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.