Direct One Step Method, Leaf Segments, Incubated Overnight, Enzyme Mixture, Teased Gently, Liberate the Protoplasts, Debris Settles, Sucose Solution

Scholarships Dictionary Visa Details Study Abroad Exam Results Online Test Jobs

	Home
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 >> Direct One Step Method

Direct (one step) method.

In one step method, the leaf segments are incubated overnight (15-18th) with enzyme mixture at 25°C and teased gently to liberate the protoplasts. The mixture is filtered through a fine wire gauze to remove leaf debris, transferred to 13 × 1000 mm screw capped tubes and centrifuged at 100g for 1 min.

The protoplasts form a pellet and supernatant removed. The process is repeated three times and protoplasts washed with 13% Sorbitol solution, which is later replaced by 20% sucrose solution. The protoplasts suspension is centrifuged at a speed of 200g for 1 min. The cleaned protoplasts, which will now float (debris settles down), can be pipetted out and bulked.

	Home
Induced Fusion NaNo₃-treatment Treatment with Calcium Ions ca⁺⁺ at High pH Polyethylene Glycol PEG Treatment Electrical Fusion Selection of Fused Protaplasts Chromosome Status of Somatic Hybrids Interspecific Hybrids Produced Through Protoplast Fusion Intergeneric Hybrids Produced Through Protaplast Fusion Use of Protoplast Fusion and Somatic Hybrids Somatic Hybrids for Gene Transfer Asymmetry in Somatic Hybrids for Gene Transfer Somatic Hybrids for Cytoplasmic Male Sterility Cytoplasmic Hybrids or Cybrids Genetic Modification Transformation of Protoplasts