Preparation method of cassava mesophyll cell protoplast
By using specific enzymatic solution and conditions to perform enzymatic decomposition of cassava leaves under dark conditions, the problem of failure of cassava protoplast separation is solved, and efficient and economical preparation of cassava mesophylla protoplasts is achieved, meeting the needs of basic research and applied research.
Patent Information
- Application Number
- CN202411818645.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art is difficult to efficiently obtain protoplasts of cassava mesophylla cells. The enzymatic decomposition of the cell wall can easily lead to browning of the protoplasts, resulting in failure of separation.
A method of preparing cassava mesophyllus leaf protoplasts is adopted, including obtaining healthy cassava leaves, cutting them into strips and fragments for enzymatic decomposition under dark conditions, using a specific proportion of enzymatic solution and enzymatic conditions, followed by filtration and centrifugation, and finally resuspend the cells through protoplast washing solution to obtain cassava mesophyllus leaf protoplasts.
The enzymatic lysis time was significantly shortened, and the protoplasts of cassava mesophylla cells were efficiently obtained. The morphology was normal and the quantity was sufficient, meeting the requirements of subsequent experiments.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant cell protoplast preparation, and in particular to a method for preparing cassava mesophyll cell protoplasts. Background Art
[0002] Plant cell protoplasts play an important role in basic research and genetic breeding, and are mainly used in the fields of observation of protein subcellular localization, plant asexual propagation, gene function research, and somatic cell hybridization breeding. Cell protoplasts have been widely used in many fields of plant science research.
[0003] Cassava (Manihot esculenta Crantz) leaves contain a large amount of cyanogenic compounds, which will cause the protoplasts to brown during cell wall enzymatic hydrolysis, thus easily leading to failure in cassava protoplast isolation. For the above reasons, compared with plants that successfully obtain protoplasts, there is no method that can efficiently obtain cassava mesophyll cell protoplasts.
[0004] Therefore, how to prepare cassava protoplasts using an efficient and economical method is a difficult problem currently faced. Summary of the invention
[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing cassava mesophyll cell protoplasts. The method is simple, efficient and economical and solves the problem that the protoplasts will turn brown during cell wall enzymolysis, which easily leads to the failure of cassava protoplast separation.
[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows:
[0007] The present invention provides a method for preparing cassava mesophyll cell protoplasts, comprising the following steps:
[0008] 1) Obtaining healthy cassava leaves;
[0009] 2) cutting the cassava leaves into strip-shaped pieces, placing the pieces in an enzymolysis solution, and performing enzymolysis in the dark to obtain an enzymolysis solution; wherein the mass volume ratio of the strip-shaped pieces to the enzymolysis solution is 1:10-50 g / ml;
[0010] 3) filtering the enzymatic hydrolyzate, taking the filtrate and subjecting it to centrifugation, discarding the supernatant, and obtaining primary protoplasts;
[0011] 4) Add protoplast washing solution to the primary protoplasts, resuspend the protoplasts and centrifuge, repeat 3-5 times, and finally resuspend the protoplast cells with protoplast washing solution to obtain cassava mesophyll cell protoplasts, which are stored.
[0012] Furthermore, in step 1), the healthy cassava leaves are cassava leaves that are growing well and free of diseases and insect pests;
[0013] Alternatively, the healthy cassava leaves are sterile cassava tissue culture seedling leaves obtained using tissue culture technology.
[0014] Furthermore, in step 2), the enzymatic hydrolysis conditions are:
[0015] The temperature is 25-28°C, and the enzymatic hydrolysis is carried out in the dark at 40 rpm for 9-12 hours.
[0016] Furthermore, the enzymatic hydrolysis solution contains the following components at final concentrations: 1%-1.5% cellulase R-10, 0.5%-1.0% macerate R-10, 0.4-0.5M mannitol, 20mM MES, 20mM potassium chloride, 0.2mM calcium chloride and 0.1% BSA.
[0017] Furthermore, in the step 2), the mass volume ratio of the strip fragments to the enzymatic hydrolysis solution is 1:30 g / ml; the enzymatic hydrolysis solution contains the following components at final concentrations: 1% cellulase R-10, 0.5% macerate R-10, 0.5M mannitol, 20mM MES, 20mM potassium chloride, 0.2mM calcium chloride and 0.1% BSA.
[0018] Furthermore, in step 2), the enzymatic hydrolysis conditions are:
[0019] The temperature was 25°C and the enzymatic hydrolysis was carried out at 40 rpm in the dark for 12 h.
[0020] Furthermore, in the step 3), the centrifugal speed is 80-100g and the centrifugal time is 3-5min.
[0021] Furthermore, in the step 4), the protoplast washing solution contains the following components at final concentrations: 154 mM sodium chloride, 125 mM calcium chloride, 5 mM potassium chloride and 2 mM MES.
[0022] Furthermore, in the step 4), the centrifugal speed is 80-100g and the centrifugal time is 3-4min.
[0023] Furthermore, in the step 4), the cassava mesophyll cell protoplasts are stored at a low temperature of 6-8°C.
[0024] Beneficial effects of the present invention:
[0025] (1) The present invention provides a method for efficiently obtaining cassava mesophyll cell protoplasts, thereby providing important technical support for basic research and applied research on cassava;
[0026] (2) The preparation method provided by the present invention can significantly shorten the time required for enzymatic hydrolysis of leaves, thereby efficiently obtaining cassava mesophyll cell protoplasts;
[0027] (3) The cassava mesophyll cell protoplasts obtained by this method were normal and full in morphology without damage. (2.42±0.08)×10 8 The quality and quantity of protoplasts met the requirements of subsequent experiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of cassava mesophyll cell protoplasts obtained by enzymatic hydrolysis with different enzymatic solutions for 6 hours (hemocytometer plate);
[0029] Figure 2 Schematic diagram of cassava mesophyll cell protoplasts obtained by enzymatic hydrolysis with different enzymatic solutions for 9 hours (hemocytometer plate);
[0030] Figure 3 Schematic diagram of cassava mesophyll cell protoplasts obtained by enzymatic hydrolysis with different enzymatic solutions for 12 hours (hemocytometer plate);
[0031] Figure 4 This is a schematic diagram of the cassava mesophyll cell protoplasts obtained in Example 9 (DIC mode).
[0032] Figure 5 This is a schematic diagram of the cassava mesophyll cell protoplasts obtained in Example 21 (BF model). DETAILED DESCRIPTION
[0033] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.
[0034] The method for preparing cassava mesophyll cell protoplasts of the present invention comprises the following steps:
[0035] 1) obtaining healthy cassava leaves; wherein the healthy cassava leaves are cassava leaves that are growing well and free of diseases and insect pests;
[0036] Alternatively, the healthy cassava leaves are sterile cassava tissue culture seedling leaves obtained by tissue culture technology;
[0037] 2) cutting the cassava leaves into strips, placing them in an enzymatic hydrolysis solution, and then placing them in a shaker at a temperature of 25-28° C. and 40 rpm in the dark for 9-12 hours to obtain an enzymatic hydrolysis solution; wherein the mass volume ratio of the strips to the enzymatic hydrolysis solution is 1:30 g / ml; and the enzymatic hydrolysis solution contains the following components at final concentrations: 1%-1.5% cellulase R-10, 0.5%-1.0% macerate R-10, 0.4-0.5M mannitol, 20mM MES, 20mM potassium chloride, 0.2mM calcium chloride, and 0.1% BSA;
[0038] 3) filtering the enzymatic hydrolyzate, centrifuging the filtrate at 80-100 g for 3-5 min, discarding the supernatant, and obtaining primary protoplasts;
[0039] 4) adding protoplast washing solution to the primary protoplasts, resuspending the protoplasts and centrifuging at a speed of 80-100g for 3-4 minutes, repeating 3-5 times, and finally resuspending the protoplast cells with the protoplast washing solution to obtain cassava mesophyll cell protoplasts, and storing them at a low temperature of 6-8°C; wherein the protoplast washing solution contains the following components at final concentrations: 154mM sodium chloride, 125mM calcium chloride, 5mM potassium chloride and 2mM MES.
[0040] Based on the above method, different enzymatic hydrolysates and enzymatic hydrolysis conditions were selected to design the preparation methods of Examples 1 to 24. The specific enzymatic hydrolysate formulas and enzymatic hydrolysis conditions are as follows:
[0041] Table 1 Effect of different enzymolysis conditions on the yield of cassava mesophyll cell protoplasts in Examples 1 to 24
[0042]
[0043]
[0044] like Figures 1 to 5 As shown in the figure: the preparation methods of Examples 1 to 24 can significantly shorten the time required for enzymolysis when enzymolysis is performed on leaves, thereby efficiently obtaining cassava mesophyll cell protoplasts; the cassava mesophyll cell protoplasts are normal and full in morphology without damage, wherein the enzymolysis solution selected in Example 21 (containing the following final concentrations of each component: 1% cellulase R-10, 0.5% macerate R-10, 0.5M mannitol, 20mM MES, 20mM potassium chloride, 0.2mM calcium chloride and 0.1% BSA) has an enzymolysis time of 12h; the effect is the best, and (2.42±0.08)×10 8 A protoplast.
[0045] It can be seen from this that the present invention provides a set of methods for efficiently obtaining cassava mesophyll cell protoplasts, thereby providing important technical support for basic research and applied research on cassava.
[0046] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.
Claims
1. A method for preparing cassava mesophyll cell protoplasts, characterized in that: The following steps are involved: 1) Obtaining healthy cassava leaves; 2) cutting the cassava leaves into strip-shaped pieces, placing the pieces in an enzymolysis solution, and performing enzymolysis in the dark to obtain an enzymolysis solution; wherein the mass volume ratio of the strip-shaped pieces to the enzymolysis solution is 1:10-50 g / ml; 3) filtering the enzymatic hydrolyzate, taking the filtrate and subjecting it to centrifugation, discarding the supernatant, and obtaining primary protoplasts; 4) Add protoplast washing solution to the primary protoplasts, resuspend the protoplasts and centrifuge, repeat 3-5 times, and finally resuspend the protoplast cells with protoplast washing solution to obtain cassava mesophyll cell protoplasts, which are stored.
2. The method for preparing cassava mesophyll cell protoplasts according to claim 1, wherein: In the step 1), the healthy cassava leaves are cassava leaves that are growing well and free of diseases and insect pests; Alternatively, the healthy cassava leaves are sterile cassava tissue culture seedling leaves obtained using tissue culture technology.
3. The method for preparing cassava mesophyll cell protoplasts according to claim 1, wherein: In the step 2), the enzymatic hydrolysis conditions are: The temperature is 25-28°C, and the enzymatic hydrolysis is carried out in the dark at 40 rpm for 9-12 hours.
4. The method for preparing cassava mesophyll cell protoplasts according to claim 1 or 3, characterized in that: The enzymatic hydrolysis solution contains the following components at final concentrations: 1%-1.5% cellulase R-10, 0.5%-1.0% malonase R-10, 0.4-0.5M mannitol, 20mM MES, 20mM potassium chloride, 0.2mM calcium chloride and 0.1% BSA.
5. The method for preparing cassava mesophyll cell protoplasts according to claim 4, wherein: In the step 2), the mass volume ratio of the strip fragments to the enzymatic hydrolysis solution is 1:30 g / ml; and the enzymatic hydrolysis solution contains the following components at final concentrations: 1% cellulase R-10, 0.5% macerate R-10, 0.5M mannitol, 20mM MES, 20mM potassium chloride, 0.2mM calcium chloride and 0.1% BSA.
6. The method for preparing cassava mesophyll cell protoplasts according to claim 5, characterized in that: In the step 2), the enzymatic hydrolysis conditions are: The temperature was 25°C and the enzymatic hydrolysis was carried out at 40 rpm in the dark for 12 h.
7. The method for preparing cassava mesophyll cell protoplasts according to claim 1, characterized in that: In the step 3), the centrifugal speed is 80-100g and the centrifugal time is 3-5min.
8. The method for preparing cassava mesophyll cell protoplasts according to claim 1, characterized in that: In the step 4), the protoplast washing solution contains the following components at final concentrations: 154 mM sodium chloride, 125 mM calcium chloride, 5 mM potassium chloride and 2 mM MES.
9. The method for preparing cassava mesophyll cell protoplasts according to claim 1 or 8, characterized in that: In the step 4), the centrifugal speed is 80-100g and the centrifugal time is 3-4min.
10. The method for preparing cassava mesophyll cell protoplasts according to claim 9, characterized in that: In the step 4), the cassava mesophyll cell protoplasts are stored at a low temperature of 6-8°C.