Method for separating and purifying protoplast of rice
A protoplast, separation and purification technology, applied in the field of bioengineering plant cell culture, can solve the problems of large damage to protoplasts, difficulty in preparing monocotyledonous plant protoplasts, easy to mix with a large number of cell debris, etc. Other convenient effects of molecular biology
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Embodiment 1
[0045] Enzymatic isolation of rice protoplasts. Wash the suspension-cultured rice callus once with culture medium, wash 2-3 times with CPW salt solution, place in CPW salt solution for pretreatment for 5-10min, and finally absorb CPW salt solution, add enzymatic hydrolysis solution, 27 ° C in the dark for 3-7 hours on a shaking table, with a rotation speed of 30-50 rpm. Filter the enzymolysis mixture through a 300-mesh filter, collect the filtrate, centrifuge at 500 rpm for 5 minutes, discard the supernatant, add 10ml of W5 solution, and gently flick the precipitate.
Embodiment 2
[0047] Purification and isolation of rice protoplasts. Use the sucrose aqueous solution isotonic with the culture medium as a solvent, add Percoll solution, and prepare mixed solutions with different concentrations of 3%, 5%, 8%, 12%, 15%, 20% (W / V), and use a dropper to Pipette 5ml of each gradient into the centrifuge tube slowly along the tube wall in order of density from large to small, and lay layers from high concentration to low concentration to form a gradient separation solution. Add 10ml of the enzymatically hydrolyzed cell suspension to the top of the Percoll density separation medium in a 50ml centrifuge tube with a pipette, and centrifuge at 800rpm for 8min. It can be seen that the protoplasts are concentrated in the middle of the centrifuge tube, showing a diffuse distribution. Use a sterile pipette to collect the suspension in the concentrated area of protoplasts in the middle of the centrifuge tube into a new centrifuge tube, add W5 solution to wash once, 500...
Embodiment 3
[0049] Comparison of Percoll density gradient centrifugation and sucrose self-sedimentation. One part of the cell suspension after enzymatic hydrolysis was purified and separated by the method in Example 2, and the other part was purified and separated by the sucrose self-sedimentation method. In the centrifuge tube, use a pipette to slowly add 10ml of cell suspension to the top of 40mL of W5 solution; place it in a refrigerator at 4°C for more than 30 minutes to allow it to settle automatically. Transfer the enriched layer solution to a 50mL centrifuge tube, add 10mL W5 solution, mix well; centrifuge at 100×g for 5min, remove the supernatant; add 10mL W5 to resuspend protoplasts, centrifuge at 100×g for 5min, remove the supernatant; add 1.5-3mL W5 Resuspend the protoplasts and place on ice for 30 min.
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