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

Inactive Publication Date: 2019-02-22
QINGDAO YUANCE GRP CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, it is relatively difficult to prepare protoplasts from monocotyledonous plants. In the past, rice protoplasts were mostly isolated from callus cells in suspension culture, such as leaves and young stems. Low
[0004] In addition, during the separation process of plant protoplasts, the density gradient centrifugation method usually used requires more than 4 centrifugation operations and the cells are centrifuged to the bottom of the tube, which is more harmful to the protoplasts, and the protoplasts obtained by centrifugation are easy to centrifuge. A large amount of cell debris is mixed, which interferes with the transformation and regeneration in the subsequent process

Method used

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  • Method for separating and purifying protoplast of rice

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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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Abstract

The invention discloses a method for separating and purifying protoplast of rice. The method comprises following steps: A, pretreatment in a CPW salt solution; B, enzymolysis; C, dilution and precipitation; D, centrifugation; E, resuspending. The step A of pretreatment in the CPW salt solution further comprises steps as follows: callus tissue obtained by fluid suspension culture is collected and slightly flicked, tissue blocks are enabled to be dispersed to the greatest extent, the tissue blocks are cleaned once by a culture solution and cleaned 2-3 times by the CPW salt solution, and a product is put in the CPW salt solution for pretreatment. With the adoption of the method of density gradient centrifugation based on a silica gel particle suspension (namely, a Percoll layering liquid) which is treated by polyvinylpyrrolidone, damage to cells is greatly reduced, time is shortened, protoplast cells with high activity can be obtained by separation, cell sizes are kept relatively consistent, and convenience is brought to follow-up transformation and biological operation of other molecules.

Description

technical field [0001] The invention relates to the technical field of bioengineering plant cell culture, in particular to a method for separating and purifying rice protoplasts. Background technique [0002] Protoplasts generally refer to the exposed cytoplasmic mass after removing the cell wall of plant cells. Because the cell wall is removed, protoplasts are more easily used for genetic engineering operations, and the introduction of foreign genes or vectors has been widely used in cell hybridization, genetic transformation, Research on gene expression and regulation, cell metabolism analysis, etc. [0003] However, it is relatively difficult to prepare protoplasts from monocotyledonous plants. In the past, rice protoplasts were mostly isolated from callus cells in suspension culture, such as leaves and young stems. Low. [0004] In addition, during the separation process of plant protoplasts, the density gradient centrifugation method usually used requires more than 4 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 刘佳音米铁柱张国栋刘鹏飞王晶邹丹丹王克响
Owner QINGDAO YUANCE GRP CO LTD
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