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In Vitro Culture Techniques : The Biotechnological Principles

 
     
 
Protoplast Fusion in Fungi
The essence of protoplast fusion in fungi is the improvement of strains to be used for commercial purposes. By this method compatibility barriers between .several species can be overcome] In recent years, several companies have realized to exploit this technique as a breeding tool for strain development in fungi. For example, two titre strains of Cephalosporium acremonium were crossed and cephalosporin C was improved. But attempts to improve citric acid production in A. nigev were unsuccessful (Peberdy, 1989).
 

Method for isolation of protoplasts is the same as described for plant cells. Generally, PEG is used for inducing protoplast fusion. But many of isolated protoplasts die due to PEG treatment. This results in low rate of survival which facilitates to detect them by using selection techniques.
 

Electrofusion, a two stage process, is another method for inducing protoplast fusion. In this method, the protoplast membranes are brought into close contact by dielectrophoresis and a field pulse is used to cause the fusion event. For the first time, it was applied in S. cerevisiae (Halfmann et. al, 1982). 

The fungal protoplast techniques are applied in the following four major areas to get the crosses (i) between isogenic strains which may provide an opportunity for genetic mapping, (ii) between different strains or isolates of a species primarily for breeding, (iii) between apparently incompatible strain of the same species, and (iv) between different species and genera.
 

Intraspecific protoplast fusion
Intraspecific protoplast fusion is the cross between the same species in an individual which involves the isogenic strains or the non-isogenic ones. The true value of protoplast fusion as a mean for establishing parasexual crosses has been realized so far in a few fungi. For example, in C. acremonium this technique offers the only way of carrying out crosses  and genetic analysis. Difficulty arises through the conventional methods because of production of heterokaryons and somatic diploids. However, through this technique the fusion products were not the heterokaryons but the haploid recombinants. Protoplast fusion technique made it possible to produce a preliminary genetic map of 8 linkage groups for C. acremonium. Genes which enhance the production of antibiotics have been identified and allied to specific linkage groups. Another example is the yeast, Candida, of which biotechnological potential would be of great use. The other examples are : Absidia glauca, Candida maltosa, Pleurotus ostreatus, Aspergillus niger, A. sojae, Fusarium graminearum, F. lycopersici, Penicillium verruculosum, Trichoderma, harzianum, T. reesei, etc.
 

Interspecific protoplast fusion
Interspecific protoplast fusion is the crosses between two different species. Interspecific protoplast fusions are of much importance in the area where new products are to be produced. Due to new genetic set up many noval secondary metabolites such as, antibiotics may be produced. Some of the examples where interspecific hybrids were produced through protoplast fusion are: S. cerevisiae x S. fermentali, S. cerevisiae x S. lipolytica, S. cerevisiae x S. rowcil, P. chrysogenum x P. notatum, P. chrysogenum x P. citrinum, P. chrysogenum x P. baarnense, Asnergillus nidulans x A. rugulosus, Ganoderma applanatum x G. lucidum, Pleurotus ostreatus x P. florida.
 

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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