PLANT TISSUE CULTURE MEDIA

CULTURE MEDIA

A large number of preparations have been used for plant tissue culture in the laboratory. These preparations are called culture media. The plant requires simple inorganic molecules for its growth. Most of the molecules that are required for plant growth are synthesized by the plant from the simple inorganic molecule absorbed from the soil. As we grow plants in the laboratory these simple inorganic molecules are provided to the plant by culture media.

The culture media are classified as simple media and complex media. The simple media have all the nutrients required for plants that are measured and calibrated for better growth of plants in the laboratory.

In complex media, we do not know the exact quantity of nutrients present in the media. They may contain tomato extract, potato extract, coconut water, and other such components. The exact quantity and quality of molecules present in them are unknown.

Simple media have inorganic salt, sugar, vitamin, and hormone added in a known quantity. Now many preparations are available in powder form that can be used to prepare culture media.

The different components of culture media

The culture media have inorganic molecules, organic nutrients, vitamins, carbon sources, and hormones. 12 inorganic molecules that are essential for plant growth. These nutrients are grouped as macronutrients and micronutrients.

Macronutrients are the nutrients that are required in large quantities. Nitrogen, carbon, phosphorus, potassium, calcium, are the main macronutrients.

Micronutrients are required in small amounts, but they are also essential for the growth of plants. The micronutrients are usually required for many biochemical reactions. The micronutrients used in culture media are copper, ferric, zinc, magnesium, Boron, Cobalt, and Nickel.

Organic nutrients

The organic nutrients that are required for plant growth in culture media are vitamins, amino acids, and growth hormones. The plants can synthesize vitamins that are required for their growth by themselves, but animals require some of the vitamins to be supplemented in their diet. Plant when cultured in the laboratory requires vitamin to be added to their culture media.

VITAMINS

Vitamins used in culture media are thymine, pyridoxine, Myo-inositol, pantothenic acid, Vitamin C, D, and E. Myoinositol is a natural constituent of plant and pyridoxine is the crucial factor for the functioning of the cell membrane. This also acts as a secondary messenger. Vitamin E is an antioxidant and vitamin C prevents the blackening of tissues in culture.

AMINO ACIDS

Amino acids are synthesized by plants under normal conditions. Amino acids are provided directly to the plant tissues in a culture medium, and plants absorb and utilize them. Amino acids in nutrient media act as the source of Nitrogen to the plant. Cysteine is included in culture media as an antioxidant, and it controls the oxidation of phenolics and prevents the blackening of tissue.

CARBON SOURCE

Sucrose, glucose, and Fructose are used as a carbon source in plant tissue culture. Monocots usually require dextrose for their growth in culture media. However, dicots better perform in presence of sucrose. When sucrose is autoclaved, it is naturally broken down into glucose and fructose, which, ensures the availability of carbon sources to the plant. Further, sucrose is required for the differentiation of cells into xylem and phloem. So, for the development of vascular tissue in plant tissue culture sucrose is needed.

GROWTH HORMONES

Auxin, cytokinin, gibberellins, ethylene, and abscisic acid are required for the normal growth and development of plants. These growth hormones are supplemented into culture media.

Auxin

It is required for growth of internode, apical dominance, abscission, and rooting in tissue culture. IAA and indole 3 butyric acid, naphthalene acetic acid, 2,4-D, IBA are widely used for rooting, in interaction with cytokinin. For shoot proliferation 2,4D and 2,4,5-T are effective. Callus growth occurs in presence of 2,4-D. Auxins are usually dissolved in ethanol or in dilute NaOH.

Cytokinin

It is concerned with cell division, modification of apical dominance, and shoot differentiation. The most common cytokinin are benzyl amino purine, iso- pentenyl-adenine, furfuryl amino purine. Cytokinin is dissolved in HCl or in NaOH.

Gibberellins

There are 20 known Gibberellins. GA3 is most commonly used in culture mediums.

Ethylene

Tissue produces ethylene on its own and its production increases under stress conditions. Ethylene is also produced in the medium due to heat, oxidation, or sunlight. Ethylene induces somatic embryogenesis in Maze ( Vain et al., 1989).

Abscisic acid is required for the normal growth and development of somatic embryos.

GELLING AGENT

the culture media can be used as liquid broth which is usually used during single-cell culture. but for the formation of plantlets, semi-solid culture media is used. For this gelling agents are used such as Agar, agrose, gelrite.

Agar

Agar is produced from Gelidium amansii. It is a natural polymer that is used in 0.8 to 1 percent of the concentration.

Agarose

Agarose consists of β-D(1-3) galactose 3,6-anhydrous α-L(1-4) galactopyranose, linked into a polymer chain of 60-120 monosaccharides unit with Sulphur side group. Agarose has a high cost. It is used only when the high-strength gel is required. Agarose is used in the concentration of 0.4 %.

Gelrite

Gelrite or phytagel, gellan gum, a linear polysaccharide produced from the bacteria Pseudomonas rhamnose and cellobiose molecules. It consists of potassium, sodium, calcium, and magnesium. But it is free from organic impurities. However, it may form clumping so, when prepared, it should be continuously added by stirring.

pH

The pH of the medium is usually adjusted between 5.0 to 6 before sterilization.

In general pH of higher than 6.0 give a fairly hard medium, and pH 5.0 does not allow satisfactory gelling. The pH of the medium usually changes at different stages of preparation. So, pH is adjusted after adding the gelling agent. pH also changes when we culture plants in the medium. As a ratio of NH4⁺ and NO3^- Ion determines the pH of medium change. pH influences the availability of different iron in the medium.

DIFFERENT COMPONENTS OF CULTURE MEDIA AND THEIR FUNCTION

NUTRIENT	SOURCE	CONSTITUENT OF (ROLE)	FUNCTION
NITROGEN	NO₃, NH₄	Amino acid, proteins, hormone, and chlorophyll	Growth of plant
Phosphorus	PO₄	DNA, RNA, ATP, etc	Normal growth, plant become sickly in absence of phosphorus
Potassium	KCl, KH₂PO₄		Cell division, protein synthesis, chlorophyll synthesis
Sulfur	Na₂NO₄	Protein and amino acid
Calcium	CaCl₂	Membrane and cell wall	Promote callose formation thereby inhibiting cell extension, regulating hormone function
Magnesium	MgSO₄.7H₂O	Component of chlorophyll and co-factor of many reactions, low pH inhibits Mg absorption.	Normal chlorophyll and normal metabolic reactions
Manganese, Zinc, Iron	MnSO₄, ZnSO₄, Fe-EDTA,	Cofactor, chlorophyll, Mn and Fe interfere with the absorption of each other.	Deficiency of result in chlorosis reduced lignifications
Boron	H₃BO₃		Shoot tip necrosis
Vitamin, vitamin C, D, E, and B		Metabolism, reduce stress, prevent tissue blackening	Thymine Biosynthesis of amino acid, vitamin E acts as an antioxidant, riboflavin inhibit callus formation
Hormone Auxin Cytokinin Gibberellin Abscisic acid and ethylene	IAA, 2,4-D 2-ip, GA3		Growth and differentiation
Carbon source Sucrose Glucose and fructose			Xylem and phloem differentiation

PREPARATION OF MEDIUM

Here the preparation of medium is given for plant tissue culture.

Dissolve a given amount of prepared powder of media in 80% quantity of tissue-grade water.

Add vitamin and hormone from the stock solution.

Add sucrose.

Add gelling agent in the remaining 20% of water.

Heat the solution to obtain a clear solution or till the gelling agent dissolve completely.

Mix the two solutions to obtain the final quantity of medium.

Maintain the pH to 5.0 to 6.0.

Autoclave the medium.

Pour the medium into the culture vessels.

Allow it to cool.

Culture plant tissue in it, under aseptic condition.

SCIENCEZY