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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Callus Formation and its Culture
In nature, callus develops by infection of microorganisms from wounds due to stimulation by endogenous growth hormones, the auxins and cytokinins. However, it has been artificially developed by adopting tissue culture techniques. Explant, a 2-5mm sterile segment, excised from a stem, tuber or root is transferred into nutrient medium and incubated at 25-28°C in an alternate light and dark regime of 12h. Nutrient medium supplemented with auxins induces cell division. Soon the upper surface of explant is covered by callus. A callus is an amorphous mass of loosely arranged thin walled parenchyma cells developing from proliferating cells of the parent tissue (Dodds and Roberts, 1985). The unique feature of callus is that the abnormal growth has biological potential to develop normal root, shoots and embryoids ultimately forming a plant.

Callus formation is governed by the source of explant, nutritional composition of medium and environmental factors. Explants of meristematic tissues develop cells more rapidly than thin walled and lignified cells of tissue. Callus is formed through 3 developmental stages: induction, cell division and differentiation.

During induction metabolic rate of cells is stimulated, duration of which depends on physiological status, and nutritional and environmental factors. Owing to increased metabolic rate, cells accumulate high contents and finally divide to form many number of cells. Cellular differentiation and expression of certain metabolic pathways start in the third phase leading to secondary products. Some times callus appears of different colors, for example, yellow, white, green or red.

Within the cell population of callus, the genetic instability results in variations in phenotypes which may be attributed to developmental (epigenetic) or genetic basis. Epigenetic changes involve selecting gene expression. They are stable and heritable at cellular level (Binns, 1981). When callus has grown on nutrient medium after a long time it becomes essential to subculture it within 28 days on a fresh medium. Otherwise there develops nutrient depletion in original medium which results in paucity of water and accumulation of toxic metabolites.

 

Content

Totipotency

Historical background

Requirements for cell and Tissue Cultures

 

A tissues culture laboratory

 

Nutrient media

 

 

Inorganic chemicals

 

 

Growth hormones

 

 

Organic constitutents

 

 

Vitamins

 

 

Amino acids

Culture of plant materials

 

Explant culture

 

Callus formation and its culture

 

Organogenesis

 

Root culture

 

Shoot culture and micropropagation

 

Cell culture

 

 

Benefits from cell culture

 

Somatic embryogenesis

 

Somaclonal variation

 

Protoplast culture

 

 

Isolation

 

 

Regeneration

 

Protoplast fusion and somatic hybridization

 

 

Fusion products

 

 

Method of somatic hybridization

 

Anther and pollen Culture

 

 

Culturing techniques

 

In vitro androgenesis (direct and indirect androgenesis)

 

Mentor pollen technology

 

Embryo culture

 

Embryo rescue

 

Protoplast fusion in fungi

 
     
 
 
     



     
 
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