Micropropagation also referred as tissue culture is used to multiply plants such as those that have been genetically modified or bred through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction.
In short, steps of micropropagation can be divided into 4 stages.
Micropropagation begins with the selection of plant material to be propagated. The plant tissues are removed from an intact plant in a sterile condition. Clean stock materials that are free of viruses and fungi are important in the production of the healthiest plants. Once the plant material is chosen for culture, the collection of explant(s) begins and is dependent on the type of tissue to be used; including stem tips, anthers, petals, pollen and other plant tissues. The explant material is then surface sterilized, usually in multiple courses of bleach and alcohol washes, and finally rinsed in sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed on a growth medium, typically containing sucrose as an energy source and one or more plant growth regulators (plant hormones). Usually the medium is thickened with agar to create a gel which supports the explant during growth. Some plants are easily grown on simple media, but others require more complicated media for successful growth; the plant tissue grows and differentiates into new tissues depending on the medium. For example, media containing cytokinin are used to create branched shoots from plant buds.
Multiplication is the taking of tissue samples produced during the first stage and increasing their number. Following the successful introduction and growth of plant tissue, the establishment stage is followed by multiplication. Through repeated cycles of this process, a single explant sample may be increased from one to hundreds and thousands of plants. Depending on the type of tissue grown, multiplication can involve different methods and media. If the plant material grown is callus tissue, it can be placed in a blender and cut into smaller pieces and recultured on the same type of culture medium to grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones are often added that cause the plantlets to produce many small offshoots. After the formation of multiple shoots, these shoots are transferred to rooting medium with a high auxin\cytokinin ratio. After the development of roots, plantlets can be used for hardening.
This stage involves treating the plantlets/shoots produced to encourage root growth and "hardening." It is performed in vitro, or in a sterile "test tube" environment.
"Hardening" refers to the preparation of the plants for a natural growth environment. Until this stage, the plantlets have been grown in "ideal" conditions, designed to encourage rapid growth. Due to the controlled nature of their maturation, the plantlets often do not have fully functional dermal coverings. This causes them to be highly susceptible to disease and inefficient in their use of water and energy. In vitro conditions are high in humidity, and plants grown under these conditions often do not form a working cuticle and stomata that keep the plant from drying out. When taken out of culture, the plantlets need time to adjust to more natural environmental conditions. Hardening typically involves slowly weaning the plantlets from a high-humidity, low light, warm environment to what would be considered a normal growth environment for the species in question.
In the final stage of plant micropropagation, the plantlets are removed from the plant media and transferred to soil or (more commonly) potting compost for continued growth by conventional methods.
This stage is often combined with the "pretransplant" stage.
In Meristem culture the meristem and a few subtending leaf primordia are placed into a suitable growing media. An elongated rooted plantlet is produced after some weeks, and is transferred to the soil when it has attained a considerable height. A disease free plant can be produced by this method. Experimental result also suggest that this technique can be successfully utilized for rapid multiplication of various plant materials, e.g. Sugarcane, strawberry.
A callus is mass of undifferentiated parenchymatous cells. When a living plant tissue is placed in an artificial growing medium with other conditions favorable, callus is formed. The growth of callus varies with the homogenous levels of auxin and Cytokinin and can be manipulated by endogenous supply of these growth regulators in the culture medium. The callus growth and its organogenesis or embryogenesis can be referred into three different stages.
In embryo culture, the embryo is excised and placed into a culture medium with proper nutrient in aseptic condition. To obtain a quick and optimum growth into plantlets, it is transferred to soil. It is particularly important for the production of interspecific and intergeneric hybrids and to overcome the embryo .
In protoplast culture, the plant cell can be isolated with the help of wall degrading enzymes and growth in a suitable culture medium in a controlled condition for regeneration of plantlets. Under suitable conditions the protoplast develops a cell wall followed by an increase in cell division and differentiation and grows into a new plant. The protoplast are first cultured in liquid medium at 25 to 28 C with a light intensity of 100 to 500 lux or in dark and after undergoing substantial cell division, they are transferred into solid medium congenial or morphogenesis in many horticultural crops respond well to protoplast culture.
Micropropagation has a number of advantages over traditional plant propagation techniques:
Micropropagation is not always the perfect means of multiplying plants. Conditions that limits its use include:
The major limitation in the use of micropropagation for many plants is the cost of production; for many plants the use of seeds, which are normally disease free and produced in good numbers, readily produce plants (see orthodox seed) in good numbers at a lower cost. For this reason, many plant breeders do not utilize micropropagation because the cost is prohibitive. Other breeders use it to produce stock plants that are then used for seed multiplication.
Mechanisation of the process could reduce labour costs, but has proven difficult to achieve, despite active attempts to develop technological solutions.