Composition of Plant Culture Media | Biotechnology

Plant culture media contains: 1. Macronutrients 2. Micronutrients 3. Adjuvant 4. Carbon Source.

1. Macronutrients:

Macronutrients or macroelements are basically inorganic salts. There are six major ele­ments indispensable for growth of plants—nitrogen, phosphorus, potassium, calcium, magne­sium and sulphur. Carbon, hydrogen and oxygen are obtained from the environment. In culture media concentration of these elements are well balanced depending on the formulations de­signed for morphogenesis.

Nutrients are generally taken up by the tissue recruiting passive or active mechanism at the cost of utilizing energy currency and their uptake is greatly influenced by the pH, temperature and the physiological status of culture media. As per the recommenda­tions of the International Association for Plant Physiology, inorganic salts are classified into major and minor elements based on their requirement by plants.

Requirement of the elements above 0.5 mM/L are referred to as macro or major elements and those whose requirement is less than 0.5 mM/L are categorized as microelements. All macroelements play a prolific and com­prehensive role in the growth of plants. 

Nitrogen is required for the synthesis of amino acids, proteins and nucleic acids. Calcium is an important component of the cell wall. Similarly magnesium is a key component of chlorophyll and also participates in photosynthesis.

Persistent efforts have been made in the improvisation of tissue culture media, particu­larly in the composition of macro-elements. One of the significant modifications in the improve­ment of media is the introduction of nitrogen in the form of ammonium (NH⁺₄) along with higher concentration of NO^–₃ and K⁺ to accomplish better callus growth, multiple shoot and embryogenesis.

In culture media, specific ion is provided by dissolving more than two inorganic salts, for example, nitrogen ion NH⁺₄ is contributed by both ammonium nitrate and ammonium chloride. Similarly, NO^–₃ ion is provided by ammonium nitrate and potassium nitrate after dissociation of ions in the water.

In the course of improvisation of media formulations, greater attention has been empha­sized for nitrogen salts and studies were heavily focused on its concentration. Introduction of reduced nitrogen in the form of NH⁺₄ together with the use of higher level of NO^–₃ and K⁺ ions enabled enriched media to produce callus as well as direct and indirect organogenesis.

One of the remarkable contributions in the designing of plant tissue culture media is the publication of well-balanced salt strength media by Murashige and Skoog to achieve optimum growth. Greater emphasis was shown by Murashige and Skoog to the level of nitrogen in the form of NH⁺₄ and NO^–₃. These two ions together constitute 60 mm total nitro­gen.

Although nitrogen could be supplied alone in the form of NO3, its presence alone drifts the pH towards alkalinity. Therefore, keeping the media pH in control, addition of reduced nitro­gen is absolutely essential. Presence of both the ions provides buffering strength to the culture media.

The increase of total nitrogen in MS medium was almost five times higher than total nitrogen used in Miller’s medium in 1956, 15 times than that in Hilderbrandt medium (1946) and 19 times than that in White’s medium (1943) (Table 6.1).

Similarly, K⁺ level were boosted to 20 mm which was 19 times higher than earlier media. Although MS medium was specifically designed for tobacco culture, its nutrient recipe enclaved in the media provided that it can potentiate to culture diversified nature of tissues belonging to wide range of plant species comes under monocot and dicot.

2. Micronutrients:

The essential microelements formulated in the culture media are copper (Cu), manga­nese (Mn), iron (Fe), zinc (Zn), iodine (I), and molybdenum (Mo) and cobalt (Co). Concentration of these elements varies according to the media and the type of tissue chosen for culture. Almost all microelements are indispensable for growth in vivo and in vitro systems.

Some of the other plant tissue culture media commonly employed are B₅ media devel­oped by Gamberg et al. (1968), Shenk and Hilderbrandt, SH (1972), Nitsch (1969) and certain modified MS medium like Linsmayer and Skoog (1965) were published subsequently. Gamberg B₅ media was specifically designed for the culture of soyabean cells. Suspension culture uses (NH₄)₂SO₄ as sole nitrogen source.

But utility of this media was later extended to other crop plants. SH medium was popularly used medium for callus culture of both monocotyledonous and dicotyledonous plants. Most of the microelements like Fe, Mn, Zn, Cu and Co are the com­ponents of plant cell proteins of metabolic importance. Five of these elements execute variety of functions in chloroplasts. Iron and copper are the components of electron transport chain.

Iso­lated root culture indicates requirement of Zn for normal development of roots. Manganese is generally used between 25 and 150 mM. Presence of Mn can initiate number of buds on the explant. Although aluminium and nickel were included in several media, their beneficial roles are however yet to be established.

Several researchers paid special attention to the role of iron in culture media. Iron is an essential microelement. It is generally supplemented in the form of ferrous sulphate or ferric citrate, but iron salts added in this form undergoes precipitation frequently and thereby is unavailable to growing tissue.

As a consequence, pH of the media drifted towards alkalinity. Therefore, in order to ensure availability of iron to the cultured tissues, chelated form of iron like Na₂ EDTA complex is supplied in the culture media. Addition of EDTA chelates iron to form FeEDTA complex, which greatly improves the availability of element to the plant tissue.

Most frequently used vitamins in plant culture are thiamine (B₁), nicotinic acid (niacin), and pyridoxine (B₆) in conjugated and non-conjugated form. Addition of small amount of myo-inositol (100-1000 mg/L) is beneficial for growth and morphogenesis. These are essen­tial biosynthetic pathways leading to the formation of essential cell constituents. Addition of amino acids into the culture media provides plant cells with immediate availability of nitrogen source. Generally L-glutamine, L-asparagine, tryptophenol, glycine is supplied in most culture media for the benefit of growth and morphogenesis.

3. Adjuvant:

Plant culture media on several occasions are supplemented with various types of adjuvant like coconut milk, yeast extract, casein hydrolysate and tomato juice. Coconut milk is beneficial for embryo and ovule culture. Orchid seed culture, commonly known as flasking technique, requires coconut milk in culture media. The beneficial role of coconut milk is due to its diversi­fied nutritional complexities.

Some of the main ingredients present in coconut milk are amino acids, vitamins and hormones like zeatin, IAA and gibberellins, besides sugar and organic acids (Table 6.2). Utility of coconut milk in culture media has limited use due to undefined nature of nutrient content and inconsistent results on many occasions.

4. Carbon Source:

Although plant cells are autotrophic and manufacture their own energy source, their numbers are limited. Therefore, it is indispensable to incorporate carbon source in the media for the culture of cells, tissues and organs. Sucrose is generally preferred to other carbon sources due to its efficient utilization by cells and tissues. Apart from sucrose other favourable candi­dates used as carbon source are glucose, maltose and raffinose. Other carbon sources like galac­tose and fructose are less frequently used.

Sucrose is generally used between the concentrations of 2—4%. Sucrose, when present in the culture media executes multiple functions like efficient utilization of nitrate and ammonia. Its influence on efficiency of cytokinin for cell division is exemplary. Sucrose is known to be involved in the differentiation of vascular elements in callus tissue.

The osmotic effect of su­crose in culture solution has been well documented. The rate of cell growth and shoot regenera­tion were optimal in culture media containing 3% sucrose. Increase in sucrose level exceeding optimal level, can elevate osmotic potential of the media, in turn progressively declines shoot regeneration.