Micropropagation Stages

The Whole objective of micropropagation is to produce large number of plants, which are able to survive under natural environmental conditions.Each micropropagation process has to pass through following stages:-

Stage 0:- Management of Donor plant/s (Source of explant)

Stage 1:- Establishment of aseptic cultures

Stage 2:- Multiplication of shoots and/or elongation

Stage 3:- Induction of roots

Stage 4:- Hardening, acclimatization and transfer of plants in soil



Stage 0:- Management of Donor plant/s (Source of explant)

It is not an essential stage of micropropagation but desirable in many cases.Particularly in tree species, to obtain suitable and responsive explants, the plants are lopped during off-season of growth and when new growth appears, the juvenile branches are used to obtain explants. Such explants are also free from contamination and easy to establish in culture.


Stage 1:- Establishment of aseptic cultures

The process of micropropagation starts with establishment of aseptic cultures and this is the most critical phase determining succcess. Explants have t be treated with suitable chemosterilant to make them free from contamination. For each type of explants and plant species, a procedure of surfave sterlization has to be standardized in order to obtain high percentage of aseptic but most proliferating cultures. While standardizing the procedure the TCDC formula is applied.

T = Selection of the TYPE of chemical to be used.

c = Effective CONCENTRATION of the selected chemical.

D = The required DURATION of the treatment.

c = A COMBINATION of the treatment if required.

Explants like root tubers, rhizomes, corms and nodal segments obtained from old branches of trees pose serious problem of contamination and may require a harsh treatment of longer duration involving one or more chemicals.

Stage 2:- Multiplication of shoots and/or elongation

The shoots which are produced via any of the following pathways of micropropagation can be multiplied for an indefinite period by repeated subculturing (transfer of multiplying shoots on fresh medium):

(a) Stimulated axillary bud proliferation

(b) De novo adventitious shoot bud differentiation

(c) Callus organogenesis

In case of somatic embryogenesis both shoot and root develop simultaneously (bipolarity) and therefore, there is no separate phases of shoot multiplication and rooting. Cytokinins are generally incorporated individually or in combination with small quantity of auxin/s to stimulate shoot multiplication. Benzyl amino purine (BAP) has been considered to be a better cytokinin as compared to kinetin (Kn) to obtain such response. The multiplied ahoot clusters may be divided into single or small group of shoots and transferred to fresh medium for further proliferation. The rate of multiplication can be controlled by the concentation of the cytokinin and the particular type used.

Stage 3:- Induction of roots

Except somatic embryogenesis all other pathways of micropropagation require a separate root induction phase. Shoots of suitable size are excised from multiplying shoot cultures and inoculated on a medium containing root inducing harmone. Auxins like indole-butyric acid (IBA), naphthalene acetic acid (NAA) and indole-acetic acid (IAA) are generally used in their various concentrations and combinations to induce roots in in vitro developed shoots. Indole-butyric acid (IBA) is the most commonly used root inducing harmone. The temperature is maintained around 26-28'c round the clock. All these factors together, make tissue culture plants adapted to luxury of culture conditions. Such plants when transferred ex vitro can not withstand the transplantation shock and die. In order to solve this problem, the tissue cultures plants are subjected to gradual and systematic hardening and acclimatization procedures enabling them to establish in soil successfully.

In this in vitro hardening procedure the culture bottles are kept in culture room in aseptic environment with their caps closed. In this step only sucrose and nutrients are withdrawn. The tissue culture plants resume normalcy with respect to photosynthetic activity and become autotrophic. Such bottles are subsequently shifted to greenhouse environment which maintains a reducing humidity gradient (80-55 %), a temperature regime of 28-32'c and a light intensity which is about ten times higher than the culture room.

Stage 4:- Hardening, acclimatization and transfer of plants in soil

The plants grown in tissue culture live in luxury and become adapted to such environment. In tissue culture media sucrose is provided as a source of carbon and this makes culture to grow either hetero- or mixoautotrophically. Although, the plants remain green, they carry out negligible photosynthetic activity. Presence of high moisture content in the culture vessel results into abnormal development (deformed guard cells) and behaviour (sluggish) of stomata. Leaves produced in culture also lack cuticle. In culture room, the light intensity is very low as makes the stomatal functioning normal and the plants are able to survive in  low humidity conditions. 

When the plants attain a reasonable height, they are transferred to polybags containing normal garden soil. Plants in polybags acclimatize to greenhouse environment and become suitable for transfer to either field or nursery. Adopting the above procedures ( or with modification, wherever necessory) a large number of tissue culture plants can be established in soil.