Plant Propogation and Nursery Management

Culture Techniques and Medium

Culture Techniques:-
Various culture techniques such as (i) meristem culture (ii) callus culture (iii) shoot bud regeneration (iv) somatic embryogenesis (v) ovule culture (vi) embryo culture (vii) anther culture and (viii) protoplast culture are employed in micropropagation.

1. Meristem culture: Meristem culture involves culture of both shoot-tip and axillary-bud. The use of small shoot-tips comprising of the apical dome with one or two leaf primordia (0.1-0.5 mm) is the basis for the technique known as meristem-tip-culture, pio¬neered by Morel in the 1950s. Meristem tip culture is now being routinely used, mainly in horticultural crops, for the elimination of virus from infected material. Virus apparently either does not easily invade or rapidly multi¬ply in the young meristematic tissue. A simple nutrient medium consisting only of salts, sucrose and vitamins is used in order to minimize the formation of callus. Gibberellic acid is often needed to promote adequate growth and NAA may be required to stimulate root formation.
2. Callus culture: A piece of sterile plant tissue with living cells is transferred¬ to a culture medium to induce callus proliferation. Sub-culturing is then done onto a medium with or without altered growth regulator concentrations, ultimately resulting in the induction of adventitious or¬gans or embryos. In the last stage, regenerated plants are removed from in vitro culture and slowly exposed to outer environment so that the plants can be fully autotrophic.
3. Cell culture: The cells are maintained in suspension cultures so as to produce free cells and are then sub-cultured to regenerate complete plant from single cells. This technique is now useful to induce variability in plant cells and to select desirable cell variants and regenerate complete plants from these variants.
4. Embryo culture: It involves aseptic excision of the embryo and its transfer to a suitable medium for development under optimum culture conditions. After the embryo has grown into a plantlet in vitro, it is transferred to sterile soil or vermiculite and grown to maturity in a green house. This technique is useful in the production of intetspecific and intergeneric hybrids which could not be otherwise accomplished and also in overcoming embryo abortion.
5. Protoplast culture: From different sources, protoplasts (the plant without any rigid cellulose wall but with plasma membrane only allowed to fuse to form a somatic hybrid) are cultured in suitable media to regenerate the cell wall and are again cultured in suitable medium for differentiation and morphogenesis.
6. Anther culture: The culture of anthers is of considerable value to breeders as it is possible to produce haploid plants which reveal recessive alleles. These haploid plants can be used for the production of homozygous diploids, thus avoiding generations of inbreeding. Added benefits, such as small flowers and prolonged flowering time, might be ensured from the use of haploid plants as they are usually smaller than their diploid counterparts and being sterile there will be no pollination-induced senescence. Anther culture has been used in Pelargonium spp. to eliminate virus, in Lilium spp. to produce haploid plants and in Gerbera to obtain different flower colour.
7. Somatic embryogenesis: The greatest potential for clonal multiplication is through somatic embryogenesis, where technically a single isolated cell can produce first an embryo, then a complete plant. Somatic embryogenesis and plantlet regeneration has been report¬ed in various species of horticultural plants by using mid-rib, leaf and stem callus on modified MS basal medium supplemented with 1.0 – 2.0 mg/l 2,4-D and 0.25-0.50 mg/l BA or kinetin.

Culture medium

• Success in the technology and application of tissue culture methods depends on the selection of proper culture medium, which meets the nutritional requirements of cultured cells and tissues. The basic components of all nutrient media are inorgan¬ic salts, carbohydrates, vitamins, growth regulators, agar (for solid medium) and water. Other components including organic nitrogenous compounds, organic acids and complex substances can be important but are optional.
• The inorganic nutrients required in macro-amounts are N,P, K, Ca, S and Mg. The optimum concentration of each nutrient for achieving maximum growth rates varies considerably. The essential nutrients required in micro-molar concentration include Fe, Mn, Zn, B, Cu and Mo. The concentration of inorganic nitrogen in the culture medium varies from 25 to 50 mM. Nitrate is commonly used in the range from 25-40 mM and the amount of ammonium varies between 2 and 20 mM. Potassium is supplied in the range of 20 mM or higher and the optimum concentrations of P, Mg, Ca and S for most tissues are 1-3 mM.
• The standard carbon source is sucrose or glucose at a concentration of 2-3%. Other carbohydrates which have been tested include fructose, lactose, maltose, galactose and starch but these compounds are generally much inferior to sucrose or glucose as a carbon source. Most media contain myo-inositol. There is no absolute requirement of myo-inositol, but its inclusion (100 mg/l) generally improves tissue growth. MS medium is the most popularly used nutrient medium in tissue culture.
• The nutrient media are supplied with certain vitamins. There is an absolute requirement (0.1-0.4 mg/1) for thiamine. Growth is also improved by the addition of nicotinic acid and pyridoxine; sometimes biotin, vitamin B12, folic acid, choline chloride, riboflavin and ascorbic acid are also added to the culture medium, depending on the type of tissue to be cultured.
• The pH of the culture medium is an important factor for maintenance of growth of cultured plant tissues. In general, plant tissues require an acidic pH, and an initial pH of 5.5 - 5.8 is optimum. The physical form of the culture medium is also an important factor determin¬ing the success or failure of tissue culture. Both liquid and solid form of culture medium are useful for initiation and proliferation of callus tissue and initial organogenesis, but for further growth of organs solid medium is essential. When culturing in a liquid medium, gentle agitation of the liquid is necessary for aeration of culture medium, rotating apparatus is used for this purpose.