Tissue culture, Steps of tissue culture technology
Explant selection (Ex-plant selection): The first and most important step in tissue culture technology is explant selection and isolation. First the explants are selected. While selecting the explant, it should be kept in mind that it should be healthy, vigorous, disease free and of improved variety. Besides, care is taken to ensure that the desired features are present in it. Later, the necessary parts such as apical bud, side bud or leaf part, dividing cells, meristem, pollen, ovule, embryo, nucellus, protoplast etc. are separated from the explant.
Preparation of culture media: Another important step in tissue culture technology is the preparation of culture media. Usually this culture media is made up of essential plant nutrients. The culture media is prepared with the main and secondary plant nutrients, vitamins, phytohormones, agar agar, glucose, sucrose (2-4%), fructose, maltose etc. in a container. This culture medium prepared with basic nutrients is called basal medium. The pH of the growing medium is kept between 5.5 and 6.0. The most commonly used culture media are MS medium (Murashige & Skoog, 1962) and B5 (Gamborg et al, 1968) medium.
Sterilization: Both culture media and explants used in tissue culture technology must be sterilized. The culture media is taken in a conical flask or large test tube. Then, both the culture medium and the explants were placed in an autoclave at 121°C for 20 minutes under 15 pounds of pressure. As a result it becomes germ free.
Placing explants in culture medium: Sterile explants are placed in sterile culture medium. The knives, tongs and hands used during this time are disinfected with ethyl alcohol. This work is done under a laminar air flow machine in a sterile environment.
Callus creation and increase in number (Callus create): After placing the explants in the culture medium, the container is kept in a controlled environment. The room temperature is kept at 17-20°C, light intensity is 3,000-5,000 lux or 1000-3000 lux and relative humidity is kept between 70-75%. Within a few days, the cell divides repeatedly to form a multicellular nucleus. This multicellular body is called a callus. After 5-7 days numerous buds or microflora are produced in the callus. Buds grow due to totipotency of cells. New seedlings are then produced from the buds.
Plantlets production: Buds are cut from the callus with a sterile knife. The buds are then placed in seedling producing culture medium. Auxin is applied in the culture medium for root formation. As a result, seedlings are produced within a few days. The method of making seedlings from explants through artificial cultivation is in-vitro culture.
Plant transfer on Tub: When the roots of the seedlings are well formed, the seedlings are carefully placed on the agar soil of the tub. The tub is kept in a light and airy place. The tub is watered occasionally. Within a few days, the seedlings grow and become suitable for planting in the field.
Plant transfer in field: Tubs with seedlings are sometimes kept in natural environment. In this the plants can adapt to the natural environment. Later, healthy, vigorous and disease-free seedlings are planted in the field.