Growth and morphogenesis of plant in vitro condition require proper composition of culture media. The culture media could be solid media or liquid media. The discovery of IAA by Went and kinetin by Skoog sparks the discovery of culture media. Skoog and Miller in 1957 demonstrated that totipotency and regulation of morphogenesis is through the combination of auxins and cytokines. There are some principle components of any culture media these are
- Carbon source
- Growth regulators
- Organic substances
- Gelling agent
Nutrients There are two types of nutrients; macronutrients which should be present in amount greater than 0.5mM in a media and micronutrients which are required in less amount (<0.5mM).
- Macronutrients Some of the examples of Macronutrients are N, P, K, Ca, Mg, S (25mm/L)
- Micronutrients are required in less amount but are essential for the growth of plants. These elements are called minor elements or trace elements. The examples of micronutrients are boron (B), copper (Cu), iron (Fe), chloride (Cl), manganese (Mn), molybdenum (Mo) and zinc (Zn).
Carbon sources The plant tissue culture media, though sucrose is very frequently used (at the concentration of 2- 5%), there are many other carbohydrates that are also used, like lactose, galactose, maltose and starch but they are less effective than either sucrose or glucose. Glucose is more effective than fructose as it is used by the cells in the beginning. Other sources that can be used as alternatives, to reduce the cost of medium, are supplements of sugarcane molasses, banana extract and coconut water. These substrates also act as sources of vitamins and inorganic ions required for growth.
Auxin is synthesized in shoot apices, leaf primordial and developing seeds from amino acid tryptophan. Auxin passes from shoot tip to the region of elongation. Auxin movement is polar. It helps in elongation of both roots and shoots. e.g. IAA- universal natural auxin, IBA and IAA are also natural auxins. Synthetic auxins- 2, 4-D (2, 4- dichlorophenoxy acetic acid), NAA, IBA etc. IBA is both natural and synthetic auxin.
- Rooting Auxin stimulates root formation on the stem cutting g. IBA, NAA.
- Parthenocarpy Application of auxins (IAA, IBA) and conjugate auxins (g. IBA-alanine) to unpollinated pistils make them develop into seedless fruits or parthenocarpes.
- Flowering NAA and 2, 4- D are often employed for inducing flowering in litchi and pineapple.
- Fruits Auxins enhance sweetening of fruits g. IBA.
- Dwarf shoots Application of NAA increases the number of dwarf shoots as well as number of fruits.
Gibberellin possesses a gibloane ring structure. It promotes growth of intact stem as well as leaves. They are synthesized in apical shoot buds, root tips and developing seeds. They are weakly acidic growth hormones which increase intermodal length of genetically dwarfed plants (Pea, corn). Gibberellins transport occurs through simple diffusion as well as through conducting channels.
- Overcoming dormancy Gibberellin can be employed for breaking seed and
- Flowering GB3 can be used in inducing offseason flowering in many long day plants as well as plant requiring vernalization.
- Delayed Ripening Ripening of citrus fruits can be delayed with the help of gibberellins. This is useful in storing the fruits.
- Fruit Growth Application of GB3 increases the number and size of several fruits. g. grape, tomato.
Cytokinins are defined as compounds having highly specific hydrophilic group or adenine and other non specific lipophilic group. Cytokinins promote cytokinesis either alone or in conjugation with auxins. Roots seem to be major source of cytokinin synthesis.Coconut milk is rich source of cytokinin.
Synthetic cytokinin- kinetin
Natural cytokinin- zeatin
- Senescence Cytokinins delay the senescence of leaves and other organs. They also increase resistance to extremes of temperature and disease.
- Tissue culture Important for tissue culture because beside cell division they are also involed in morphogenesis.
- Shelf life Increases the shelf life of vegetables.
- Resistance Application of cytokinin is helpful in developing resistance to pathogens and extremes of temperature in plants.
Ethylene is a gaseous harmone which stimulates transverse or isodiametric growth but retards the longitudinal one. It is produced in plants from the amino acid methionine. Maximum synthesis of ethylene occurs during climatric ripening of fruits and tissues undergoing senescence.
- Fruit Ripening Ethylene lamps are used for fruit ripening purpose.
- Feminising Effect External supply of very small quantity of ethylene increases the number of female flowers and hence fruits in cucumber.
- Sprouting and storage organs Rhizomes, corns and other storage organs can be made to sprout early by exposing them to ethylene.
- Trophic movement makes plant ageotropes.
- Increases abscission of plant.
- Under water stress- leaves form ethylene which stimulates ABA- stomata closure.
ABA is mildly acidic growth hormone which functions as general growth inhibitor by counter acting other hormones (auxin, gibberellins and cytokinins) or reaction mediated by them.Its production occurs under stress conditions like drought, water logging and other adverse environmental conditions.
- Antitranspirant Application in minute quantity to leaves shall reduce transpiration to a great extent through partial closure of stomata.
- Flowering Induces flowering in short day plants kept under unfavorable photoperiods.
- Rooting Use of ABA promotes rooting in many stem cuttings.
- Dormancy ABA can be used in prolonging dormancy of buds, storage organs and seeds.
- Vitamins are produced endogenously. Vitamins are needed for metabolic processes in tissue culture. Since tissue synthesize vitamins in low concentration, therefore, they have to be supplemented from outside. Thiamine, nicotinic acid, pyrodoxin and calcium pentothenate are the vitamins which are commonly supplements.
- Amino acids Culture tissues are usually capable of synthesizing amino acids for various metabolic processes but we need to supplement them for stimulating cell growth in protoplast cultures and for establishing cell lines. These amino acids are carried easily by cells because they act as catalysts in many reactions. The important amino acids are L-glutamine, L-asparagine, L-glycine, L-arginine and L-cysteine.
- Other organic substances Culture media are supplemented with other organic materials that are usually extracted and are of undefined nature, for example, coconut milk, orange juice, tomato juice, mashed banana, malt and yeast extract, casein hydrolysates, potato extract. Usually 5-20% of coconut milk and 0.05-1% casein hydrolysates are used in tissue culture. Potato extract is used in anther cultures.
Growth of cultured tissues is greatly influenced by the hardness of culture medium. There are number of gelling agents used like agar, agarose and gellan gum. In plant tissue culture, agar has been used as a solidifying agent since long. The type of agar or gelling agent used can influence the growth of the tissue in culture. There are several advantages of agar, over the other gelling agents; like it get mixed with water, gets easily melt at the temperature range of 60-100°C and forms a gel that is stable at all the feasible incubation temperature. It do not get reacted with media constituents and is not digested by plant enzymes.