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Role of tissue culture for elimination of viral disease 
By Allah Jurio Khaskheli
M.Sc. Agri. (Hons.)
Department of Biotechnology
Sindh Agriculture University Tandojam

Almighty Allah has created human being as supreme to all other livings, Ashraf-ul-Makhlukat. He has bestowed human being with great capabilities. The man has made immense progresses in biological and physical science, due to this supreme quality.

Tissue culture is the most effective and important discovery of human being in the field / world of biological sciences. It is an establish fact that plant provides about 80% requirement of our daily life. It is also fact that man and animals also depend on plants. Unfortunately, nowadays, our crop plants are highly affected by many dangerous and damageable diseases such as bacterial, viral, and fungal diseases. The most virulent diseases, which cause great losses in our crop plants, are the viral diseases. Unfortunately, there is no any chemical control against viral diseases, to eliminate the virus from our crops; but in the light if modern world, the method of tissue culture is going to solve this problem.

Viral plant diseases are very common, which causes great economic losses in our cultivated crops, in the form of reduction in quantity and quality of yield, all over the world. The plants, which are infected by viral diseases, show many symptoms, which depend on the types of disease attack. Some of these symptoms are leaf distortion e.g. Curling of leaf, leaf yellowing (either of the yellowing of whole leaf or in a pattern of strips or blotches), or other growing distortions, which include the stunting of whole plant, and abnormalities in flower or fruit formation.

The most of the viruses are generally inhabits the process of photosynthesis, which results in blank of chlorophyll in the plants and as a result, the plants become disable to prepare their food. The example of common viral diseases, which causes tremendous losses in our crop plants are; CLCV (cotton leaf curl virus), sugarcane leaf virus, BBTV (banana bunchy top virus), TMC (tobacco mosaic virus) etc. The most of the growers, even general scientists/experts, just gave an idea that viral dieses can be controlled by controlling the viral vectors, through restricting the spread of an organism, from infected one to another host. The few examples, of the generally viral vectors are insect pests of plants, which are fed on different plant parts, such as aphid, white fly etc.

Now, in these days, in the modern world, the method of tissue culture is considered as the most effective method for the control of viral diseases. The plantlets are grown in the Plant Tissue Culture Laboratories (PTCL), for the production of disease free plants through tissue culture techniques, under intensive care and control conditions. The baby plants are nourished with artificial medium, containing all the nutrients required for the growth of plants, in laboratory condition.

Food is the basic requirement of every organism. Similarly, the first thing for the elimination of viral diseases from the plants through tissue culture is to provide proper food, which is called medium, an artificial food. It is prepared in the laboratory.

Mostly MS medium is used in tissue culture laboratory. In 1962, two scientists, Murashige and Skooge used first time MS medium, contains all the necessary components, which are needed for the growth of plants in vitro conditions (in laboratory conditions) i.e. micronutrient elements, macro nutrients elements, vitamins, growth regulators, glucose, proteins and also the agar (a type of sea weed for solidifying the media). Carbon is the structural backbone of the organic compounds in the make up the living cells. The carbon source includes glucose, sucrose and lactose. The nitrogen is also basic need, which increases ratio of chlorophyll pigments in the plants, which is essential for the processes of photosynthesis nitrogen. It is needed for the synthesis of such molecules e.g. amino acid, DNA, RNA and ATP. The trace elements like zinc, iron ,copper ,manganese ,etc are also required in very minute amounts, for the proper growth of plants. The artificial nutrient medium can also be changed according to the nature of plants.

First of all, healthy and young portion of the target plants are collected from the field. The collected portion should be free from virus. Amputated healthy portion of the plant is called ex-plant. It is washed thoroughly to remove dust particles and disease organisms. After washing, the ex-plant is sterilized with 70% sodium hypo chloride or normal bleach solution for 20 to 25 minutes. Sterilized exp-plant and all other necessary equipments /materials are transferred in laminar air flow cabinet.

Under laminar air flow cabinet, again wash and sterilize the ex-plant with double distilled and sterilized water, to remove sodium hypo chloride from the plant, to avoid the chance of contamination (attack of microorganism). The required portion of exp-plant is cut (i.e. growing part) with the help of sterilized knife and scissor. The cutting portion of explants is transferred in to MS medium, which is already prepared in the tissue culture laboratory. After the process of culturing, the culture bottle is transferred into the growth room, where the temperature, humidity and light are already under control, artificially. The plants are kept in dark room for 3 to 4 weeks until the process of differentiation start. In the process of differentiation, the roots and shoots are formed. After shooting, the newly plants are sub-cultured. Sub-culture is process, in which cultured plants are again cultured in a new medium under aseptic condition under laminar air flow cabinet, near about similar to as described for the first culture preparation. The process of sub-culturing is repeated 3 to 5 time for complete elimination of virus from plants, after which, the plant are transferred into the glass house. This process of transferring plants in to glass houses is called hardening and weaning process. This process is very necessary for the survival of tissue cultured plants, because, when the plants are coming from tissue culture laboratory, they are more sensitive to natural light, humidity and temperature. Therefore, the process of hardening and weaning makes these plants stable/ string, to survive in natural conditions. Finally, these plants are transferred from green house to the field.

The plants required 3 to 6 month for development into complete virus free plants, through tissue culture techniques.;

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