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  Section: General Biotechnology / Plant Biotechnology
 
 
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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Cell (Suspension) Culture
Cell suspension is prepared by transferring a fragment of callus (about 500 mg) to the liquid medium (500 ml) and agitating them aseptically to make the cells free in medium. It is difficult to have suspension of single cell. However, the suspension includes single cell, cell aggregates (varied number of cells), residual inoculum and dead cells (Dodds and Roberts, 1985). King (1980) has described that a good suspension consists of a high proportion of single cells than small cluster of cells. It is more difficult to have a good suspension than to find optimum environmental factors for cell separation.

King and Street (1977) described the techniques of cell separation by changing the nutritional composition of medium. No standard technique for separation of cells from callus has been recommended. When cells are transferred into a suitable medium they divide after lag phase (no cell division) and linearly increases their population. After some time, based on nutrient level, the rate of cell division decelerates until it comes to stationary phase (see Fig. 14.2) At this stage, to keep the cells viable, it is essential to subculture the cells. By using plate technique, cell lines can be raised where a mass of cells is spread over medium. Further, growth of plated cells depends on cell density.

Street (1977) suggested that cell density should be determined before subculturing. Growth of culture depends on a critical cell density below which culture will not grow, for instance, for a clone of Acer pseudoplatanus 9-15 x 103 cells/ ml are required. Generally cultures are agitated on orbital shaker or magnetic stirer for good result. Thus different types of works can be carried out by using cell suspensions.

The cultured cells of a higher plant is inherently dualistic. On one hand it possesses necessary genetic informations for existence on cell level (reproduction, growth, mature state, programmed death). On the other hand, the cultured cells retain supplementary informations that determine the production of substances which are important for integrative functions and biocoenotic interactions of the plants. The informations determining the progression to a programme for the development of a whole plant are also redundant for existence at cell level (Butenko, 1985). Cell culture systems have been employed in numerous morphological analyses by varying the origin of cells and physicochemical factors (White, 1963; Street, 1973).

Kurz and Constabel (1979) have described the properties of cultured plant cell suspensions in common with culture of microorganisms as (i) they grow in sterile environment, (ii) they are homogeneous in size, (iii) they have a doubling time which is longer than that of microorganisms but considerably shorter than cells in situ, and (iv) they can be grown on a large scale.

 

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


Benefits from Cell Culture
Cell suspension cultures have many advantages over the callus cultures as below :
(i) The suspension can be pipetted
(ii) They are less heterogeneous and cell differentiation is less pronounced
(iii) They can be cultured in volumes up to 1
(iv) They can be subjected to more stringent environmental controls
(v) They can be manipulated for production of natural products by feeding precursors (Kurz and Constabel, 1979).
 
     
 
 
     



     
 
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