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.

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Abstract

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:

Item Type:	Article
Subjects:	Plant sciences
Divisions:	UNSPECIFIED
Depositing User:	Dr. Aparna Maitra Pati
Date Deposited:	30 Dec 2011 12:22
Last Modified:	30 Dec 2011 12:22
URI:	http://ihbt.csircentral.net/id/eprint/865

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