Micropropagation is in vitro regeneration of plants from organs tissues, cells or protoplast with aim of obtaining clones which are virus free or is to preserve germplasm by cryopreservation. The main functions of micropropagation are
i. Propagation of plants.
ii. Rapid multiplication of new varieties prior to their propagation by conventional methods. It provides a mean of germplasm storage for maintenance of disease-free stock.
In this technique, a small piece of plant tissue (explant) is taken from the donor plant and cultured on a nutrient medium providing sterile conditions. By altering the composition of the medium and the environmental conditions (temperature, light regime, etc.) the development of this piece of tissue can be directed along different patterns and finally the whole plant can be regenerated. They are called as clones, as the offspring produced after the cultured growth has same genetic make-up as that of the parent explants
The methods mainly used for micropropagation are
i. Callus culture followed by organogenesis or embryogenesis.
ii. Proliferation of auxiliary or adventitious buds resulting from repeated subculture on multiplication media containing cytokinin.
iii. Somatic embryo has been used for micropropagation of several plant species and in some species, they are the only route available for this purpose e.g. oil palm, date palm etc.
iv. Microcutting from auxiliary buds of apically dominant shoots grown on hormone free or low cytokinin.
Stages of Micropropagation
Selection and maintenance of stock plants for culture initiation: The first steps in micropropagation involve selection and growth of Stock plants under controlled and hygienic conditions to reduce the risk of contamination.
Initiation and establishment of aseptic culture on appropriate medium
Explant isolation:– Virtually any part of the plant can be used as explant like vegetative parts (Shoot tip, meristem, leaves, stems, roots) or reproductive parts (Anthers, pollen, ovules, embryo, seed, spores). Shoot tip and auxiliary buds are most often used. Size of explant, age of the stock plant, physiological age of explant, developmental age of explant these are some of the factors which decide the success rate of stage I.
Surface sterilization:– Explants are surface sterilized by treating it with disinfectant solution of suitable concentration for a specific period. Ethyl alcohol, bromine water, mercuric chloride, silver nitrate, sodium hypochlorite, calcium hypochlorite etc. can be used as disinfectant.
Washing:– Washed with water.
Multiplication of shoots: In this stage, rapid multiplication of the regenerative system is carried out for obtaining large number of shoots. About 4.3 X 107 shoots can be produced from a single starting explant in a year.
Cultures obtained from stage I are placed on a suitable medium containing an increased proportion of cytokinin to produce numerous shoots. This stage can be repeated a few cycles until a desired number of shoots are developed to carry out for rooting.
Rooting of regenerated shoots: In stage III, shoots from stage II are prepared to transfer to soil. Shoots are separated manually from clusters and transferred on a rooting medium that contains a suitable concentration of auxin. Elongation of shoots prior to rooting, rooting of shoots, and prehardening cultures to improve survival are some of the activities carried under this stage. Sometimes, shoots are directly established in the soil as micro-cuttings to develop roots.
Transfer of plantlets to sterilized soil for hardening under greenhouse environment.
• It is a fast method of propagation, producing thousands of plantlets in a matter of months.
• Healthy plant material is ensured since soil and disease-causing organisms are excluded during the propagation cycle.
• The method is programmable to meet specific targets of time and quantity because it is independent of seasonal changes and the weather.
• Micropropagation saves an enormous amount of care usually required by cuttings and seedlings (watering, weeding, spraying etc.)
• Excess material produced can often be stored over long periods.
• Species and cultivars can be stored in small spaces.
• This technique is a complex procedure and it has varied procedures depending on the plant type.
• The experiments of tissue culture must be handled by highly trained people as the procedure requires special and careful observations.
• As all the plants are genetically similar, there is reduction in genetic diversity.
• Monoculture is produced due to tissue culture and all the progeny may be vulnerable to the same infections or diseases.