Nutrient media

The intact plants can make their own food but the in vitro culture of plant parts or cells requires a variety of nutrients and suitable physical conditions for their growth. The composition of plant tissue culture medium depends upon the type of plant tissues or cells that are used for culture. No single medium can be used for all types of plants and organs, so the composition of the culture medium for each plant material has to be worked out. A typical nutrient medium consists of the following components:

a) Inorganic nutrients (both micro- and macro-elements - C, H, O, N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B, Mb), The six elements namely nitrogen, phosphorus, potassium, calcium, magnesium and sulfur are the essential macronutrients for tissue culture. The ideal concentration of nitrogen, and potassium is around 25 mmol l-1 while for calcium, phosphorus, sulfur and magnesium, it is in the range of 1-3 mmol l-1.
Among the micronutrients, iron requirement is very critical. Chelated forma of iron and copper are commonly used in culture media.

b) A carbon source and energy source (usually sucrose) )- Plant cells and tissues in the culture mediumare heterotrophic and therefore depend on the external carbon for energy. Among the various energy sources, sucrose is the most preferred. During the sterilization of the medium, sucrose gets hydrolysed to glucose and fructose and the plant cells utilize first the glucose and than the fructose. The other carbohydrates such as lactose, maltose, galactose etc have been used in culture media but with limited success.

c) Organic supplements vitamins (e.g. nicotinic acid, thiamine, pyridoxine and myo-inositol), amino acids (e.g. arginine) The plant cells in culture are able to synthesize vitamins just like natural plants, but in suboptimal quantities which does not support proper growth of cells in culture. Therefore the medium is supplemented with vitamins to achieve good growth of cells. Similarly amino acids are added to the cell cultures to stimulate the cell growth and estabilish the cell lines. Organic acids especially the intermediates of krebs cycle e.g. citrate, malate, succinate, pyruvate also enhances the growth of plant cells. Sometimes antibiotics (e.g. streptomycin, kanamycin) are also added to the medium to prevent the growth of the microorganisms.

d) Growth regulators (e.g. auxins, cytokinins and gibberellins Plant hormones play an important role in growth and differentiation of cultured cells and tissues. The growth hormones included in culture media involve: auxins, cytokinins, and gibberellins. The auxins facilitate the cell division and root differentiation. The cytokinins induce cell division and differentiation and the gibberellins is mainly used to induce plantlet formation from adventive embryos formed in culture. Auxins induce cell division, cell elongation, and formation of callus in cultures. 2,4-dichlorophenoxy acetic acid is one of the most commonly added auxins in plant cell cultures. Cytokinins, promotes RNA synthesis and stimulate protein and enzyme activities in tissues. Kinetin and benzyl-aminopurine are the most frequently used cytokinins in plant cell cultures.
The ratio of auxins and cytokinins play an important role in the morphogenesis of culture systems. When the ratio of auxins to cytokinins is high, embryogenesis, callus initiation, and root initiation occur. For axillary and shoot proliferation, the ratio of auxins to cytokinins is kept low. Among the gibberellins, gibberellin A3 (GA3) is the most commonly used for tissue culture. GA3 enhances callus growth and induces dwarf plantlets to elongate.

e) Solidifying agents like agar. Generally a gelling agent agar (a polysaccharide obtained from red algae, Gelidium amansil) is added to the liquid medium for its solidification. . The agar obtained from seaweeds provides solid surface for the growth of cells because in the liquid medium, the tissue will be submerged and die due to lack of oxygen. Cells are grown in suspension medium with out agar but such cultures are aerated regularly either by bubbling sterile air or by gentle agitation. Some other less frequently used solidifying agents are biogel (polyacrlyamide pellets), phytagel, gelrite, and purified agarose.

f) Other compounds like casein hydrolysate, coconut milk, malt extract, yeast extract, tomato juice, etc. may be added for specific purposes.

g) pH - An optimum pH (usually 5.7) is also very important. At pH higher than 7.0 and lower than 4.5, the plant cells stop growing in cultures. The most extensively used nutrient medium is MS medium (developed by Murashige and Skoog in 1962).

Major types of media

White’s medium - is one of the earliest plant tissue culture media

MS medium - formulated by Murashige and Skoog (MS) is most widely used for many types of culture systems

B5 medium - developed by Gamborg for cell suspension and callus cultures and at present it’s modified form used for protoplast culture

N6 medium - formulated by Chu and used for cereal anther culture
Nitsch’s medium developed by Nitsch and Nitsch and used for anther culture

Preparation of media

The methodology for media preparation involves preparation of stock solutions (in the range of 10x to 100x concentrations) of highly purified chemicals and demineralized water. The stock solutions are stored in glass or plastic containers and frozen till further requirement. Now a days, plant tissue culture media are commercially prepared, and are available in the market as dry powders. The culture media is usually sterilized in an autoclave at 1210C and 15 psi for 20 minutes. Hormones and other heat sensitive organic compounds are filter sterilized and added to the autoclaved medium.

Maintenance of Aseptic Environment

It is very important to maintain aseptic environment during the in vitro culture of plant cells and tissues. Following are some of the methods adopted for sterilization:

(a) Sterilization of Glassware- The glassware can be sterilized in a hot air oven at 160-1800C for 2-4 hours.

(b) Sterilization of instruments- The metallic instruments are incinerated by dipping them in 75% ethanol followed by flaming and cooling.

(c) Sterilization of nutrient media- The culture media are transferred into glass container, plugged with cotton or sealed with plastic closures and sterilized by autoclaving at 15 psi for 30 min. The autoclaving denatures the vitamins, plant extracts, amino acids and hormones therefore the solution of these compounds are sterilized by using Millipore filter paper with pore size of 0.2 micrometer diameter.

(d) Sterilization of plant materials- The surface of the plant material is made sterile by using disinfectants e.g. sodium hypochlorite, hydrogen peroxide, mercuric chloride, or ethanol. The transfer of sterile plant material on to the nutrient medium is done under the cabinet of laminar airflow.

(e) Sterilization of Culture room and transfer area- the floor and walls of the culture room should be washed with detergent followed by 2% sodium hypochlorite or 95% ethanol. The sterilization can also be done by exposure to UV light. The cabinet of laminar air flow is sterilized by exposing to UV light for 30 min. and 95% ethanol 15 minutes before starting the work.