Enzymatic Method, Isolation of Mesophyll Protoplasts, Enzymatic Treatment, Protoplasts Isolated, Sterilization of Leaves, Peeling Epidermis

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Protoplast Culture Regeneration and Somatic Hybridization Protoplast Culture Regeneration and Somatic Hybridization - Introduction Isolation of Proplasts Mechanical Method Enzymatic Method Direct One Step Method Sequential Two Step Method Purification of Isolated Protoplasts Sedimentation and Washing Flotation Viability and Plating Density of Protoplasts Testing Protoplast Viability Minimum Plating Density MPD and Culturing Individual Protaplasts Protaplast Culture and Regeneration of Plants Isolation of Subprotoplasts Protoplast Fusion and Somatic Hybridization Techniques of Protoplast Fusion

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Enzymatic method

The enzymatic method is used now almost invariably for the isolation of protoplasts, since it gives large quantities of protoplasts, without cell breakage and with minimum of osmotic

Isolation of Mesophyll Protoplasts Using Leaves with Epidermis Peeled Off

1. Leaves	6. Protoplasts in washing medium
2. Sterilization of leaves	7. Centrifugation
3. Removal of epidermis by peeling	8. Cell debris
4. Peeled leaves in enzyme solution	9. Protplasts
5. Released Protoplasts in enzyme solution	10. Isolated Protoplasts

shrinkage. 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

Diagram of the principle steps in enzymatic isoltion of protoplasts; leaves are removed and cut into fine strips, incubated in enzyme mixture. The enzyme is then removed and the protoplasts are ready for purifiation

	A. Shoot Culture	B. Remove Leaves	C. Cut Leaves

G. Protoplasts Ready for Purification	F. Centrifuge 500g 1 min	E. Isolated Protoplasts	D. Incubate in Enzyme

sterilized by first dipping them into 70% ethyl alcohol for about a minute and then treating them with 2% solution of sodium hypochlorite for 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 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 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 any one 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 are first isolated using macerozyme and 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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