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Protoplast Isolation and Culture, In Plant Biotechnology: Methods in Tissue Culture and Gene Transfer (Eds.R Keshavachanadran and Peter KV), Universities Press (India)Pvt.Ltd., India,

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Title Protoplast Isolation and Culture, In Plant Biotechnology: Methods in Tissue Culture and Gene Transfer (Eds.R Keshavachanadran and Peter KV), Universities Press (India)Pvt.Ltd., India,
 
Creator Pati, P K
Sharma , Madhu
Ahuja, Paramvir Singh
 
Subject Plant sciences
 
Description Protoplast technology, which includes the
isolation, culture and fusion of plant protoplast
leading to the production of whole plant, not only
establishes the totipotency of plant protoplasts
but also opens up new vistas with far reaching
implications.
The term ‘protoplast’ was first used by Hanstein
in 1880. It refers to all the components of a plant
cell except the cell wall. As the cell wall interferes
with the most cellular genetic modifications,
there is a need to remove the cell wall and isolate
plant protoplasts. Klercker 1 first isolated plant
protoplasts of Stratiotes aloides by microsurgery
on plasmolyzed cells. Protoplasts obtained by this
procedure were mainly used for physiological
studies. However, limited yield, excessive damage,
and restricted suitability of the technique to
specific tissues, were the major drawbacks. With
refinements in the technique, protoplasts were
isolated in large numbers by enzymatic removal of
cell wall as pioneered by Cocking. 2
For enzymatic isolation of plant protoplasts,
the basic structure of plant cell wall was taken
into consideration. The primary components of
a plant cell wall have been identified as cellulose,
hemicellulose and pectic substances (see Fig. 11.1).
Cellulose is a simple, linear polymer of D-glucose
with β-1,4 linkage. The molecular weight varies
between 50,000 and 2,500,000 Da in different
species. Hemicelluloses are less well defined and
appear to be mixed polymers of glucose, galactose,
mannose, arabinose and xylose. 3 Most of these
carbohydrates are linked with both β-1,4 and
β-1,3 linkages. The middle lamella, which holds
the cells together, is rich in pectin. Pectin is a
polymer of methyl-D-galacturonate connected
with α-1,4 linkages with molecular weights of
25,000 to 360,000 Da. 4 Hence, in enzymatic
method, source tissues were treated with cell
wall degrading enzymes such as cellulases,
hemicellulases and pectinases. Initially, a two-step
procedure was adopted, first producing single
cells and then releasing protoplasts. 5 However,
with further refinement, a single-step procedure 6
was developed for cell separation and cell wall
degradation where tissues were treated with a
mixture of hydrolytic enzymes. Protoplast yield
was high and now this is the most frequently used
method. Moreover, the use of hydrolytic enzymes
offered advantages like:
 
Date 2008
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://ihbt.csircentral.net/865/1/Chapter-11%5B1%5D.pdf
Pati, P K and Sharma , Madhu and Ahuja, Paramvir Singh (2008) Protoplast Isolation and Culture, In Plant Biotechnology: Methods in Tissue Culture and Gene Transfer (Eds.R Keshavachanadran and Peter KV), Universities Press (India)Pvt.Ltd., India,. Tissue Culture and Transfer, 11. pp. 115-132.
 
Relation http://ihbt.csircentral.net/865/