Components Of Plant Tissue Culture Media
The conventional means of food production are not sufficient to cater to the needs of growing populations. So, new breeding techniques like tissue culture are helpful for the development of high-yielding and disease-resistance varieties. Plant tissue culture is a fast and effective way to multiply plant tissue and cells to produce a desired product. Tissue culture is the technique of growing cells, tissues, or organs in a sterilized culture medium under controlled environmental conditions. It is clear from the definition of tissue culture that we require artificial media for in-vitro culture. Some components of plant tissue culture media are inorganic salts, water, plant tissue culture media, vitamin, micro-nutrients, etc. Basically, the plant tissue culture media should contain the same nutrients as required by the whole plant.
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Naturally, plants grow in soil but to obtain specific changes in varieties of plants, they are made to grow in culture media. Media is a substance solid, semi-solid, or liquid necessary for the nurturing of the explant. The part of the plant used for tissue culture is called an explant. Plant tissue culture media is crucial for the plant growth and development of cells in-vitro. It provides rapid multiplication, meristem culture that are free from virus and other infections, shoots and root regeneration, etc. Media varies depending on their concentrations as well as constituents. The components of plant tissue culture media are :
- INORGANIC SALTS
- ORGANIC COMPOUNDS
- PLANT GROWTH HORMONES
- GELLING AGENTS
Components of Plant Tissue Culture Media
Plant tissues in culture require a continuous supply of certain inorganic chemicals to provide essential macro and micronutrients. The inorganic salt formulations can vary as per the requirements. These inorganic salts or mineral elements are specific and are directly involved in the metabolism of the plant. The element must be absolutely necessary for supporting the normal growth of the plant and its reproduction. The essential elements which are required in relatively large amounts are termed macronutrient elements. These include carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, calcium, potassium, and magnesium. In addition to the macronutrient elements, plant cells require traces of certain micronutrients. All plants require some micronutrients which are iron, manganese, zinc, boron, copper, molybdenum, and chlorine. Stocks are always prepared with glass-distilled or demineralized water. Macro and micronutrients are generally added to the medium as salts so that it is more easily available for plants.
The plant growth and culture require some complex organic supplements. So, fruit juices and fresh coconut milk are some of the common organic supplements.
Vitamins perform catalytic functions in enzyme systems and are required only in trace amounts. Usually, thiamine is the only essential vitamin for all plant tissue culture media. Whereas, niacin and pyridoxin may stimulate growth. The need for thiamine in tissue cultures is especially evident at low levels of cytokinin. Some other vitamins in plant tissue culture media include ascorbic acid, biotin, folic acid, riboflavin, and calcium pantothenate. It is good to store vitamin stock in the freezer.
AMINO ACIDS :
Amino acids are not common in plant tissue cultures except for glycine. We use certain amino acids in the plant tissue culture medium that are beneficial for plant cell growth. Some of the amino acids that most frequently produce positive results are L-aspartic acid, L-asparagine, L-glutamic acid, etc.
Activated charcoal absorbs many organic, and inorganic molecules, toxins, and inhibitory compounds. So it removes contaminants from agar. It is important to note the type of activated charcoal we add to the culture because the adsorptive characteristics are dependent on the manufacturing process.
Green cells in culture are generally not photosynthetically active and require a carbon source. Other carbohydrate sources, such as fructose and starch are also useful. Sucrose is majorly required as an energy source.
ANTIBIOTICS IN PLANT TISSUE CULTURE MEDIA
Because of excessive contamination problems with certain plant explants, we incorporate fungicides and bactericides in the culture medium. Transformation experiments using Agrobacterium make it necessary to incorporate antibiotics into the medium. Several antibiotics are toxic to the explant but, at the same time control or eliminate the Agrobacterium. Commonly used antibiotics are vimentin, carbenicillin, cefotaxime, and augmentin.
PLANT GROWTH REGULATORS IN CULTURE MEDIA
The plant growth and development of plants are coordinated by hormones, also called plant growth regulators (PGRs), through various signalling pathways. The addition of these PGRs to plant tissue culture media may have different kinds of effects depending upon their concentration and the ratio between different PGRs.
The type and concentration of plant growth regulators will vary according to the cell culture purpose. An auxin ( IAA, NAA, 2,4-D, or IBA) is necessary for most plant cells for division and root inhibition. At high concentrations, auxin can suppress morphogenesis. The auxin 2,4-D is widely useful in callus induction: IAA, IBA, and NAA are useful for root induction.
Cytokinins (kinetin, BA, zeatin, and 2iP) promote cell division, shoot proliferation, and shoot morphogenesis. Thidiazuron has cytokinin activity and is commercially useful as a cotton defoliant. It is effective in low concentrations to stimulate shoot formation. Cytokinin stocks can be stored for several months in the refrigerator. Kinetin and zeatin are thermostable.
Gibberellins are useful to induce organogenesis, particularly adventitious root formation. Since it can inhibit callus growth and auxin-induces adventitious root formation, we use gibberellin (GA3) frequently in plant cell culture. This growth hormone is essential for the induction of lateral shoots.
Abscisic acid (ABA), a plant hormone involved in leaf and fruit abscission and dormancy, so, useful in embryo culture. ABA is heat stable but light sensitive and its stock solutions are prepared in water. The partial conversion of the 2-cis isomer of ABA to the 2-trans isomer of lower biological activity occurs in the light.
In the case of a semi-solid or solid matrix, the gelling agent is used to prepare the matrix. The most common gelling agent is agar. Agar provides no possible presence of toxic substances, growth stimulants, or vitamins. It is a mixture of high molecular weight polysaccharides agarose and agaropectin that are obtained from red algae and some organic and inorganic compounds.
Agar binds to the water molecules to form gel but it is aerated even after solidification. This makes it possible for the tissue to grow and remain aerated. Some other gelling agents are pectin, alginate, modified starch, carrageenan, etc.
pH OF PLANT TISSUE CULTURE MEDIA
The pH of plant tissue culture media determines the acidity or alkalinity of the solution, hence the viability and proper functioning of the solution. The cells may disrupt if the pH is too high or too low, so it is acceptable between 5.5 to 6. Below 5.5, the agar will not gel properly and above 6.0, the gel may be too firm. Media pH generally drops by 0.6 to 1.3 units after autoclaving. Cultures of some plant tissues cause a pH drop over time due to the production of organic acids or nitrogen utilization. pH is influenced by the inorganic salts, carbohydrate source, gelling agent, activated charcoal, and medium storage method.
Thus, these are the components of plant tissue culture media essential for the in-vitro culture of plant tissues. Many advances are possible in this field as the advantages and drawbacks of many components are unknown.