Plant Tissue Culture : Equipment, Steps and Techniques

Equipment and different materials required for the process of tissue culture

Equipment those are required for the process: 

• Washing bucket
• Balance
• pH meter
• autoclave
• microscope
• microtome
• Refrigerator
• Laminar air flow cabinet
• centrifuge machine
• microwave oven
• shaker
• burner, etc.

Glassware:

Test tubes, conical flanks, cylindrical flanks, beakers, Petridis, measuring cups, pipettes, seeded glass rods, bottles, etc.

Chemicals:

Various inorganic salts, growth hormones, vitamins, amino acids, charcoal yeast, alcohol, spirits, carbohydrates, etc., are used to prepare the culture medium. The other materials that are used are:

• Scissors
• Tweezers
• Surgical Bread
• Scissors
• Spatula
• Filter Paper
• Aluminum Foil
• Plastic containers, etc.

Sterile environment

Tissue culture requires a completely sterile environment to do the process correctly. If the culture medium is contaminated in any way, the medium cannot be used anymore. Culture medium containers and other glassware are sterilized by placing them in a hot air oven from 160°C to 180°C for 1-2 hours for the process to be done properly. Instruments such as forceps, needles, scalpels, etc., are sterilized properly by heating them using a burner and immersing them in 95% alcohol.

The different types of tissue culture

Tissue culture methods are other, such as:

1 . Axillary bud culture

2. Meristem culture

3. micropropagation

4. Seedling production through callus culture

5. Somatic embryogenesis

6. Haploid through pollination culture

7. Plant production

8. Protoplast culture, etc.

The steps (or technique) of the Tissue Culture Procedure: 

The tissue culture procedure can be described in the following steps:

Step 1 : The selection of mother plant or explant

An explant is a part of a plant that is separated from a plant to use in the procedure of tissue culture. Hence, the selection of an explant is, therefore, a very important issue. Generally, the top bud of the stem and the lateral bud is used as an explant, or the leaf top can also be used. The plant from which the explant is or will be taken is called the mother plant. The mother plant must be free from diseases or other problems and must be of excellent quality for a better procedure. For tissue culture, tissue is collected from healthy, diseased, and well-characterized plants, and the collected tissue is called explant.

Step 2: The preparation of culture medium

 For tissue culture work, a culture medium must first be prepared at the beginning. This medium is prepared by combining all the chemical elements required for plant nutrition and growth. Different types of main elements and secondary elements (macro and microelements), vitamins, sucrose (2 to 4%), phytohormones, etc., need to be in this medium. Coagulating material, such as agar, must be mixed in the right amount to thicken the medium for the procedure. A culture medium rich in basic elements is called a basal medium, and the pH of the medium must be kept between 5.5 to 5.8.

Step 3: Sterilization

 The culture medium contains nutrients, so the bacteria can easily grow in it. But the culture medium and the explants must be sterilized for the procedure. Therefore, the medium is poured into a conical flask or test tube, and the mouth of the container is closed with sterile cotton to prevent air from entering. The vessel is then sterilized in an autoclave for the process to be done properly. The medium is placed in an autoclave at a specified temperature (121°C), pressure (15 lb), and time (20 minutes). In-vitro culture is the method of making grafts from explants by the process of artificial cultivation in glass containers in the laboratory under sterile conditions.

Step 4: The placement of explants or tissue in medium

Explants are sterilized (including hands, forceps, etc., with alcohol) and placed in a fully sterile glassware medium.

Step 5: Callus formation and multiplication

After placing the explants and tissues in the medium, the pot is placed in a room controlled by electric light (3,000-5,000 lux), temperature (17-20°C), and relative humidity (70-75%). After a few days, the tissue repeatedly divides to become a single cell mass in the process. The corpus callosum is a shapeless mass of tissue, and after the explant is placed in the medium and kept under light and heat control, the shapeless clump of tissue that forms is a callus. Numerous buds are formed from the callus at a time during the process.

Step 6: Transplantation and seedling production in root production medium

The buds are carefully cut and placed in a root production medium, where each bud root grows into a full-fledged plant during the procedure.

Step 7: Transplanting seedlings into tubs

When a suitable number of well-formed roots have developed, full-grown seedlings are removed from the culture pot and carefully transferred to tubs in a slow process. In this way, plant production is done through tissue culture.

Step 8: seedlings storing

 In the natural environment, i.e., in the field, seedlings are kept in humid conditions with transfer tubs. Still, transplanted seedlings are sometimes kept outside the room to acclimatize to the natural environment outside. Once the full-grown seedlings are alive and strong, they are transplanted into the soil at one point in their natural environment at the end.

Tissue culture technology is currently being applied to many types of research. The procedures of tissue culture vary depending on the method of application and purpose, and what nature and size of tissue from which plant to use and what kind of culture medium to use will depend entirely on the purpose of the culture. The procedure described may vary depending on the type of application.

Application of tissue culture technology in plant breeding and advanced innovation:

Since the discovery of the tissue culture method, many problems in the field of plant breeding have been solved easily. So, breeders have achieved many successes by applying this method to various plants.

Micropropagation

For plants that cannot produce seeds (e.g., Thuja, Sea Banana), for those plants, tissue culture can be used to produce and market seedlings. Suppose it is necessary to produce many seedlings from a good variety of flower, fruit, or grain-producing plant. In that case, it is possible to produce a large number of seedlings by tissue culture from a plant of that good variety. The seedlings grown in this way are exactly like their parent plants. Hence, the production of plants is with similar characteristics. Tissue culture technology is a very effective process for this method of producing many seedlings from very small tissues, also known as micropropagation.