Tissue Culture

Tissue culture : Tissue culture requires separation of cells, tissues or organs of a plant and allowing them to grow in aseptic nutrient media under controlled light and temperature. The cultured parts termed explants, require energy (Usually a carbohydrate like sucrose) and salts (Both macro and micro nutrients) apart from vitamins and the amino acid glycine. When a tissue from an organ is cultured, It grows into undifferentiated tissue called "callus". The callus can be differentiated into shoot, root or complete plants by manipulating the concentration of auxin and cytokinin.

Tissue culture technique is based on totipotent nature of plant cell or phenomenon of totipotency, i.e., each and every plant cell has inherent capacity to develop into complete plant.

The concept of totipotency was given by Haberlandt (1902) and practical application of totipotency was shown by Steward (1932), when he developed a complete carrot plant from a single cell obtained from root of wild carrot. The advantages of tissue culture in the improvement of crop plants are:

Explant $\to$ Disinfection $\to$ Culture medium $\to$ Callus $\to$ Plantlet (Embryoids).

The following techniques of tissue culture are useful in crop improvement. 

(i) Micropropagation : Propagation through tissue culture is called micropropagation. Production of large number of individuals in vitro in a limited space which can be employed for agriculture, horticulture and forestry. e.g., Potato, Bananas, Begonia, Carnation, Chrysanthemum and Gerbera.

(ii) Somatic embryogenesis : Somatic cells are cultured in electric shakers to obtain single cell suspension. When the number of cells has increased to a maximum depending upon the amount of medium, the culture is made stationary. Each cell starts differentiating into an independent embryo showing all the stages of embryo development such as globular heart shaped and torpedo shaped stages. They are called "embryoids". Somatic embryoids can give rise a complete plant having normal root system. Success has been achieved in carrot, celery and alfalfa. 

(iii) Raising of disease free plants : The virus free clones can be obtained from a virus infected plant by tissue culture since virus is translocated through sieve tubes, the apical meristem of virus infected plant remain free of virus. The shoot apex of such plant can be cultured.

(iv) Androgenic haploids : These are haploid plants raised from pollen grains by anther culture technique. The first example of androgenic haploid was reported by Guha and Maheshwari (1964) from anther culture of Datura innoxia. Haploid plants are always pure because they are having one gene for each trait, i.e., no dominant and no recessive. If such a gene undergoes mutation, it can be easily expressed. In China, this technique has produced Jinghua-I (winter wheat) and Guan-18 (Rice variety). These two are superior, high yielding and disease resistant varieties.

(v) Rescue of hybrid embryos : The hybrid embryos produced as result of interspecific or intergeneric crosses normally collapse due to incompatibility. These embryos can be isolated from female plants and rescued by growing on synthetic medium.

(vi) Induction and selection of desirable mutants : The single cell cultures raised in਎lectric shakers are allowed to grow in static cultures where the cells divide to form colonies. These cells are treated with chemical or physical mutagens to induce mutations. The desirable mutants are selected and multiplied.

(vii) Somaclonal variations : The spontaneous variations which appear in cells or tissues in artificial medium are known as somaclonal variations. The variants having desirable traits such as tolerance to pests, pesticides, diseases and environmental stresses are selected and exploited for agricultural purpose.

(viii) Somatic hybridization : Cells of two plants belonging to different varieties, species and even genera are first treated with pectinase and cellulase enzymes. The enzymes dissolve away the walls. The naked protoplasts of the two are made to fuse by electrofusion (high frequency alternating electric field with short current pulses) and chemofusion through sodium nitrate or polyethyleneglycol (PEG). It produces hybrid protoplasts. The latter may have a single fusion nucleus (synkaryon) or two unfused nuclei (heterokaryon). Sometimes one of the two nuclei degenerates. In that case the hybrid protoplast is called cytoplasmic hybrid or cybrid (heteroplast). Pomato is a somatic hybrid between tomato and potato and is example of intergeneric hybrid.

(7) Genetic engineering : This is the latest method of crop improvement in which instead of involving whole chromosomal set (genome), manipulation of a segment of DNA (gene) is done.

Recombinant DNA technology is connected with obtaining desired DNA sequences from different organisms with the help of restriction endonucleases, joining them together to produce a new combination, chimeric or recombinant DNA (rDNA) that is then incorporated to produce the required product. There is cutting and pasting of DNA fragments. Because of it, recombinant DNA technology is also called gene splicing.

A plant in which a specific character has been introduced is called transgenic. The first transgenic commercial crop was tobacco. It was made tolerant to certain herbicides used for removing weeds. Transgenic tomato has been made resistant to horn-worm larvae.

Many transgenic plants such as tomato, cotton, tobacco, etc., have been developed which are resistant to certain specific insects and pests.

Genetic engineering is helpful in producing tomatoes with delayed ripening, mangoes with less ethylene production and potatoes with 20�% more starch content.

Prospects of genetic engineering

Genetic engineering has put us in a threshold of a new form of medicine, “gene therapy” to find cures for crippling diseases like haemophilia and phenylketonuria.

Introduction of genes coding for vitamins, hormones etc., in higher animals opens up new vistas.

Possibility of transfer of nitrogen fixing genes from bacteria or blue green algae to major food crops is bound to enhance food production.

Production of new plants and animals tailored to new characteristics is now a reality.

Thorough study of the nature and functions of the heredity material is possible because of their technique leading to location of specific genes within the chromosomes and a deeper insight with in to when and where enzymes are made.