GDSYZX-United/PROJECT/preparation arabidopsis protoplast

Preparation of the Arabidopsis protoplast

Place:Biology Experiment Teaching Center of Sun Yat-sen University

Experimental apparatus:Shaking table, 50 ml centrifuge tube, filtrating equipment, analytical balance, measuring cylinder

Experimental drugs:Cellulase hydrolysate, W5 solution

Procedure:

  1. the configuration of Cellulase hydrolysate
    Cellulase 0.225g
    Mecerozyme R10 0.045g
    mannitol 1.09g
    KCl 1.5g
    MES 4.18g
    water 12ml
    cooled down to room temperature after waiting in 55℃ water 10min.
    CaCl2 1.11g
    BSA 1ml
    water 2ml
    filtrating

  2. Pick Arabidopsis leaves and cut out them with 0.5-1mm, then immerse it in 5ml Cellulase hydrolysate.

  3. Put them in shaking table for 30min in the dark, then waiting for another 3h without shaking. When it comes to green and then gently shake it.

  4. (Adding 5ml W5 solution and then filtrating.)

  5. The protoplast was observed under the microscope

Result:

There is no protoplast in Cellulase hydrolysate (it didn’t come to green), and the cell wall of young leaves were not damaged.

Introspection:

  1. Cutting leaves without using the blade but by hand , or deal it with a scalpel pulling, which may damaged the cells.
  2. The shaking table should not exceed 80r/min.

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Filtrate during the configuration of Cellulase hydrolysate

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Cut out the leaves

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Immerse Arabidopsis leaves in 5ml Cellulase hydrolysate.

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Prepare the microscopic slides

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Cellulase hydrolysate under microscope (nothing).

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Plant cells under microscope.