STAGE 1: Initiation phase
The initiation phase is the first phase of tissue culture. Here, the tissue of interest is obtained and introduced and sterilized in order to prevent any microorganism from negatively affecting the process. It is during this stage that the tissue is initiated in to culture.
STAGE 2: Multiplication stage
The multiplication phase is the second step of tissue culture where the in vitro plant material is redivided and then introduced in to the medium. Here, the medium is composed of appropriate components for growth including regulators and nutrients. These are responsible for the proliferation of the tissue and the production of multiple shoots.
STAGE 3: Root formation
It is at this phase that roots are formed. Here, hormones are required in order to induce rooting, and consequently complete plantlets.
General procedure for plant tissue culture:
- The appropriate mixture (such as the MS mixture) is mixed with distilled water and stirred while adding the appropriate amount of sugar and sugar mixture. Here, sodium hydroxide or hydrochloric acid is used to adjust the pH – Contents used here will depend on the plant to be cultured and the number of tissues to be cultured.
- Agar is added to the mixture, heat and stirred to dissolve.
- After cooling, the warm medium is poured into polycarbonate tubes (to a depth of about 4 cm).
- With lids sitting on the tubes, the tubes are placed in a pressure cooker and sterilized for 20 minutes.
- Cut the plant part in to small pieces (about 1cm across). On the other hand, such parts as the African violet leaves can be used as a whole.
- Using detergent and water, wash the plant part for about 20 minutes.
- Transfer the plant part in to sterilizing Clorox solution, shake for a minute and leave to sock for 20 minutes.
- Using a lid, gently discard the Clorox and retain the plant part in the container and then cap the container.
Transferring the plant material to a tissue culture medium:
- 70 percent alcohol should be used for the sterilization of the equipment used and containers.
- Open the container and pour sterile water to cover half the container.
- Cover with a sterile lid again and shake the container for 2 to 3 minutes in order to wash the tissue and remove the bleach.
- Pour the water and repeat this three times.
- Using sterilized gloves, remove the plant part from the container and on to a sterile Petri dish.
- Using a sterile blade cut the plant material to smaller pieces of about 2 to 3 mm across avoiding the parts that have been damaged by bleach.
- Using sterile forceps, place a section of the plant in to the medium.
- Replace the lid/cap and close tightly.
This procedure will result in the development of a callus, which then produces shoots after a few weeks. Once the shoots develop, then the plant section may be placed in the right environment (well lit, warmth etc) for further growth.
Technique for Plant in Vitro Culture:
- Micropropagation – This technique is used for the purposes of developing high- quality clonal plants (a clone is a group of identical cells). This has the potential to provide rapid and large scale propagation of new genotypes.
- Somatic cell genetics – Used for haploid production and somatic hybridization
- Transgenic plants – Used for expression of mammalian genes or plant genes for various species it has proved beneficial for the engineering of species that are resistant against viruses and insects.