow the sprouting of eyes,
or buds, on stored potatoes, kill broad-leaved weeds, and
prevent the premature falling of fruit and flower petals.
Growth substances are also used to produce seedless
fruits, such as tomatoes, figs, and watermelons, and to
stimulate root growth in plant cuttings.
Gibberellins are other important plant-growth hormones;
more than 50 kinds are known. Gibberellins, which form in
the seeds, young leaves, and roots, are also responsible
for protein synthesis, especially in the main stem of the
plant. Unlike auxins, gibberellins form in the seeds,
young leaves, and plant roots before flowing upward into
the stem. They control the elongation of stems, the
creation of proteins, and they cause the germination of
some grass seeds by initiating the production of enzymes
that break down starch into sugars to nourish the plant
embryo. Gibberellins are used to increase fruit size,
increase cluster size in grapes, delay ripening of citrus
fruits, speed up flowering of strawberries, and stimulate
starch break down in barley used in beer making.
More than 50 different gibberellins have been isolated and
identified; the most common is gibberellic acid.
Scientists have developed chemical compounds, called
growth substances, that are based on gibberellin hormones
and can cause the elongation of flowering stems in certain
plants during their growth period. These synthetic growth
substances can also substitute for the natural hormone in
plants where a genetic malfunction has blocked the
production of gibberellins. Growth substances are also
used to increase the yield and size of certain crops, such
as seedless grapes.
Cytokinins promote the growth of lateral buds, acting in
opposition to auxin; they also promote bud formation.
Cytokinins form in the roots and move up to the leaves and
fruit to maintain growth, cell differentiation, and cell
division. Cytokinins are used to maintain the greenness of
plant parts, such as cut flowers.
In addition, plants produce the gas ethylene through the
partial decomposition of certain hydrocarbons. Ethylene,
another inhibitor, also causes abscission, perhaps by its
destructive effect on auxins, and it also stimulates the
ripening of fruit. Thus, ethylene regulates fruit
maturation and abscission.
Among the growth inhibitors is abscisic acid, which
promotes abscission, or leaf fall; dormancy in buds; and
the formation of bulbs or tubers, possibly by preventing
the synthesis of protein.
Brassinosteroids act with auxins to encourage leaf
elongation and inhibit root growth. Brassinosteroids also
protect plants from some insects because they work against
some of the hormones that regulate insect molting.
Salicylates stimulate flowering and cause disease
resistance in some plants.
Jasmonates regulate growth, germination, and flower bud
formation. They also stimulate the formation of proteins
that protect the plant against environmental stresses,
such as temperature changes or droughts.