Method for preparing and transforming 84K poplar callus protoplast and application of 84K poplar callus protoplast

Abstract

The invention discloses a method for preparing and transforming callus protoplast of populus 84K and application of the method, and belongs to the technical field of genetic transformation of woody plants. In order to solve the technical problem that a high-efficiency and high-activity preparation method of the 84K poplar protoplast is lacked in the prior art, the method takes callus of 84K poplar as a material, and optimizes a whole set of preparation process including enzymatic hydrolysate components, enzymatic hydrolysis conditions, osmotic pressure and purification steps to prepare the 84K poplar protoplast. The efficient and reliable protoplast preparation and transformation method specially designed for the 84K poplar callus is obtained. The protoplast cells obtained by the method are relatively high in activity and transformation efficiency, and can meet the requirements of subsequent researches in the aspects of molecules, cells, heredity, gene function verification, molecular breeding and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of woody plant genetic transformation, and particularly relates to a method for preparing and transforming 84K poplar callus protoplasts and application thereof. BACKGROUND

[0002] 84K poplar is a hybrid clone of Populus alba L. Populus alba ) and Populus adenophora Maxim. Populus glandulosa ), which has the outstanding advantages of fast growth, good wood properties, strong resistance, and wide adaptability, and plays an important role in ecological environment management and urban greening. At the same time, as a typical woody model plant, 84K poplar has an irreplaceable position in forest molecular breeding and basic biology research.

[0003] However, the inherent long life cycle and highly hybrid genetic background of poplar pose significant challenges in in-depth research at the biochemical, molecular, and various omics (such as genome, transcriptome, and proteome) levels. Under this background, protoplast, as a single-cell system without cell wall, provides an ideal technical platform for efficient research on gene function, signal transduction, and molecular regulation network of poplar at the cellular level.

[0004] Using 84K poplar protoplast system, multi-dimensional molecular biology research can be efficiently carried out. By transient transformation technology, exogenous genes can be introduced into protoplasts to quickly analyze the expression pattern and regulation mechanism of genes; with the help of fluorescent protein labeling combined with confocal microscope observation, the distribution dynamics of target proteins in cells can be accurately analyzed, and then the biological function can be inferred; in the study of protein interaction, techniques such as bimolecular fluorescence complementation can intuitively present protein interaction in living cells, providing clues for analyzing complex signal regulation networks; in addition, CRISPR / Cas9-based gene editing technology can be used for targeted gene mutation in protoplasts, providing an effective tool for directional improvement of wood plant traits.

[0005] Although the 84K poplar protoplast system has broad prospects in theoretical research, its efficient and high-activity preparation is still a key bottleneck restricting the development of related research. The dense structure of poplar cell wall is rich in pectin, cellulose, and hemicellulose, and other complex polysaccharides, resulting in low enzymolysis efficiency; at the same time, protoplasts without cell wall protection are extremely fragile, and are easy to lose activity or even rupture due to factors such as osmotic pressure imbalance, mechanical damage, or oxidative stress during isolation and purification. Therefore, systematic optimization of the whole preparation process including enzyme solution components, enzymolysis conditions, osmotic pressure stabilizers, and purification steps is crucial for obtaining high-quality protoplasts.

[0006] To overcome the above challenges, the selection of appropriate starting materials is the first step to optimize the preparation process. Compared with directly using organs with high differentiation degree such as leaves, using callus (especially uniform suspension cell lines) as the starting material for protoplast preparation has multiple irreplaceable advantages: first, callus cells divide vigorously, and the physiological state is highly uniform, which can provide a protoplast population with consistent genetic background and low differentiation degree, greatly improving the repeatability of the experiment; second, protoplasts prepared from light-colored embryogenic callus do not contain chloroplasts, which can completely avoid the background interference of chloroplast autofluorescence on fluorescence labeling experiments (especially subcellular localization studies); third, callus can be scaled up and continuously expanded in culture bottles under sterile conditions, which is not limited by season and external environment, can stably and massively supply experimental materials, significantly improves the preparation efficiency and reduces the cost, and is especially suitable for research and application scenarios that require high-throughput operation.

[0007] In summary, although the 84K poplar protoplast system has great potential in functional genomics research, there is currently a lack of a complete, stable, and repeatable standardized method from high-quality callus induction to efficient enzymatic preparation, to high-activity protoplast acquisition and subsequent transformation. The existing technology cannot meet the strict requirements of high-throughput transformation, microscopic observation, and gene editing applications while ensuring high yield and high activity of protoplasts. Therefore, developing a high-efficiency and reliable protoplast preparation and transformation method specifically designed for 84K poplar callus has become a key technical problem that needs to be broken through in the field, and has important scientific value and application prospects for promoting cell and molecular level research of poplar and other woody plants. SUMMARY

[0008] To solve the technical problem that the prior art lacks an efficient and high-activity preparation method for 84K poplar protoplasts, the present application uses 84K poplar callus as the material, and optimizes the whole preparation process including enzyme solution components, enzymatic conditions, osmotic pressure, and purification steps, to obtain a high-efficiency and reliable protoplast preparation and transformation method specifically designed for 84K poplar callus.

[0009] To solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a method for preparing and transforming 84K poplar callus protoplasts, which comprises the following steps: S1, culturing of 84K poplar callus: taking 1-month-old healthy 84K poplar sterile tissue culture seedlings, selecting dark green healthy leaves, cutting 3-5 times vertically along the main leaf vein, and placing them in 84K poplar callus induction medium, 23-27℃ dark culture for 3-4 weeks, inducing dedifferentiation, and obtaining white and yellowish moist callus; S2, extraction of 84K poplar protoplasts: 0.3-0.7 g of callus obtained in S1 was put into 5 mL of enzyme solution, and after being gently blown apart, enzyme hydrolysis was carried out under the conditions of avoiding light and low-speed shaking to obtain enzyme hydrolysate; S3, purification of 84K poplar protoplasts: a 70 μm cell filter screen was wetted with pre-cooled W5 solution, and the enzyme hydrolysate obtained in S2 was filtered into a sterile centrifuge tube to obtain protoplasts, which were washed 2-4 times with pre-cooled W5 solution; finally, W5 solution was added for storage to obtain a protoplast suspension; S4, transient transformation of 84K poplar protoplasts: the protoplast suspension obtained in S3 was placed on ice for 30 min, centrifuged, and the W5 supernatant was removed; pre-cooled MMg solution was added to resuspend the protoplasts, which were centrifuged and the supernatant was removed; MMg solution was added to dilute the protoplast suspension to 10 5 μg / μL of GFP plasmid was mixed with the protoplast suspension at a volume ratio of 1:10, and PEG4000 transformation solution was gently added, and after gentle mixing, transformation was carried out under the condition of avoiding light.

[0010] In an embodiment of the present application, the composition of the 84K poplar callus induction medium in S1 is as follows: 2.41 g / L WPM, 20 g / L sucrose, 1 mg / L 2,4-D, 100 mg / L Kinetin, 5.5 g / L agar, and the balance is water, with a pH of 5.9.

[0011] In an embodiment of the present application, the main components of the enzyme solution in S2 include cellulase R-10 with a mass fraction of 1.5%, macerozyme R-10 with a mass fraction of 0.40%, pectinase Y-23 with a mass fraction of 0.50%, and 0.50 M mannitol.

[0012] In an embodiment of the present application, the temperature of the enzyme hydrolysis in S2 is 25°C, and the time is 4 h.

[0013] In an embodiment of the present application, the rotation speed of the low-speed shaking in S2 is 60-100 rpm.

[0014] In an embodiment of the present application, the concentration of mannitol in the W5 solution in S3 is 0.485 M.

[0015] In an embodiment of the present application, the washing in S3 is adding W5 solution to the protoplasts, resuspending, centrifuging at 100 g for 2-3 min, and removing the supernatant; the acceleration and deceleration of the centrifugation process is 3.

[0016] In an embodiment of the present application, the mass concentration of PEG4000 in the PEG4000 transformation solution in S4 is 30%.

[0017] In one embodiment of the present application, the time for the transformation of S4 is 20-25 min.

[0018] In one embodiment of the present application, the conditions for the centrifugation of S4 are all 100 g centrifugation for 1 min.

[0019] A second object of the present application is to provide the use of the above method in screening plant functional genes or regulatory elements.

[0020] A third object of the present application is to provide the use of the above method in cultivating high-quality transgenic plants.

[0021] The present application has the following beneficial effects: The present application takes 84K poplar callus as material, and obtains a high-efficiency and reliable protoplast preparation and transformation method specially designed for 84K poplar callus by optimizing the whole preparation process including enzyme solution components, enzyme hydrolysis conditions, osmotic pressure and purification steps. The method selects 84K poplar callus with a growth period of 3 to 4 weeks as material, and uses 1.5% cellulase R-10, 0.5% pectinase Y-23, 0.4% isolated enzyme R-10 and 0.5 M D-mannitol as main components of the enzyme solution to avoid light hydrolysis for 4.0 h, so that 2.5×10 6 After the protoplasts are stored in the W5 solution containing 0.485 M D-mannitol for 16 h, the survival rate can reach 85%. In the transient transformation test, the PEG4000 transformation solution with a PEG4000 mass concentration of 30% and the transformation condition of 25 min can make the transformation efficiency reach 25.6%.

[0022] The protoplast extraction method developed in the present application is suitable for the dissociation of 84K poplar callus protoplasts, and the protoplasts obtained after purification have high cell activity and transformation efficiency, which can meet the needs of subsequent researches in the aspects of molecular, cellular, genetic and gene function verification and molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a 84K poplar callus induction process chart; wherein, A is the induction day 0, B is the induction day 7, C is the induction day 14, D is the induction day 24, E is the induction day 30; Figure 2 It is a result chart of the influence of mannitol concentration in the enzyme solution on the number of 84K poplar protoplasts; Figure 3 It is a result chart of the influence of mannitol concentration in the enzyme solution on the activity of 84K poplar protoplasts; Figure 4Fig. 1 is a diagram of the state of protoplasts obtained by enzymolysis of callus using an enzymolysis solution containing 0.50 M mannitol; wherein, Figure 4 Fig. 1A is a 4x magnification diagram of Fig. 1, Figure 4 Fig. 1B is a 10x magnification diagram of Fig. 1, Figure 4 Fig. 1C is a 40x magnification diagram of Fig. 1; Figure 5 Fig. 2 is a diagram of the trypan blue viability detection of 84K poplar protoplasts; wherein, Figure 5 Fig. 2A is a diagram of the state of protoplasts after staining with trypan blue (20x magnification), Figure 5 Fig. 2B is a diagram of the state of protoplasts after staining with trypan blue (40x magnification), the red arrow is a protoplast with high viability, and the green arrow is a protoplast with low viability or damage; Figure 6 Fig. 3 is a diagram of the effect of enzymolysis time on the yield of 84K poplar callus protoplasts; wherein, Figure 6 Fig. 3A is a diagram of protoplasts obtained when the enzymolysis time is 3 h (10x magnification), Figure 6 Fig. 3B is a diagram of protoplasts obtained when the enzymolysis time is 4 h (4x magnification), Figure 6 Fig. 3C is a diagram of protoplasts obtained when the enzymolysis time is 5 h (10x magnification), Figure 6 Fig. 3D is a diagram of the effect of different enzymolysis times on the yield of protoplasts; Figure 7 Fig. 4 is a diagram of the effect of the concentration of mannitol in W5 solution on the purification and preservation of 84K poplar protoplasts; wherein, Figure 7 Fig. 4A is a diagram of the state of protoplasts obtained by purifying 84K poplar protoplasts using W5 solution containing 0.485 M mannitol (40x magnification), Figure 7 Fig. 4B is a diagram of the state of 84K poplar protoplasts after being preserved in W5 solution containing 0.485 M mannitol for 16 h (10x magnification), Figure 7 Fig. 4C is a diagram of the state of 84K poplar protoplasts after being preserved in W5 solution without mannitol for 16 h (10x magnification), Figure 7 Fig. 4D is a diagram of the effect of the concentration of mannitol in W5 solution on the number of protoplasts after purification of 84K poplar protoplasts, Figure 7 Fig. 4E is a diagram of the effect of the concentration of mannitol in W5 solution on the survival rate of 84K poplar protoplasts after being preserved for 16 h; Figure 8 Fig. 5 is a diagram of the state of 84K poplar protoplasts after being preserved in W5 solution for 20 h; wherein, Figure 8 Fig. 5A is a diagram of the state of 84K poplar protoplasts after being preserved in W5 solution containing 0.485 M mannitol for 20 h (10x magnification), Figure 8B in FIG. 1 is the state diagram of 84K poplar protoplasts after 20 hours of preservation in W5 solution containing 0.485 M mannitol (20 times magnification), Figure 8 C in FIG. 1 is the state diagram of 84K poplar protoplasts after 20 hours of preservation in W5 solution without mannitol (10 times magnification), Figure 8 D in FIG. 1 is the state diagram of 84K poplar protoplasts after 20 hours of preservation in W5 solution without mannitol (20 times magnification); Figure 9 FIG. 2 is a result diagram of transient expression of 84K poplar protoplasts; wherein, A1 is the bright field after transformation (20 times magnification), A2 is the bright field of successfully transformed protoplasts (40 times magnification), A3 is the bright field of unsuccessfully transformed protoplasts (40 times magnification), B1 is the fluorescence field after transformation (20 times magnification), B2 is the fluorescence field of successfully transformed protoplasts (40 times magnification), B3 is the fluorescence field of unsuccessfully transformed protoplasts (40 times magnification), C1 is the superposition of the bright field and the fluorescence field after transformation (20 times magnification), C2 is the superposition of the bright field and the fluorescence field of successfully transformed protoplasts (40 times magnification), and C3 is the superposition of the bright field and the fluorescence field of unsuccessfully transformed protoplasts (40 times magnification). DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. It should be noted that the following embodiments are only used to explain the present application but not to limit the scope of the present application. The following embodiments are only a part of the embodiments of the present application but not all the embodiments. Those skilled in the art can refer to the content herein, appropriately improve the process parameters to achieve the purpose of the present application. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein to realize and apply the present application technology without departing from the content and scope of the present application. In the art, other technicians will not make creative efforts, and the embodiments obtained by them are protected by the present application.

[0025] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0026] The culture medium and solution preparation method involved in the present application are as follows: Preparation of plant culture medium: 84K poplar callus induction medium (CIM) preparation: 2.41 g / L WPM, 20 g / L sucrose, 1 mg / L 2,4-D, 100 mg / L Kinetin, 5.5 g / L agar, pH adjusted to 5.9.

[0027] Enzymatic solution preparation: 84K poplar callus protoplast enzymatic solution preparation (10 mL): 1.5% (w / v) cellulase R-10, 0.40% (w / v) macerozyme R-10, 0.50% (w / v) pectolyase Y-23, 200 μL 1 M KCl solution, 400 μL 0.5 M MES solution (pH: 5.7), 1 mL 1% BSA solution, 100 μL 1 M CaCl2solution, 35 μL β-mercaptoethanol, and different concentrations of mannitol (0.40 M, 0.45 M, 0.50 M, 0.55 M, 0.60 M), sterile water to constant volume. According to the above reagent formula, add mannitol, preheated MES solution and KCl solution in a 10 mL centrifuge tube in turn, add three enzyme powders, mix gently up and down, do not vortex, reduce foam to avoid enzyme activity reduction; place the enzymatic solution in a 55°C water bath for 10 min, place it at room temperature, add β-mercaptoethanol, CaCl2solution and BSA solution in turn, and the rest of the volume is supplemented with sterile water. After mixing the enzymatic solution, filter the enzymatic solution through a 0.22 μm filter membrane into a six-well cell culture plate.

[0028] W5 solution preparation (50 mL): 7.6 mL 1 M NaCl solution, 6.25 mL 1 M CaCl2solution, 250 μL 1 M KCl solution, 200 μL 0.5 M MES solution (pH: 5.7), 0.045 g glucose, and different concentrations of mannitol (0 M, 0.480 M, 0.485 M, 0.490 M, 0.495 M), finally sterile water to constant volume.

[0029] MMg solution preparation (10 mL): 4 mL 1 M mannitol solution, 0.5 mL 0.3 M MgCl2solution, 80 μL 0.5 M MES-K solution, finally sterile water to constant volume.

[0030] PEG4000 transformation solution preparation (5 mL): different mass fractions (20%, 30%, and 40%) of PEG4000 solid, 1 mL 1 M mannitol solution, 500 μL 1 M CaCl2solution, finally sterile water to constant volume.

[0031] Enzymatic solution and PEG4000 transformation solution were prepared and used immediately after filtration and sterilization; W5 solution and MMg solution were stored in a 4°C refrigerator after sterilization, and other solution mother liquor also needed to be sterilized with 0.22 μm filter screen. The MES solution was heated at 70°C for 2-3 min before adding to the enzymatic solution.

[0032] Example 1: Step one, culture of 84K poplar callus: Take 1-month-old healthy 84K poplar aseptic tissue culture seedlings, select dark green healthy leaves, cut 3-5 times vertically along the main leaf vein, and place in 84K poplar callus induction medium (CIM) for dark culture at 25°C for 30 days to induce dedifferentiation and obtain white and yellowish moist callus.

[0033] Step two, extraction of 84K poplar protoplasts: Take 0.5 g of callus and put it into 5 mL of enzymatic solution, and use 1 mL of pipette gun to gently blow it apart (Note: the tip of the gun should be cut off about 1 cm). Finally, enzymatic digestion was carried out at 25°C, avoiding light, and low speed (80 rpm) shaking for 4 h to obtain the enzymatic digestion product.

[0034] Step three, purification of 84K poplar protoplasts: Use pre-cooled W5 solution to wet the 70 μm cell filter screen, gently filter the enzymatic digestion product into a sterile round-bottom centrifuge tube, add 1 volume of pre-cooled W5 solution, and mix well. Centrifuge at 100 g for 3 min (other parameters of the centrifuge: temperature is 4°C; acceleration and deceleration is 3), remove the supernatant as much as possible, add 2 mL of pre-cooled W5 solution, resuspend the protoplasts; centrifuge at 100 g for 2 min, remove the supernatant, and add 2 mL of pre-cooled W5 solution; repeat the previous step, finally add 2 mL of W5 solution for storage, and obtain the protoplast suspension.

[0035] Determination of protoplast yield using a hemocytometer: Use a 25x16 type hemocytometer, add 10 μL of protoplast suspension (remove the tip of the gun) from the side, count the total number of protoplasts in the counting area, and repeat not less than three times. Calculate the protoplast yield using the protoplast yield calculation formula: Protoplast yield (number / g·FW) = total number of protoplasts in the counting area x 10000 x dilution factor / fresh weight of the material.

[0036] Identify protoplast activity by staining with trypan blue: Mix the protoplast suspension with 0.4% trypan blue solution at a volume ratio of 9:1, take 10 μL and add it to the hemocytometer for live and dead cell counting. Calculate the protoplast activity using the protoplast activity calculation formula: Protoplast activity (%) = number of protoplasts not stained / (number of protoplasts not stained + number of protoplasts stained blue) x 100%.

[0037] Step four, transient transformation of 84K poplar protoplasts: the purified protoplast suspension was placed on ice for 30 min, centrifuged at 100 g for 1 min, and the W5 supernatant was removed; 2 mL of pre-cooled MMg solution was added to resuspend the protoplasts, which were centrifuged at 100 g for 1 min, and the supernatant was removed; an appropriate amount of MMg solution was added to dilute the protoplast solution to 10 5 mL; 2 mL of a centrifuge tube was prepared, 10 μL of 1.5 μg / μL GFP plasmid was added, 100 μL of protoplast suspension was added, 5 mL of PEG4000 transformation solution was added gently, and the mixture was mixed gently and inverted up and down. The transformation was carried out in the dark for a certain period of time. The transformation was terminated by adding an equal volume of pre-cooled W5 solution, mixing, centrifuging at 100 g for 1 min, and removing the supernatant as much as possible; 100 μL of W5 solution was added, centrifuged at 100 g for 1 min to remove the supernatant, and the operation was repeated twice; finally, 1 mL of W5 solution was added, and the mixture was placed in a cell counting plate and stored in the dark for 12-16 h. The expression of GFP in the protoplasts transfected with the plasmid was observed.

[0038] Test Example 1 According to the method of step one in Example 1, the leaves of 84K poplar were induced to dedifferentiate to prepare calli, as shown in Figure 1 According to the method of step two in Example 1, the calli were enzymatically digested using enzymatic digestion solutions containing different concentrations of mannitol, and then the yield and activity of the obtained protoplasts were determined according to the methods for determining the yield and activity of protoplasts provided in Example 1.

[0039] As shown in Figure 2 , when the concentration of mannitol in the enzymatic digestion solution was 0.5 M, the number of 84K poplar callus protoplasts reached the highest value, which could reach 2.475×10 6 / g·FW, and there was a significant difference (P<0.05) compared with the treatments with 0.40 M, 0.45 M, 0.55 M, and 0.60 M mannitol in the enzymatic digestion solution. Figure 3 As shown in Figure 4 , when the concentration of mannitol in the enzymatic digestion solution was 0.5 M, the activity of 84K poplar callus protoplasts reached the highest value, which could reach 73.5%. The protoplasts obtained by enzymatic digestion of calli using an enzymatic digestion solution containing 0.50 M mannitol are shown in Figure 5 . The trypan blue viability detection chart of 84K poplar protoplasts is shown in , and the protoplasts with high viability and good integrity were not stained blue, while the protoplasts with low viability and fragmentation were stained with trypan blue due to the pressure existing in the process of enzymatic de-walling and washing, which reduced the stability of the membrane or lost integrity.

[0040] According to the method of step one in example 1, the dedifferentiation of 84K poplar leaves was induced to prepare callus, and then the callus was enzymolyzed according to the method of step two in example 1, the concentration of mannitol in the enzymolysis solution was 0.5 M, and the enzymolysis time was set to be 3 h, 4 h, 5 h and 6 h respectively, and then the yield of protoplasts was detected after enzymolysis, and the results are shown in Figure 6 .

[0041] It can be seen from Figure 6 that with the increase of enzymolysis time, the yield of protoplasts shows a trend of first increasing and then decreasing, and reaches the highest at 4 h, which is significantly different from the yield of protoplasts obtained at other enzymolysis times. If the enzymolysis time is 3 h, the protoplast separation is not sufficient, and there are still small cell groups in the enzymolysis solution; if the enzymolysis time is too long, the 84K poplar protoplasts will be broken and shrunk due to the existence of enzymolysis pressure. When the enzymolysis time is 4 h, most of the cell groups have been sufficiently enzymolyzed, and most of the protoplasts are intact, so the enzymolysis time of 4 h is the best for the separation of 84K callus protoplasts.

[0042] Test example 3: According to the method of step one in example 1, the dedifferentiation of 84K poplar leaves was induced to prepare callus; according to the method of step two in example 1, the callus was enzymolyzed, the concentration of mannitol in the enzymolysis solution was 0.5 M, and the enzymolysis time was set to be 4 h; according to the method of step three in example 1, the protoplasts were purified, wherein the concentration of mannitol in the W5 solution was adjusted to be 0 M, 0.480 M, 0.485 M, 0.490 M and 0.495 M respectively; and then the number and survival rate of protoplasts were calculated after purification.

[0043] It can be seen from Figure 7 and Figure 8 that the purification and preservation effect of 84K poplar protoplasts in W5 solution containing 0.485 M mannitol is the best, and the survival rate can reach 85% after 16 h of preservation. A large number of 84K poplar protoplasts are damaged after 16 h of preservation in W5 solution without mannitol, and only 46% of the survival rate, which is not suitable for the incubation and preservation of 84K poplar protoplasts. After 20 h of preservation in W5 solution containing 0.485 M mannitol, a large number of 84K poplar protoplasts are still intact, while only a small number of 84K poplar protoplasts are intact after 20 h of preservation in W5 solution without mannitol.

[0044] Test example 4: The dedifferentiation of 84K poplar leaves was induced to prepare callus according to the method of step one in Example 1; the callus was enzymolyzed according to the method of step two in Example 1, the concentration of mannitol in the enzymolysis solution was 0.5 M, and the enzymolysis time was set to 4 h; the protoplasts were purified according to the method of step three in Example 1, wherein the concentration of mannitol in the W5 solution was 0.485 M; the transient transformation of 84K poplar protoplasts was performed according to the transient transformation method provided in step four in Example 1, wherein the mass concentration of PEG4000 in the PEG4000 transformation solution was 30%, and the transformation time was set to 15 min, 20 min and 25 min, respectively.

[0045] As shown in Table 1, when the transient transformation time of 84K poplar protoplasts was 25 min, the transient transformation efficiency was the highest, about 25.62%, but there was no significant difference with the transformation time of 20 min. It is possible that the transformation time is too short, so that most of the plasmid cannot enter the cell through the plasma membrane; if the transformation time is too long, the activity of the protoplasts will be reduced due to the toxicity and stress of the PEG4000 solution, thereby reducing the transformation efficiency.

[0046] Table 1 Influence of transformation time on the transient transformation efficiency of 84K poplar protoplasts

[0047] Note: The data in the table are mean ± standard deviation, and different letters in the same column represent significant differences (p<0.05).

[0048] Test Example 5: The dedifferentiation of 84K poplar leaves was induced to prepare callus according to the method of step one in Example 1; the callus was enzymolyzed according to the method of step two in Example 1, the concentration of mannitol in the enzymolysis solution was 0.5 M, and the enzymolysis time was set to 4 h; the protoplasts were purified according to the method of step three in Example 1, wherein the concentration of mannitol in the W5 solution was 0.485 M; the transient transformation of 84K poplar protoplasts was performed according to the transient transformation method provided in step four in Example 1, wherein the transformation time was set to 25 min, and the mass concentration of PEG4000 in the PEG4000 transformation solution was set to 20%, 30% and 40%, respectively.

[0049] From table 2, with the increase of PEG4000 concentration in PEG4000 transformation solution, the transient transformation efficiency of 84K poplar protoplast presents the trend of first rising and then falling. When the mass concentration of PEG4000 in PEG4000 transformation solution is 30%, the transient transformation efficiency is the highest. When the concentration of PEG4000 in PEG4000 transformation solution is too high, the permeability of cell membrane is not changed greatly, and most of plasmids cannot enter the cells through the plasma membrane; and when the transformation time is too long, due to the decrease of protoplast activity caused by high-concentration PEG4000 transformation solution, the transformation efficiency is reduced. Therefore, when the mass concentration of PEG4000 in PEG4000 transformation solution is 30%, the transformation efficiency is the best.

[0050] Table 2 Influence of PEG4000 concentration on the transient transformation efficiency of 84K poplar protoplast

[0051] Note: The data in the table are mean ± standard deviation, and different letters in the same column represent significant difference (p<0.05) From Figure 9 It can be known that, since the empty plasmid carrying GFP can be expressed at any site in the cell, the whole 84K poplar protoplasts successfully transformed present bright green, and the untransformed protoplasts only have spontaneous green fluorescence of chloroplast under the observation of fluorescence field.

[0052] In conclusion, the extraction method of protoplast developed by the application is suitable for dissociation of 84K poplar callus protoplast, and the protoplast obtained after purification has high cell activity and transformation efficiency, which indicates that the protoplast dissociated by the method can meet the needs of subsequent researches in aspects of molecular, cell, genetic, gene function verification and molecular breeding.

[0053] Although the application has been disclosed as above with preferred embodiments, it is not intended to limit the application, and anyone skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, therefore, the protection scope of the application should be defined by the claims.

Claims

1. A method for preparing and transforming 84K poplar callus protoplasts, characterized in that, Includes the following steps: Culture of S1 and 84K poplar callus: Take healthy 1-month-old aseptic tissue culture seedlings of 84K poplar, select dark green healthy leaves, cut 3-5 times vertically to the main vein, place them in 84K poplar callus induction medium, and culture in the dark at 23-27℃ for 3-4 weeks to induce dedifferentiation and obtain white-yellow moist callus. Extraction of S2 and 84K poplar protoplasts: Take 0.3-0.7 g of callus tissue obtained from S1, put it into 5 mL of enzymatic hydrolysis solution, gently blow it apart, and then carry out enzymatic hydrolysis under light-proof and low-speed shaking conditions to obtain the enzymatic hydrolysis product. Purification of S3 and 84K Yang protoplasts: Wet a 70 μm cell filter with pre-cooled W5 solution, filter the enzymatic hydrolysis product obtained in S2 into a sterile centrifuge tube to obtain protoplasts, wash the protoplasts 2-4 times with pre-cooled W5 solution; finally, add W5 solution for preservation to obtain protoplast suspension; Transient transformation of S4 and 84K Yang protoplasts: The protoplast suspension obtained in S3 was placed on ice for 30 min, centrifuged, and the supernatant of W5 was removed; pre-cooled MMG solution was added, the protoplasts were resuspended, centrifuged, and the supernatant was removed; MMG solution was added to dilute the protoplast suspension to 10. 5 1.5 μg / μL of GFP plasmid was mixed with protoplast suspension at a volume ratio of 1:10, and then gently added to PEG4000 transformation solution. After gentle mixing, the mixture was transformed under dark conditions.

2. The method according to claim 1, characterized in that, The composition of the 84K poplar callus induction medium described in S1 is as follows: 2.41 g / L WPM, 20 g / L sucrose, 1 mg / L 2,4-D, 100 mg / L Kinetin, 5.5 g / L agar, with the balance being water, and the pH being 5.

9.

3. The method according to claim 1, characterized in that, The main components of the enzymatic hydrolysate described in S2 include 1.5% cellulase R-10, 0.40% dissociation enzyme R-10, 0.50% pectinase Y-23, and 0.50 M mannitol.

4. The method according to claim 1, characterized in that, The enzymatic hydrolysis in S2 is carried out at a temperature of 25°C for 4 hours.

5. The method according to claim 1, characterized in that, The washing described in S3 involves adding W5 solution to the protoplasts, resuspending them, centrifuging at 100 g for 2-3 min, and removing the supernatant; the centrifugation process is accelerated and decelerated by 3.

6. The method according to claim 1, characterized in that, The concentration of mannitol in the W5 solution described in S3 is 0.485 M.

7. The method according to claim 1, characterized in that, The mass concentration of PEG4000 in the PEG4000 conversion solution described in S4 is 30%.

8. The method according to claim 1, characterized in that, The conversion time described in S4 is 20-25 min.

9. The application of the method according to any one of claims 1-8 in screening plant functional genes or regulatory elements.

10. The application of the method according to any one of claims 1-8 in the cultivation of high-quality transgenic plants.

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