Question

describe the step involved in tissue culture technique​

Answer 1

Explanation:

Step # 1. Inoculation of Explant:

Successful control of contamination largely depends upon the precautions taken to prevent the entry of microorganisms at the time of transferring the sterilised explants on the nutrient medium. Dust, hair, hands and clothes are the potential sources of contami­nation. The inoculating chamber should be dust free the operator should wear sterile headgear and clothes (aprons) before entering the culture area.

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The hands should be wiped with 95% alcohol and the transfer area also should be cleaned and wiped with 95% alcohol before starting the transfer process. Talking or sneezing should be avoided during transfer of explant into the media. The neck or mouth of culture container should be flamed, the transferring instruments also to be flamed and dipped in alcohol.

Care should be taken so that the explant should not touch the edge of culture vessel, and after trans­ferring the mouth should be closed by cap or by cotton plug and petridishes to be sealed by ‘Parafilm’. During transfer it is also to ensure that the plant tissue should be exposed to the media properly.

Step # 2. Incubation of Culture:

After inoculation, the cultures are incubated in culture room or in a BOD incubator at 25±2°C temp. For certain plant or for some particular culture type below or above 25°C is needed.

Some tissues grow well under low light condition (approx. 1000 lux), for regenera­tion light and dark periods are needed, and for regenerated plantlet well lighted (approx. 3000 lux) condition and 16h light with 8h dark period is needed. The illumination in the culture room is provided by cool white fluorescent light placed approx. 18″ above the cul­ture racks.

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Low humidity causes the quick desiccation of culture medium and high humidity is favourable for contamination of culture medium. Specific relative humidity (20-98%) is to be maintained in the culture room and uniform air circulation is to be done properly.

For cell suspension culture agitation and aeration is secured by the use of shaker sys­tems; open platform orbital shakers or the orbital incubators fitted with fluorescent lights to provide different day/night regimes.

Step # 3. Sub-Culturing:

The growth and development of tissues cultured in vitro are generally monitored by observing the cultures at regular intervals in the culture room or incubators.

Based on the observations either with hand-lens or with the aid of simple microscope under aseptic con­ditions, the explants may be required to transfer to new media (freshly prepared) or with new ingredients or hormone composition depending on the state of growth of cell or tissue.

The same precautions and full aseptic conditions are maintained during the transfer process also. The delaying of this process may lead to inhibition of proper development of tissues and also delaying the regeneration of plantlets.

In case of suspension culture the change of media or fresh inoculation at quick intervals is needed and also for callus culture the sub-culturing of the callus tissue is needed to get the callus tissue in dividing conditions.

Step # 4. Transplantation of the Regenerated Plant:

Plants regenerated from in vitro tissue culture are transplanted to soil in pots. Prior to transfer to pots the acclimatization of these regenerated plants are needed. The plants at this time develop adequate root systems and cuticular leaf surface structure so that it can withstand the field environmental condition.

The process of acclimatization needs the humid chamber and a slow process to make the plantlet habituated from high humid con­dition to normal atmospheric humidity. The greenhouse or the growth chamber should have artificial light system also which includes a mixture of fluorescent and incandescent lamps designed to provide balanced wavelengths of light for plant growth and photosyn­thesis.

Answer 2

Describe the various steps involved in tissue culture.

1) Selection of plant:

• The plant which has to be cultured in vitro is selected.

2) Isolation of explant:

• Explant is any part of plant excised out for tissue culture.

3) Sterilization of explant:

• Explant is surface sterilized so as to avoid contamination.

4) Inoculation of explant:

• The explant is inoculated on the nutrient medium.

5) Incubation:

• After inoculation, the cultures are incubated so as to provide proper conditions for their growth & regeneration like temperature, moisture etc.

6) Initiation of callus:

• Callus is a mass of undifferentiated cells formed by the dedifferentiation of plant cells or the explant.
• Callus further regenerates to form roots & shoots & eventually the complete plant.

7) Sub-culturing:

• The cultured cells or tissues are transferred regularly to new nutrient medium called subculturing.

8) Regeneration:

• Regeneration is the formation of organized structures like roots, shoots, flower buds etc from the cultured cells

9) Hardening:

• The plantlets are removed from the cultures and prepared for soil transfer & this transfer to fields is known as hardening.