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
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
- Pick Arabidopsis leaves and cut out them with 0.5-1mm, then immerse it in 5ml Cellulase hydrolysate.
- 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.
- (Adding 5ml W5 solution and then filtrating.)
- The protoplast was observed under the microscope
- Cutting leaves without using the blade but by hand , or deal it with a scalpel pulling, which may damaged the cells.
- The shaking table should not exceed 80r/min.
Filtrate during the configuration of Cellulase hydrolysate
Cut out the leaves
Immerse Arabidopsis leaves in 5ml Cellulase hydrolysate.
Prepare the microscopic slides
Cellulase hydrolysate under microscope (nothing).
Plant cells under microscope.