MICROPROPAGATION
Micropropagation is the propagation of plants by using plant cells, tissues, and organs with the help of tissue culture technique. Tissue culture is a technique of maintaining, growing and multiplying cells, tissues, organs and plants on artificial medium in suitable containers under aseptic controlled environmental conditions.
The first successful culture experiment was carried out by White (1932). He cultured and subcultured tomato root on artificial medium having yeast extracts. In 1939, three workers, White, Nobecourt and Gautheret independently perfected the technique of tissue culture. They were able to develop callus. Callus is an irregular undifferentiated and inorganised mass of actively dividing cell.
In micropropagation, small pieces of plant organs or tissues are grown in a container in suitable nutrient medium under sterilized and controlled conditioned. The tissue grows into a mass of undifferentiated cells called callus. The callus later differentiates into plantlets which are separated and grown into full-size normal plants in the nurseries or in the fields. The plant part used in tissue culture is called explant.
Micropropagation involves the following steps:
Selection of an explant: Explant is the plant part that is used for initiating a culture. Plant parts such as cotyledons, hypocotyl, stem, leaf, shoot apex, root apex, young inflorescence, petals, anthers, young embryo, pollen grains etc., can be induced to form callus. Selection of a specific explant is often necessary for successsful plant regeneration. For example, embryonic tissue is required for culture of cereals and shoot apices are suitable for micropropagation of woody plants.
Aseptic conditions: Absolute aseptic conditions are necessary for obtaining healthy plantlets. Therefore, all equipments, culture medium, glasswares and explants are properly sterilized to avoid growth of microorganisms. All types of apparatus, including containers, forceps, needles and the nutrient medium are sterilized in an autoclave (temperature over 120 degrees centigrade) for 15 to 30 minutes. Inoculation chamber is disinfected with ultraviolet rays. Culture room is kept dust free, disinfected at a temperature of 20 to 25 degrees centigrade. Thermolabile substances such as hormones and vitamins are sterilized by filtering their solution through a bacterial filter. Sterilization of explant is done by various chemical agents, such as, absolute alcohol, calcium or sodium hypochlorite, bromine water, hydrogen peroxide, mercuric chloride, antibiotics etc.
The choice of chemical and duration of treatment depends on the sensitivity of the material to be sterilized. The effectiveness of the sterilizing agents can be increased by adding a very small amount of detergent to the sterilizing agent. It is, however, essential to wash the explant thoroughly with distilled water after sterilization in order to remove the traces of sterilizing agent which may otherwise seriously affect the growth of the culture.
Nutrient or culture medium: The nutrient of culture medium is selected depending upon the plant material to be cultured. The basic components of all nutrient media for plant tissue culture are inorganic nutrients, carbohydrates, vitamins, growth regulators, solidifying agent (for example, Agar) and water. The nutrient medium may be liquid, semisolid or solid. The semisolid or solid media are prepared by heating the nutrient solution with less than 1% agar or gelatine. The nutrient solution contains 2 to 4% sugar, vitamins, minerals and growth regulators. The growth regulators can be replaced by organic complexes like coconut milk, banana pulp or yeast extract. The pH of the medium is adjusted to 5.0 to 6.0.
The desired amount of sterilized culture medium is poured in an appropriate culture vessel such as a vessel, flask, tube, petri dish, etc. These are then plugged with nonabsorbent and sterilized cotton wool. The process is carried out under aseptic condition in the culture laboratory.
The next step is inoculation or transfer of the explant. These are placed on suitable nutrient medium in culture vessel under sterilized condition such as inoculation chamber or laminar flow cabinet.
The cultures are maintained under controlled conditions of temperature and light, the temperature is controlled between 20 and 25 degree centigrade. After four to six weeks, the plant tissue gets changed to an unorganized and undifferentiated mass of actively dividing cell called callus. Darkness favours callus formation. After some time as the dry matter increases, the amount of nutrients in the medium decreases and the medium also gets reduced due to evaporation. The tissues growing in the culture, therefore, begin to die. Hence, the tissues are regularly transferred to new containers with fresh media. This is called subculturing.
Differentiation or morphogenesis occurs when the callus is exposed to light or provided with proper dosage of auxin and cytokinin. The root formation occurs in higher concentration of auxin while the shoot formation occurs when the medium has higher concentration of cytokinin. When both the hormones are available in sufficient quantity, both shoots and roots appear. The regenerated plants are transferred to greenhouse or field conditions after their acclimatization.
Advantages and limitations of micropropagation
Advantages of micropropagation: In the recent years, micropropagation is increasingly used for rapid clonal multiplication of several economically important plants. Seeds of some economically important plants take a very long time to germinate and establishment of their young seedling is affected by several factors. Micropropagation is helpful in such case as a large number of plants can be obtained in very short span of time. Since meristematic tips of plants are usually virus free, so it is possible to get virus-free plants by this method. Such virus-free plants materials are useful for long-term storage. They can be put alive for several years by sub culturing periodically. Several ornamentals (for example, carnations, chrysanthemums, orchids, asparagus) and economically important plants (for example, potato, sugarcane, tapioca) are multiplied by micropropagation.
Limitation of micropropagation: Micropropagation technique cannot be applied for all plant species. Plantlets of many economically important plants obtained through micropropagation fail to establish when transferred to soil. When the callus is obtained by culturing relatively mature and differentiated cells, it often produces polyploids. As a result, the cloned plants may not possess the desired character.