Question

write the main steps in tissue culture?​

Answer 1

STAGE 1 INITIATION

We take a piece of plant tissue (bud, leaf, stem or root) and disinfect it as much as possible using sterile water, bleach and alcohol. All of this work takes place in a laminar flow hood in the lab. This hood forces sterile air over the transfer area so there’s less chance of bacteria and fungi coming in contact with the plant tissue. Bacteria and fungi can grow quickly in the tissue culture containers as what is good for plant growth is also grow for microbial growth.

STAGE 2 MULTIPLICATION

The cleaned pieces of plant tissue are placed in sterile containers that contain media with all the ingredients needed for the tissue develop into a plant. The media contains nutrients, plant hormones, an energy source (such as sugar), water and a thickener to make the media gel. In Stage 2, we want the plants to produce several leafy shoots on a clump of cells called a base or callus tissue. After about 2 weeks, the cultures need to be transferred to new containers and media as they used up what was in the initial containers.

This is when you can see the how quickly plants can multiply. Plants grow quickly in tissue culture and they are divided each time they are transferred so 1 plant = 3 plants = 9 plants = 27 plants = 81 plants and so on until you have the number of plants you need.

STAGE 3 ELONGATION

Plants are transferred once again, in the laminar flow hood, onto a medium that makes the shoots elongate. This is the stage where the stems of the plants grow in length and begin to resemble little plants.

STAGE 4 PRE-ROOTING

This can happen in the tissue culture lab or in the greenhouse, depending on the species. The elongated shoots are trimmed off the base tissue and placed on to media that contain auxins, for rooting. Once roots are visible, the plants can then be planted into the greenhouse. Plants coming from the lab environment have been grown at high relative humidities and are very tender. They have to be watched very closely when they are moved to the greenhouse to make sure they are acclimatizing properly.

Answer 2

Main steps in Tissue Culture are:-

1. Selection of plant
2. Isolation of explant
3. Sterilization of explant
4. Inoculation of explant
5. Incubation
6. Initiation of callus
7. Sub-culturing
8. Regeneration
9. Hardening