All tissue culture media are mostly synthetic or chemically defined. A few of them have organic complex nutrients e.g., potato extract. Although, a variety of media have been formulated to date but none of them is suitable for all plant species. The composition of White, MS and B5, the most commonly used media are listed in table below
Inorganic Nutrients Apart from C, H and O, the nutrient media provide elements essential for plant growth and referred as macro- and micronutrients depending upon their concentrations.
N, P, K, Ca, S, Mg are macronutrients and are needed in concentrations (0.5 m mol l-1 or > 0.5 mM) whereas Fe, Zn, Mn, Cu, B and Mo are micronutrients and are required in concentrations (0.5 m mol l-1). The different tissue culture media provide different concentrations of inorganic nutrients. B5 and MS media are rich, especially for K and N. Generally, iron is provided as iron-EDTA complex to keep it available at higher pH. Nitrate and ammonium are used as the sole N source and presence of NH+ resist change of pH towards alkalinity. The vitamins are required for callus growth. These include inositol, thiamine, pyridoxine and nicotinic acid. Among these thiamine is most essential for optimum callus growth.
Carbon Source The most commonly used carbon source for all cultured plant materials is sucrose (20-50 gl-1 or g/l). In certain monocots, glucose may be better than sucrose. The other sugars like maltose, galactose, lactose, mannose and starch may also be used by some plants.
Growth Regulators The growth regulators (GRs) which are used in plant tissue culture include: Auxins, IAA (Indole-3-acetic acid), IBA (Indole-3-butyric acid), NAA (Napthalene Acetic Acid), NOA (Naphthoxy Acetic Acid), 2, 4-D (2, 4-dicholorophenoxy acetic acid) to support cell division and callus growth (especially 2,4-D), somatic embryo induction and rooting, cytokinins like kinetin, FAP (Furfurylamino purine), BAP (Benzylamino purine), zeatin, 2-ip (Isopentenyl adenine), TDZ (Thidiazuron, a compound having cytokinin activity) are employed to promote cell division, regeneration of shoot, somatic embryo induction and proliferation and growth of axillary buds. Abscisic acid (ABA) promotes SE and shoot bud regeneration in many species and markedly improves SE maturation. Among the 20 gibberellins known, GA3 is exclusively used to promote shoot elongation and SE germination. The concentrations used of various growth regulators are as follows: auxins (0.1-3 mg/l); cytokinins (0.1-3 mg/l); ABA up to (0.2 mg/l); and GA3 (0.1-1 mg/l).
Complex Organic Additive These additives are used only when synthetic media do not work and are not preferred now days. In earlier studies, coconut milk, casein hydrolysate and tomato juice were used.
Callus and Suspension Culture
The explants cultured on a suitable media undergo division. The mature cells differentiate to become meristematic cells and this process is called dedifferentiation. Dedifferentiation involves renewed and enhanced RNA and protein synthesis leading to the formation of new cellular components required for meristematic activity. Initially, cell divisions are confined to the cut ends, but finally it covers the entire explants. The resulting cell mass is ordinarily unorganized and is defined as Callus but actually it consists of several cell types including fibers and vascular elements.
Callus Culture needs to be sub-cultured every 3-5 weeks on agar solid media due to cell growth, nutrient depletion and medium drying. Therefore, calluses are easy to maintain and are the most widely used.
Suspension Culture Tissues and cells cultured in a liquid medium produced a suspension of single cells and cells clumps, these are called suspension cultures. Liquid cultures require continuous agitation and generally by a gyratory shaker at 100-250 rpm (revolution per minute), the aeration and dissociation of cell clumps into smaller piece are achieved. Suspension cultures grow much faster than callus cultures and need to be subcultured about every week. Suspension cultures are the only system for a large scale production of cells and somatic embryos (SEs)- (Somatic embryos are the cells that are formed from the cells that are not involved in the development of embryos). These are broadly grouped as follows
- Batch Cultures
- Continuous Cultures
- Immobilized Cell Cultures
- Batch Cultures: In a batch culture, the medium and the cells produced from explants are grown in culture flasks (100-250 ml) and fermenters
- Continuous Culture: In a continuous culture, the cell population is maintained in a steady state by regularly replacing a portion of the used or spent medium by fresh medium. These systems are of following two types
- Closed Continuous Culture: The cells are separated from the used medium taken out for replacement and added back to the culture so that cell biomass keeps on increasing and replaced by equal volume of fresh medium. The replacement volume is so adjusted that cultures remains at sub-maximal growth indefinitely.
- Open Continuous Cultures: The open cultures are two main types i.e., turbidostat or chemostat types. In a turbidostat, cells are allowed to grow upto a preselected turbidity level which is measured as OD when a predetermined volume of the culture is replaced by fresh normal culture medium
Tissue Culture Media 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).
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).
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.
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.