Sequential Two Step Method, Segment with Mixture, Macerozyme, Potassium Dextran Sulphate, Mannitol, Vaccum Infiltrated, Slow Shaking, Enzyme Mixture

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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

Home >> Plant Biotechnology and Genomics >> Protoplast Culture Regeneration and Somatic Hybridization >>Sequential Two Step Method

Sequential (two step) method.

In two step method, leaf segments with mixture A (0.5% macerozyme + 0.3% potassium dextran sulphate in 13% mannitol at pH 5.8) are vacuum infiltrated for 5 min; transferred to a water bath at 25°C and subjected to slow shaking.

After 15 min. the enzyme mixture is replaced by fresh ‘enzyme mixture A’ and leaf segments incubated for another hour. The mixture is filtered using nylon mesh, centrifuged (100g) for 1 min. and washed three times with 13% mannitol to get a pure sample of isolated cells.

These cells are then incubated with ‘enzyme mixture B’

(2% cellulase in a 13% solution of mannitol at pH 5.4) for above 90 min at 30°C. After incubation, the mixture is centrifuged at 100g for 1 min, so that protoplasts form a pellet, which is cleaned three times as in ‘one step method’ above.

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