Micropropagation procedure is divided in stages for the sake of understanding. Murashige proposed three (I to III) stages, Debergh and Maene added stage ‘0’. Currently we have accepted five stages procedure (0 to IV).
- Stage 0
- Stage I
- Stage II
- Stage III
- Stage IV
Stage 0
Selection and maintenance of stock plants for culture initiation
This stage was basically introduced to overcome the problem of contamination. Stock plants are grown under more hygienic conditions to reduce the risk of contamination.
Stage I
Initiation and establishment of aseptic culture
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.
Establishment of explant on appropriate medium – There is no one universal culture medium; however modifications of Murashige and Skoog basal medium (Murashige and Skoog, 1962) are most frequently used.
Stage II
Multiplication of shoots or somatic embryo formation (rapid) using a defined culture medium.
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. Normally, medium for stage I and II is same, but cytokinin proportion is increased for stage II to produce numerous shoots. This stage can be repeated a few cycles until an desired number of shoots are developed to carry out for rooting. Factors which can affect shoot multiplication are physiological status of plant material, culture media, culture environment.
Stage III
Rooting of regenerated shoots or germination of somatic embryos in vitro.
In this stage, shoots or shoot clusters from stage II are prepared to transfer to soil. Shoots are separated manually from clusters and transferred on a rooting medium containing an auxin. Elongation of shoots prior to rooting, rooting of shoots (individual or clumps), and prehardening cultures to improve survival are some of the activities carried under this stage. Sometimes, shoots are directly established in soil as micro-cuttings to develop roots.
Stage IV
Hardening
Transfer of plantlets to sterilized soil for hardening under greenhouse environment.
Page Advantages of micropropagation gives details on merits, demerits and problems occurring during micropropagation