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  Basic techniques of plant tissue culture

Basic techniques of plant tissue culture:

1. Culture vessels:
The culture vessels employed for plant tissue studies involves Erylenmayer flask (conical flask), petri plates and culture tubes (25 x 150mm).

2. Culture medium:
The significant media employed for all purpose experiment are Gamborg medium (B5 medium), Murashige and Skoog medium (MS medium), White medium (W medium) and Nitsch medium. The culture medium is stopped with cotton plug or aluminium foil sheet. The pH of the medium is adjusted to 5.8 the acidic range.

3. Sterilization:
Sterilization is the method used to get rid of the microbes like bacteria and fungi in the culture medium and plant tissues. Thus, it is significant to sterilize the culture medium and plant tissue.

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                                                             Diagram: Techniques of tissue culture

The culture medium could be sterilised through keeping it in an autoclave and keeping the temperature of 121°C for 15 minutes.  The plant tissue (inoculums) is to be surface sterilised.

4. Inoculation:
Transfer of explants (stem, root, leaf, etc.) on to a culture medium is called inoculation. The inoculation is performed within aseptic condition for that an apparatus called laminar air flow chamber is employed. Flamed and cooled forceps are employed for transfer of plant materials to dissimilar culture media kept in glassware.

5. Incubation:
The culture medium along with the inoculums is incubated at 26 ± 2 oC with the intensity of light at 2000 to 4000 lux (unit of intensity of light) and permitting photoperiod of 16 hour of light and 8 hours of darkness.

6. Induction of callus:
Because of activity of auxins and cytokinins, the explant is induced to create callus. The callus is an undifferentiated tissue’s unorganized mass. The technique of callus formation is that auxin induces cell elongation and cytokinin induces cell division as a outcome of which masses of cells are created.

7. Morphogenesis:
Creation of new organs from the callus under the effect of auxin and cytokinin is termed as morphogenesis. Roots and shoots are distinguished from the callus. Such types of embryos are called termed as embryos result in the Creation of young plantlet.

8. Hardening:
Displaying the plantlets to the natural environment in a stepwise way is termed as hardening. At last the plantlets are gradually transferred to the soil.

 

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