Extraction method suitable for poplar leaf protoplast with multiple genotypes

Abstract

The invention relates to the technical field of protoplast extraction, in particular to an extraction method suitable for poplar leaf protoplast with multiple genotypes, which specifically comprises the following steps: step 1, selecting poplar tissue culture seedling leaves, putting the leaves into enzymatic hydrolysate, carrying out dark culture at 24-26 DEG C for 3-4 hours to obtain enzyme-protoplast suspension, adding a W5 solution into the enzyme-protoplast suspension, and carrying out enzymatic hydrolysis for 3-4 hours to obtain enzymatic hydrolysate; filtering, centrifuging the filtrate, removing the supernatant, and mixing the lower layer with the W5 solution to obtain a protoplast suspension; step 2, performing low-temperature precipitation and centrifugation on the protoplast suspension, removing supernate, and mixing the lower-layer protoplast with a W5 solution to obtain a protoplast extracting solution;

Description

Technical Field

[0001] The present invention relates to the technical field of protoplast extraction, and particularly relates to a method for extracting protoplasts from poplar leaves applicable to multiple genotypes. Background Art

[0002] As a perennial woody plant, poplar is an ideal model plant for studying processes such as tree growth, development, senescence, and environmental adaptation. However, due to characteristics such as the long growth cycle and complex genetic characteristics of poplar, its application in biochemical, molecular, cellular, genetic, genomic, transcriptomic, and proteomic analyses is limited. Protoplasts are a general tool for studying gene function and signal pathways based on cells. Therefore, by extracting poplar protoplasts, these research limitations can be overcome, and the gene function and signal transduction mechanism of woody plants can be understood more deeply, providing a theoretical basis for the genetic improvement and breeding of woody plants.

[0003] Poplar protoplasts can be used in research on gene expression and regulation, subcellular localization, protein interaction, gene editing and genetic improvement, etc. Through transient transformation technology, foreign genes are introduced into the extracted poplar protoplasts, and their expression in cells is observed to reveal the gene expression regulation mechanism; by introducing proteins or genes with fluorescent labels into protoplasts and observing their localization in cells using a confocal microscope, the functions and interactions of proteins or genes can be understood; through techniques such as bimolecular fluorescence complementation (BiFC), the interaction of two proteins in cells is observed, providing important clues for revealing the functions and regulatory networks of proteins; in addition, through gene editing techniques such as CRISPR / Cas9, genes in protoplasts can be site-directed mutated or knocked out to achieve the directional improvement of the traits of woody plants.

[0004] In summary, the extraction and application of poplar protoplasts play an important role in plant science research. The extraction and application of poplar protoplasts not only promote the scientific research development of poplar but also provide reference for the extraction and application of protoplasts of other woody plants. By continuously optimizing and improving protoplast extraction and transformation technologies, the efficiency and accuracy of woody plant scientific research can be further improved, making an important contribution to promoting the development of woody plant scientific research.

[0005] However, the extraction of poplar protoplasts still faces significant difficulties and limitations, mainly due to the cell wall characteristics of poplar and the fragility of the protoplasts themselves. As a woody plant, poplar has more pectin in its cell wall. Pectin is a complex polysaccharide, and its presence increases the toughness of the cell wall but also makes the separation of protoplasts more difficult. In addition to pectin, the poplar cell wall also contains other components such as cellulose and hemicellulose. These components are intertwined to form a complex network structure, further increasing the difficulty of protoplast separation. The extraction process of protoplasts is affected by factors such as sample type, enzyme solution, enzyme digestion conditions, centrifugation speed, and osmotic pressure stabilizer. Moreover, the structure of the protoplasts after removing the cell wall is relatively fragile and is easily damaged by factors such as mechanical force and chemical reagents. Therefore, suitable enzyme digestion conditions are crucial for the success of protoplast extraction. In addition, due to differences in leaf thickness, hardness, and cell wall composition among different genotypes of poplar, there are also differences in the difficulty of protoplast extraction, and the enzyme digestion efficiency, protoplast purification efficiency, etc. all need to be further improved.

[0006] Therefore, providing a method for extracting protoplasts applicable to the main representative varieties of different poplar factions has become an urgent problem to be solved. Summary of the Invention

[0007] To solve the above problems, the present invention provides a method for extracting protoplasts from the leaves of multiple genotypes of poplar, which specifically includes the following steps:

[0008] Step 1: Select the leaves of poplar tissue culture seedlings and put them into an enzyme solution, and incubate them in the dark at 24-26°C for 3-4 hours to obtain an enzyme-protoplast suspension. Add W5 solution to the enzyme-protoplast suspension, filter it to obtain a protoplast suspension.

[0009] Preferably, the ratio of the leaves, the enzyme solution, the W5 solution added to the suspension, and the W5 solution mixed in the lower layer is 1 g: 10 mL: 10 mL: 10 mL. Most preferably, cut the leaves into strips 1 mm wide, incubate them in the dark with shaking at 60 rpm for 3-4 hours, and then increase the shaking speed to 80 rpm for 30 minutes.

[0010] Preferably, when adding W5 solution to the enzyme-protoplast suspension, first add 1 / 2 volume of W5 solution, filter it, then add 1 / 2 volume of W5 solution to the filter residue, mix it slightly and then filter it, centrifuge it, remove the supernatant, and mix all the lower layer liquid and the filtrate to obtain a protoplast suspension.

[0011] Preferably, the enzymatic hydrolysate includes 1.5% (w / v) cellulase R-10, 0.5% (w / v) pectinase Y-23, 0.6 mol / L mannitol, 10 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, and 0.1% (w / v) BSA based on water. Most preferably, 0.5% (w / v) pectinase Y-23 is replaced with 0.5% (w / v) macerozyme R-10.

[0012] Step 2: Perform low-temperature precipitation on the protoplast suspension, centrifuge, remove the supernatant, and mix the lower-layer protoplasts with W5 solution to obtain a protoplast extract.

[0013] Preferably, the volume ratio of the protoplast suspension to the W5 solution is 1:1.

[0014] Preferably, the low temperature is 0-4°C, and the precipitation is carried out for 30 minutes.

[0015] The present invention has the following advantages: By optimizing the enzymatic hydrolysate and purification method for plant protoplast extraction, the present invention has developed a set of extraction methods suitable for protoplasts of poplar leaves of multiple genotypes. The protoplast cells obtained by this method all have high activity and transformation efficiency, providing key technical support for quickly obtaining protoplasts and carrying out research on functional genomics, single-cell omics, synthetic biology, and molecular breeding of poplar. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0017] Figure 1 Protoplast cells of poplar leaves of different genotypes after enzymatic hydrolysis with enzymatic hydrolysate I and enzymatic hydrolysate II. Among them, A and B: Populus × euramericana cv. 741; C and D: Populus × euramericana cv. 107; E and F: Populus alba × Populus glandulosa cv. WQ20; G and H: Populus euphratica. A, C, E, and G are protoplast cells after enzymatic hydrolysis with enzymatic hydrolysate I, and B, D, F, and H are protoplast cells after enzymatic hydrolysis with enzymatic hydrolysate II. The scale bar in the figure is 100 μm.

[0018] Figure 2Protoplast cells of Populus×euramericana cv. 741-I and Populus×euramericana cv. 741-II purified by different purification methods. Among them, A: Protoplasts directly washed with W5 solution; B: Protoplasts after sucrose centrifugation and layering; C: Protoplasts of Populus×euramericana cv. 741-I purified by W5; D: Protoplasts of Populus×euramericana cv. 741-II purified by W5; E: Protoplasts of Populus×euramericana cv. 741-I purified by sucrose solution; F: Protoplasts of Populus×euramericana cv. 741-II purified by sucrose solution. a: Protoplasts of Populus×euramericana cv. 741 leaves digested by Enzyme Solution I, b: Protoplasts of Populus×euramericana cv. 741 leaves digested by Enzyme Solution II. 1: Protoplast cells under bright field condition; 2: Protoplast cells under fluorescence condition (BP460-495) after FDA staining. The scale bar in the figure is 100 μm.

[0019] Figure 3 Cells of protoplasts digested by Enzyme Solution I and II purified by W5 solution. Among them, A: Protoplast cells of Populus×popularis cv. 107-I after purification; B: Protoplast cells of Populus×popularis cv. 107-II after purification; C: Protoplast cells of Populus×euramericana cv. WQ20-I after purification; D: Protoplast cells of Populus×euramericana cv. WQ20-II after purification; E: Protoplast cells of Populus euphratica Oliv.-I after purification; F: Protoplast cells of Populus euphratica Oliv.-II after purification. 1: Protoplast cells under bright field condition; 2: Protoplast cells under fluorescence condition (BP460-495) after FDA staining. The scale bar in the figure is 100 μm.

[0020] Figure 4 Protoplast cells of leaves of different genotypes of Populus digested by Enzyme Solution I and purified by W5 solution. Among them, A: Leaf protoplasts digested by Enzyme Solution I; B: Protoplasts after natural precipitation with W5 solution; a. Populus bolleana Lauche-I, b. Populus jrtyschensis Kom.-2-I, c. Populus deltoides cv. 'Danhong'-I; C: Protoplasts of Populus bolleana Lauche-I purified by W5; D: Protoplasts of Populus jrtyschensis Kom.-2-I purified by W5; E: Protoplasts of Populus deltoides cv. 'Danhong'-I purified by W5. 1: Protoplast cells under bright field condition; 2: Protoplast cells under fluorescence condition (BP460-495) after FDA staining. The scale bar in the figure is 100 μm.

[0021] Figure 5 Results of transient transformation of GFP plasmid in protoplast cells of leaves of different genotypes of Populus. Among them, A: Populus×euramericana cv. 741; B: Populus bolleana Lauche; C: Populus jrtyschensis Kom.-2; D: Populus×popularis cv. 107; E: Populus deltoides cv. 'Danhong'; F: Populus×euramericana cv. WQ20. 1: Protoplast cells under bright field condition; 2: Protoplast cells under GFP fluorescence condition (BP460-495). The scale bar in the figure is 100 μm. Detailed implementation methods

[0022] The technical solutions in the embodiments of the invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the invention without creative efforts belong to the scope of protection of the invention.

[0023] Preparation of solutions for examples and test examples:

[0024] Preparation of Enzyme Solution I: Add 0.3 g of cellulase R-10, 0.10 g of macerozyme R-10, 2.2 g of mannitol, 100 μL of 2 mol / L KCl solution, and 2 mL of 0.2 mol / L MES solution to 15 mL of water. After complete dissolution, incubate in a water bath at 55 °C for 10 min to inactivate Dnase and protease activities. Then add 200 μL of 1 mol / L CaCl2 solution and 0.02 g of BSA, adjust the pH value to 5.7, make up the volume to 20 mL with water, and filter sterilize.

[0025] Preparation of Enzyme Solution II: Add 0.3 g of cellulase R-10, 0.10 g of pectinase Y-23, 2.2 g of mannitol, 100 μL of 2 mol / L KCl solution, and 2 mL of 0.2 mol / L MES solution to 15 mL of water. After complete dissolution, incubate in a water bath at 55 °C for 10 min to inactivate Dnase and protease activities. Then add 200 μL of 1 mol / L CaCl2 solution and 0.02 g of BSA, adjust the pH value to 5.7, make up the volume to 20 mL with water, and filter sterilize.

[0026] Preparation of W5 Solution: Add 5.13 mL of 3 mol / L NaCl solution, 12.5 mL of 1 mol / L CaCl2, 0.25 mL of 2 mol / L KCl solution, and 1 mL of 0.2 mol / L MES solution to 80 mL of water, adjust the pH value to 5.7, make up the volume to 100 mL with water, and filter sterilize.

[0027] Preparation of MMG Buffer: Add 7.29 g of mannitol, 0.3 g of MgCl2, and 2 mL of 0.2 mol / L MES to 80 mL of water, adjust the pH value to 5.7, make up the volume to 100 mL with water, and filter sterilize.

[0028] Preparation of PEG-Calcium Transfection Solution: Add 0.36 g of mannitol, 4 g of PEG, and 1 mL of 1 mol / L CaCl2 solution to a 50 mL centrifuge tube, adjust the pH value to 5.7, make up the volume to 10 mL with water, and filter sterilize.

[0029] Preparation of WI solution: Add 9.11 g of mannitol, 1 mL of 2 mol / L KCl solution, and 2 mL of 0.2 mol / L MES solution to 80 mL of water respectively. Adjust the pH value to 5.7, make up the volume to 100 mL with water, and then filter and sterilize.

[0030] Example 1

[0031] Step 1: Select about 1 g of young leaves of Populus tissue culture seedlings subcultured for 3 - 4 weeks. Without crushing the tissue, use a surgical blade to cut the leaves into 1 - mm - wide strips and transfer them to a petri dish containing 10 mL of enzyme solution. Use a pipette tip or forceps to completely immerse the leaf strips in the enzyme solution. Seal the plate (tissue culture flask) with Parafilm and wrap it with aluminum foil. Incubate the petri dish in the dark at 24 - 26 °C with shaking at 60 rpm for 3 - 4 hours, then increase the shaking speed to 80 rpm for 30 minutes to obtain an enzyme - protoplast suspension.

[0032] Then slowly add 5 mL of W5 solution to the enzyme - protoplast suspension, shake well, pre - wet a 40 - μm cell filter with W5 solution and place it on top of a 50 - mL round - bottom centrifuge tube. Keep the tube at a 45° tilt, and pass the enzyme - protoplast suspension through the filter to remove undigested leaf tissue and collect the filtrate. Add another 5 mL of W5 solution to the petri dish containing leaf strips, shake well, gently rotate the petri dish to release more protoplasts, and collect the filtrate through the filter again. Mix the two filtrates together, then place them in a 50 - mL Falcon tube and centrifuge the tube at 100 g for 5 minutes, and then remove the supernatant. Mix the lower layer with 10 mL of W5 solution to obtain a protoplast suspension.

[0033] Step 2: Purify the protoplast suspension with W5 solution and sucrose solution respectively.

[0034] (1) W5 direct washing and centrifugation method: Transfer the protoplast suspension obtained in Step 1 to a 15 - mL Falcon tube, let it precipitate naturally at 0 - 4 °C for 30 minutes, then centrifuge at 100 g for 2 minutes, remove the supernatant, and resuspend the lower - layer protoplasts in 10 mL of W5.

[0035] (2) Sucrose layering method: Add 5 mL of 0.60 mol / L sucrose solution to a 15 - mL tube, then slowly release the protoplast suspension obtained in Step 1 along the side of the test tube to the top of the sucrose solution, and then centrifuge at 100 g for 5 minutes, remove the supernatant, and transfer the surface layer of the sucrose solution containing protoplasts to a 15 - mL Falcon tube containing 10 mL of W5 to obtain a protoplast extract.

[0036] Step 3: Counting of protoplasts

[0037] Centrifuge the protoplast extract obtained in Example 1 at a speed of 100 g for 3 min, remove the supernatant, add 2 mL of W5 solution to the centrifuge tube, gently tap the centrifuge tube to resuspend the particles, and then add 8 mL of W5 solution. Centrifuge the centrifuge tube at 100 g for 3 minutes again, remove the supernatant, and mix the lower layer with 1 - 2 mL of MMG buffer. Use a cell counting chamber to calculate the number of protoplasts, and adjust the final density in the MMG buffer to 2 - 4×10 6 / mL.

[0038] Step 4, Activity detection of protoplasts

[0039] Take 100 μL of the protoplast extract prepared in Example 1 and place it in a 0.2 mL PCR tube. Add 1 μL of the 10 mg / mL FDA mother liquor, mix well, and let it stand at room temperature for 2 min. Take 40 μL and observe and count it on a cell counter under a fluorescence microscope. First, observe the morphology of protoplast cells under bright field conditions, record the number of protoplast cells that are not broken, have a complete morphology, and are brightly colored, and calculate the cell yield per unit mass of leaves. Then, use a fluorescence microscope to observe and record the number of cells emitting fluorescence in the GFP channel as the active protoplasts, and calculate the activity ratio of protoplast cells by the ratio of the number of active cells to the number of protoplast cells in the same field of view. Each experiment is repeated more than twice, and the average value of three replicates is taken for each count.

[0040] Step 5, Transformation of protoplasts

[0041] Transfer 10 μL of the plasmid containing the GFP tag (concentration 1 μg / μL) to a 10 mL round-bottom vial or a 2 mL centrifuge tube. Use a 1 mL pipette tip to transfer 100 μL of the protoplast extract (about 2×10 5 protoplasts) to the vial or centrifuge tube. Then use a 1 mL pipette tip to transfer 110 μL of the PEG transfection reagent, place it at room temperature in the dark for 15 minutes, then add 1 mL of W5 solution to terminate the reaction, centrifuge at 100 g for 5 minutes, remove the supernatant, and collect the lower layer of protoplasts. Mix the lower layer of protoplast particles with 1 mL of W5 solution, and repeat the W5 washing step twice. Resuspend the protoplasts in 2 mL of WI medium and transfer the protoplasts to a single well in a 6-well plate or a 12-well plate. Culture the transfected protoplasts in the dark for 48 hours (25 ± 2 °C). Observe the GFP expression in the DNA-transfected protoplasts.

[0042] Test Example 1

[0043] According to the method in Step 1 of Example 1, tissue culture seedlings of poplars such as Populus × euramericana cv. 741, Populus × euramericana cv. 107, WQ20 poplar, and Populus euphratica were selected as receptor materials respectively. Their leaves were enzymolyzed with Enzyme Solution I and Enzyme Solution II respectively, and the protoplasts were counted. The results are shown in Table 1 and Figure 1 .

[0044] Table 1

[0045]

[0046] As can be seen from Table 1 and Figure 1 , the cell yields (the number of protoplast cells obtained from unit weight of leaves) of poplar leaves enzymolyzed with Enzyme Solution I are all greater than those with Enzyme Solution II. Especially for Populus × euramericana cv. 107 and WQ20 poplar, the cell yields are basically 3 - 5 times more. The protoplast cell state obtained by enzymolysis with Enzyme Solution I is better, the cells are round and translucent, while a large proportion of cells in the protoplasts obtained by enzymolysis with Enzyme Solution II have ruptured and there are cell residues. Therefore, for poplar leaves of different genotypes, the effect of enzymolyzing protoplasts with Enzyme Solution I is significantly better than that with Enzyme Solution II, and Enzyme Solution I is the preferred enzyme solution for extracting poplar leaf protoplasts.

[0047] Test Example 2

[0048] Taking the protoplasts of Populus × euramericana cv. 741 leaves enzymolyzed with Enzyme Solution I (741 poplar - I) and the protoplasts of Populus × euramericana cv. 741 leaves enzymolyzed with Enzyme Solution II (741 poplar - II) as objects, two different protoplast purification methods were set for cell purification. According to the method in Step 2 of Example 1, centrifugal stratification was carried out with W5 solution and sucrose solution respectively, and the protoplasts were counted and their activities were detected. The results are shown in Table 2 and Figure 2 .

[0049] Table 2

[0050]

[0051] As can be seen from Table 2 and Figure 2 , the loss of protoplasts in 741 poplar - I and 741 poplar - II after centrifugal stratification purification with sucrose solution is the largest. The concentrations of 741 poplar - I after direct washing with W5 solution and centrifugal stratification purification with sucrose decreased from (28.10 ± 2.34)×10 6 cells / g to (25.60 ± 0.62)×10 6 cells / g and (8.34 ± 1.41)×10 6 cells / g respectively, while the concentration of 741 poplar - II decreased from (18.81 ± 1.88)×10 6 cells / g to (13.37 ± 0.84)×10 6 cells / g and (6.11 ± 0.28)×106 cells / g, indicating that the purification method of centrifugal stratification with sucrose solution has a greater impact on the yield of poplar protoplasts. In addition, there are more debris in the supernatant after washing and centrifuging 741 poplar-II with W5 solution than 741 poplar-I ( Figure 2 A), and the same situation also occurs in sucrose stratification purification ( Figure 2 B), so it is speculated that there are more dead cells in 741 poplar-II than 741 poplar-I. The subsequent detection results of the viability of protoplast cells after FDA staining also show that whether 741 poplar-II is directly washed with W5 solution or purified by sucrose stratification, the obtained protoplasts basically have no viability, while the viability of the protoplasts of 741 poplar-I purified by washing with W5 solution is 93.87%, higher than the viability of the protoplasts purified by sucrose stratification, which is 70.76%. This again shows that the protoplasts enzymolyzed by enzyme solution I are superior to those of enzyme solution II in terms of quantity and viability. Based on the above results, the purification method of directly washing with W5 solution is superior to the purification method of centrifugal stratification with sucrose solution in terms of both cell yield and viability. The purification method of directly washing with W5 solution is recommended as the preferred purification method for poplar leaf protoplasts.

[0052] The purification method of directly washing with W5 solution was used to purify the protoplasts of 107 poplar (107 poplar-I), WQ20 poplar (WQ20 poplar-I), Populus euphratica (Populus euphratica-I) enzymolyzed by enzyme solution I and 107 poplar (107 poplar-II), WQ20 poplar (WQ20 poplar-II), Populus euphratica (Populus euphratica-II) enzymolyzed by enzyme solution II, and the protoplasts were counted and their activities were detected. The results are shown in Table 3 and Figure 3 .

[0053] Table 3

[0054]

[0055] As can be seen from Table 3 and Figure 3 , there is little difference in the number of purified cells compared with that before purification. The cell viabilities of 107 poplar-I, WQ20 poplar-I and Populus euphratica-I are 79.11%, 11.28% and 34.92% respectively, but the viabilities of 107 poplar-II, WQ20 poplar-II and Populus euphratica-II are 0. This again shows that enzyme solution II is not suitable for enzymolysis of poplar leaf protoplasts, and most of the protoplasts enzymolyzed by it are dead cells or have ruptured cell membranes.

[0056] Test Example 3

[0057] In this Test Example 3, the protoplasts of Populus bolleana, Populus jrtyschensis cv. 'Zhonglin2' and Populus deltoides cv. 'Danhong' were enzymolyzed using enzyme solution I according to the method of Example 1, and the protoplast cells were purified by the purification method of directly washing with W5 solution, and were counted and their activities were detected. The results are shown in Table 4 and Figure 4 .

[0058] Table 4

[0059]

[0060]

[0061] As can be seen from Table 4 and Figure 4 it can be known that the protoplast cell concentrations of Populus bolleana, Populus jrtyschensis cv. Xibei 2 and Populus deltoides cv. Danhong after purification are (13.50±1.25)×10 6 cells / g, (13.70±0.22)×10 6 cells / g and (14.17±0.70)×10 6 cells / g respectively. Through the cell viability detection of FDA staining, the cell viabilities of the protoplasts of Populus bolleana, Populus jrtyschensis cv. Xibei 2 and Populus deltoides cv. Danhong are 92.16%, 72.58% and 32.56% respectively. It shows that Enzymolysis I is suitable for the protoplast enzymolysis of poplar leaves with different genotypes, and the method of directly washing and purifying with W5 solution is suitable for the purification of protoplasts.

[0062] Perform transient expression of protoplast transformation on the above protoplasts according to the method of Step Five of Example 1, and the results are shown in Table 5 and Figure 5 .

[0063] Table 5

[0064]

[0065] As can be seen from Table 5 and Figure 5 it can be known that there are different degrees of differences in cell activities among the protoplasts dissociated from poplars of different factions. Among them, the protoplast activity of the section Populus is the highest, and the cell viabilities of Populus × euramericana cv. 741 and Populus bolleana can reach more than 92%. The protoplast activities of the section Aigeiros and Populus euphratica are relatively low. However, the transformation efficiency of protoplast cells has a great relationship with the genotype. For example, the cell viabilities of the protoplast cells of Populus × euramericana cv. 741 and Populus bolleana are very high, but the cell transformation rates are 49.55% and 46.23% respectively. The protoplast cell activities of the section Aigeiros poplars are not high, but their transformation efficiency can reach 37%-57%. Among them, the cell viability of Populus deltoides cv. WQ20 is only 11.28%, while its transformation rate can reach 37.14%. The protoplast cell activities and transformation rates of Populus euphratica are not very high.

[0066] To sum up, the extraction method of protoplasts developed in the present invention is applicable to the dissociation of protoplasts from poplar leaves with different genotypes such as the section Populus and the section Aigeiros. The protoplast cell activities obtained after purification vary due to different genotypes, but relatively high transformation efficiencies can be obtained. It shows that the protoplasts dissociated by this method meet the research requirements for subsequent molecular, cellular, genetic, gene function verification, and molecular breeding, etc.

[0067] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting protoplasts from poplar leaves applicable to multiple genotypes, characterized in that, It includes the following steps: Step 1: Select the leaves of poplar tissue culture seedlings and put them into the enzyme solution, and conduct dark culture at 24 - 26°C for 3 - 4 h to obtain an enzyme-protoplast suspension. Add W5 solution to the enzyme-protoplast suspension, filter, centrifuge the filtrate, remove the supernatant, and mix the lower layer with the W5 solution to obtain a protoplast suspension; Step 2: Conduct low-temperature precipitation on the protoplast suspension, centrifuge, remove the supernatant, and mix the lower-layer protoplasts with the W5 solution to obtain a protoplast extract.

2. The extraction method for poplar leaf protoplasts applicable to multiple genotypes according to claim 1, characterized in that, In Step 1, the ratio of the leaves, enzyme solution, W5 solution added to the suspension, and the W5 solution for mixing the lower layer is 1 g: 10 mL: 10 mL: 10 mL, and the culture is shaking culture with a rotation speed of 60 - 80 rpm.

3. A method for extracting protoplasts from poplar leaves applicable to multiple genotypes, as claimed in claim 1, wherein When adding the W5 solution to the enzyme-protoplast suspension in Step 1, first add 1 / 2 volume of the W5 solution, filter, then add 1 / 2 volume of the W5 solution to the filter residue, filter, mix the two filtrates, centrifuge, remove the supernatant, and mix the lower layer with the W5 solution to obtain a protoplast suspension.

4. A method for extracting protoplasts from poplar leaves applicable to multiple genotypes, as claimed in claim 1, wherein The enzyme solution in Step 1 includes 1.5% (w / v) cellulase R-10, 0.5% (w / v) macerozyme R-10, 0.6 mol / L mannitol, 10 mmol / L KCl, 20 mmol / L MES, 10 mmol / L CaCl2, and 0.1% (w / v) BSA based on water.

5. A method for extracting protoplasts from poplar leaves applicable to multiple genotypes, as claimed in claim 1, wherein In Step 2, the volume ratio of the protoplast suspension to the W5 solution is 1:

1.

6. The extraction method for poplar leaf protoplasts applicable to multiple genotypes according to claim 1, wherein The low temperature in Step 2 is 0 - 4°C.

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