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Home >> Protoplast Culture, Somatic Hybrdization and Somatic Cybridization >> Enzymatic Method of Isolation of Protoplast
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Enzymatic Method of Isolation of Protoplast - The enzymatic method is almost invariably used now for the isolation of protoplasts, since it gives large quantities of protoplasts, where cells are not broken and osmotic shrinkage is minimum. However, sometimes mechanical and enzymatic methods are combined, where cells are first separated mechanically and later used for isolation of protoplasts through enzymatic treatment.

The protoplasts can be isolated from a variety of tissues including leaves, roots, in vitro shoot cultures, callus, cell suspension and pollen. Young cell suspensions are particularly ideal for isolation of protoplasts in large quantities. However, the most commonly used organs are leaves which can be employed for isolation of protoplasts using the following steps:

(i) sterilization of leaves,
(ii) peeling off the epidermis,
(iii) enzymatic treatment and
(iv) isolation and cleaning of protoplasts.

Fully expanded leaves are obtained from about 10 weeks old plants (in tobacco) and are sterilized by first dipping them into 70% ethyl alcohol for about a minute and then treating them with 2% solution of sodium hypochlorite fur 20-30 minutes. The leaves are then rinsed three times with sterile distilled water and subsequent operations are carried out under aseptic conditions (under "laminar air flow chamber').

The lower epidermis of the sterilized leaves is carefully peeled off and the stripped leaves are cut into small pieces. The peeling operation is easy, if the water supply is limited before excision or if leaves are allowed to become flaccid after sterilization. Mesophyll protoplasts can be obtained from these peeled leaf segments, while those for epidermis are obtained from the peeled epidermis.

In cereals, where it is difficult to peel off the epidermis, leaves are cut in long strips and used with enzyme mixture. When the starting material is an in vitro shoot culture, the sterilization step can be omitted and leaves can be cut into pieces under aseptic condition and used directly for enzymatic treatment.

From the peeled leaf segments, obtained as above, the protoplasts can be isolated using anyone of the two methods:

(i) direct (one step) method, in which treatment with macerozyme (or pectinase) and cellulase is done simultaneously, or
(ii) sequential (two step) method, in which cells i are first isolated using macerozyme and cells are then treated with cellulase to isolate protoplasts. In both these methods, peeled leaf segments are placed with their lower surface downwards in a Petri dish containing the enzyme mixture.

The enzyme mixture in direct method consists of 0.5% macerozyme + 2% cellulase in 13% sorbitol or mannitol at pH 5.4, while in sequential (or two step) method, two enzyme mixtures ('mixture A' and 'mixture. B'; see later) are used one after the other.

While the yield of protoplasts is higher in one step method (since both palisade and spongy mesophyll tissues are used in this method, while only palisade is used in two step method), the protoplasts isolated by two step method are better for culture studies.

 
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