Preparation method of plant protoplast

Abstract

The invention discloses a preparation method of plant protoplast, which is characterized in that the plant protoplast is prepared by using a buffer solution containing mannitol and antibiotic combination, so that the integrity of the protoplast can be increased, and cell pollution can be reduced. The method provided by the invention solves the problems that the active protoplast is directly separated from the plant tissue and the unicellular omics experiment is difficult to carry out, and provides technical support for the research of plants in the aspects of unicellular transcriptomics, genetics, developmental science and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing plant protoplasts. Background Art

[0002] Single-cell sequencing technology was first introduced in 2009 and has been developing continuously for more than a decade. In particular, in recent years, single-cell sequencing has experienced explosive development and popularity. In 2011, Nature Methods magazine listed single-cell sequencing technology as one of the most noteworthy technical fields in the next few years. In 2013, Science magazine ranked single-cell sequencing as the top of the six most noteworthy fields of the year, and Nature Methods magazine listed the application of single-cell sequencing as the most important methodological progress of 2013. In 2017, the first batch of 38 projects to be funded by the "Human Cell Atlas Project", which is comparable to the "Human Genome Project", was officially announced, opening a new era of single-cell sequencing. At present, single-cell transcriptome sequencing technology has been widely used in different types of tissues and cell lines of various species (especially humans, mice, etc.), including normal and diseased tissues. However, in the field of botany, the application of single-cell sequencing technology is still in its infancy. After nearly two years of development, single-cell sequencing technology has been applied to the research of plant cell atlas, plant organ development and other fields. At present, most of the research on plant single cells is based on cell clustering and differential gene analysis, and then analyzes cell development and its regulatory mechanism, and then deeply explores the response mechanism of cells under abiotic stress environment. With the continuous breakthrough of technical bottlenecks, it has gradually expanded to multiple species such as rice, corn, tomato, poplar and peanut, involving multiple tissue types such as roots, stems, leaves, flowers, pollen and sperm, and seed endosperm. The main methods for obtaining cells by single-cell sequencing technology include serial dilution method, micromanipulation technology, laser capture microdissection, microfluidic chip technology, fluorescence-activated cell sorting technology, etc., but the most commonly used method for obtaining cells is enzymatic hydrolysis. Enzymatic hydrolysis digests the extracellular matrix connecting cells, releases cells one by one, and then enriches and purifies the cells to finally obtain a single cell suspension. The cell wall in plant tissue is the main enzymatic hydrolysis of intercellular connections. The content of polysaccharides such as cellulose, hemicellulose, and pectin is high. The cell wall can be removed by the corresponding enzyme to obtain protoplasts.

[0003] The existing single-cell RNA-seq (scRNA-seq) technology for plants includes the following steps: sample processing, which is generally divided into sample pre-treatment, enzymatic digestion, filtration, cleaning and other steps. This process is difficult to operate in botany because plant protoplasts are difficult to obtain, difficult to separate from the original tissue, and easy to break; after sample processing, single cell capture is completed through microfluidic droplet generation technology, and then the on-machine library is constructed through subsequent library construction experiments; corresponding data are obtained through sequencing analysis.

[0004] The shortcomings of the prior art include: (1) the plant protoplast experimental method shown in the literature is difficult to operate and the description is incomplete. The experimenter needs to have sufficient theoretical knowledge and undergo professional training and practice to complete the corresponding operation content; (2) plant sample processing is difficult, and it is difficult to obtain plant protoplasts of qualified quality through conventional methods; (3) the sample processing methods used in the prior art for different plant species and tissue organs vary greatly, and there is no universal or relatively universal sample processing solution; (4) the different sample processing solutions in the prior art have a great impact on the quality of protoplasts, resulting in huge differences and instability in quality control indicators such as cell viability and clumping rate.

[0005] Therefore, there is an urgent need in the art for a relatively universal method for preparing plant protoplasts. Summary of the invention

[0006] As mentioned above, there is an urgent need in the art for a method for preparing high-quality plant protoplasts.

[0007] The inventors used a combination of mannitol and antibiotics in the process of preparing plant protoplasts, thereby increasing the integrity of the protoplasts and reducing cell contamination, and ultimately using the obtained high-quality protoplasts for single-cell omics sequencing technology.

[0008] In a first aspect, the present invention provides a method for preparing plant protoplasts, the method comprising the following steps:

[0009] S1, sample preparation: wash plant tissues with plant lysis buffer;

[0010] S2, protoplast enzymatic hydrolysis: cutting the plant tissue into thin slices and performing enzymatic hydrolysis using a plant enzymatic hydrolysis buffer;

[0011] S3, centrifugation purification: after the first centrifugation, discard the supernatant; add plant lysis buffer to the precipitate and shake the precipitate; after the second centrifugation, discard the supernatant; add plant dissociation buffer to the precipitate, resuspend the cell precipitate and obtain protoplasts;

[0012] The plant lysis buffer, the plant enzymatic hydrolysis buffer and the plant dissociation buffer contain mannitol and antibiotics.

[0013] In a second aspect, the present invention provides a kit for preparing plant protoplasts, the kit comprising reagents for performing the method described in the first aspect, the plant lysis buffer, the plant enzymatic hydrolysis buffer, and the plant dissociation buffer comprising mannitol and antibiotics.

[0014] In a third aspect, the present invention provides the use of the method described in the first aspect or the kit described in the second aspect in protoplast culture, transient transformation, cell fusion, and single-cell sequencing.

[0015] The beneficial effects of the present invention include:

[0016] (1) The dissociation schemes for different plant tissue and organ samples were unified to make the dissociation schemes universal;

[0017] (2) Clarify the operation steps in the dissociation protocol, improve the dissociation success rate, standardization, and reproducibility between different batches of experiments, and reduce the difficulty of experimental operation;

[0018] (3) Through the standard operation of the present invention, protoplasts of qualified quality that meet the requirements of single-cell omics experiment library construction can be obtained, and at the same time, quality control indicators such as protoplast viability and agglomeration rate are controlled within the qualified range. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the specific implementation manner of the present invention, the drawings in the specific implementation manner are briefly introduced below.

[0020] Figure 1 The results of single-cell sequencing experiments of cucumber callus in Example 1 of the present invention are shown, FIG. A: the dissociation results of callus 1 samples, and FIG. B: the dissociation results of callus 2 samples.

[0021] Figure 2 The results of protoplast dissociation of tomato leaves in Experimental Group 1 in Example 2 of the present invention are shown.

[0022] Figure 3 The results of protoplast dissociation of tomato leaves in Experimental Group 2 in Example 2 of the present invention are shown.

[0023] Figure 4 Shown are the results of dissociation of soybean cotyledons using 800 mM mannitol.

[0024] Figure 5 The results of protoplast dissociation of watermelon fruit in Experimental Group 1 in Example 3 of the present invention are shown.

[0025] Figure 6 The results of protoplast dissociation of watermelon fruits in Experimental Group 2 in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0026] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is only used to illustrate the present invention by way of example, and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims. In addition, those skilled in the art will understand that the technical solution of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0028] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value to one-tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are included in the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or two limits, ranges excluding either or both of those included limits are also included in the subject matter.

[0029] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can usually be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this article, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of..."

[0030] In order to better understand the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers and other numerical values ​​used in the specification and claims indicating quantities, percentages or ratios should be understood as being modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values, which may vary depending on the desired properties sought to be obtained. At a minimum, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0031] As used herein, the term "protoplast" refers to a plant cell whose cell wall has been completely or partially removed, exposing its lipid bilayer membrane. Typically, a protoplast is an isolated plant cell without a cell wall that has the potential to be regenerated into a cell culture or a whole plant.

[0032] Herein, the term "callus" refers to an undifferentiated amorphous cell mass in a plant, and may be a callus isolated from a plant tissue, a callus obtained by inducing dedifferentiation of a plant explant, or a callus proliferated by culturing the above callus.

[0033] In this article, the term "enzymatic hydrolysis" refers to the use of active enzymes to hydrolyze a specific substance. This method can be used in general biological experiments. Enzymes are proteins or RNAs produced by living cells that have high specificity and high catalytic efficiency for their substrates. In the present invention, enzymatic hydrolysis is used to digest plant cell walls and extracellular matrix.

[0034] In this article, the term "single-cell sequencing" refers to sequencing the genome of a single cell. A single cell is the basic structural and functional unit of an organism. Under the same external stimulus or physiological conditions, cells derived from the same type of cell may show cell-to-cell differences, i.e., heterogeneity.

[0035] In this article, the term "single-cell transcriptome" refers to the amplification and sequencing of the whole transcriptome at the single-cell level. The principle is to amplify the trace whole transcriptome RNA of the isolated single cell for high-throughput sequencing. In the present invention, any single-cell transcriptome sequencing technology that has been disclosed can be used for single-cell sequencing, including but not limited to TangRNA-Seq, Smart-Seq / Smart-Seq2, CEL-Seq, Quartz-seq, STRT-seqUMI technology.

[0036] As used herein, the term "UMI", or Unique Molecular Index, refers to a random, non-random, or semi-random molecular tag that can be attached to a nucleic acid molecule. When incorporated into a nucleic acid molecule, the UMI sequenced after amplification is directly counted to correct for subsequent amplification bias.

[0037] In this article, the term "genome alignment rate" refers to the ratio of reads aligned to the reference genome to all reads; the term "exon alignment rate" refers to the ratio of reads aligned to the exon region of the reference genome to all reads; the term "intron alignment rate" refers to the ratio of reads aligned to the intron region of the reference genome to all reads.

[0038] Herein, the term "library construction" refers to adding an adaptor structure to one or both ends of DNA that can be used for sequencing and library splitting.

[0039] As mentioned above, since plant protoplasts are difficult to separate from original tissues and are easily broken in the prior art, the present invention aims to provide a method for preparing high-quality plant protoplasts.

[0040] In a first aspect, the present invention provides a method for preparing plant protoplasts, the method comprising the following steps:

[0041] S1, sample preparation: wash plant tissues with plant lysis buffer;

[0042] S2, protoplast enzymatic hydrolysis: cutting the plant tissue into thin slices and performing enzymatic hydrolysis using a plant enzymatic hydrolysis buffer;

[0043] S3, centrifugation purification: after the first centrifugation, discard the supernatant; add plant lysis buffer to the precipitate and shake the precipitate; after the second centrifugation, discard the supernatant; add plant dissociation buffer to the precipitate, resuspend the cell precipitate and obtain protoplasts;

[0044] The plant lysis buffer, the plant enzymatic hydrolysis buffer and the plant dissociation buffer contain mannitol and antibiotics.

[0045] The plant lysis buffer, plant enzymatic hydrolysis buffer, and plant dissociation buffer in the present invention are all commonly used buffers in the art. Plant lysis buffers usually contain components such as buffers and reducing agents to change the permeability and activity of cells, and are used to clean plant tissues and dissolve centrifugal precipitates; plant enzymatic hydrolysis buffers usually contain components such as buffers, reducing agents, and enzymes that degrade cell walls (such as cellulase, pectinase, etc.) to degrade plant cell walls and release protoplasts; plant dissociation buffers usually contain components such as buffers and reducing agents to dissociate plant protoplasts, destroy proteins that maintain cell associations in the intercellular space, and separate cells. These buffers can be purchased directly or configured by themselves according to manufacturer instructions, journal literature content, etc.

[0046] In one embodiment, the concentration of the mannitol is 600mM, and the mannitol at this concentration can increase the osmotic pressure level and significantly improve the protoplast integrity. In one embodiment, the concentration of the antibiotic is 1%, wherein the percentage refers to the final concentration volume percentage. In one embodiment, the plant lysis buffer also includes morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, and bovine serum albumin, that is, the plant lysis buffer includes mannitol, antibiotics, morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, and bovine serum albumin. In one embodiment, the plant enzymolysis buffer also includes morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, bovine serum albumin, cellulase, and dissociation enzyme, that is, the plant enzymolysis buffer includes mannitol, antibiotics, morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, bovine serum albumin, cellulase, and dissociation enzyme. In one embodiment, the plant dissociation buffer further comprises morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, bovine serum albumin, and nuclease-free water, that is, the plant dissociation buffer comprises mannitol, antibiotics, morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, bovine serum albumin, and nuclease-free water. In a preferred embodiment, the concentration of the morpholineethanesulfonic acid is 20mM, the concentration of the potassium chloride is 20mM, the concentration of the calcium chloride is 10mM, the concentration of the dithiothreitol is 1mM, and the concentration of the bovine serum albumin is 0.1%, wherein the percentage refers to the final concentration volume percentage. It should be understood that other suitable concentrations are also applicable to the present invention to realize the preparation method of plant protoplasts.

[0047] In a second aspect, the present invention provides a kit for preparing plant protoplasts, the kit comprising reagents for performing the method described in the first aspect, the plant lysis buffer, the plant enzymatic hydrolysis buffer, and the plant dissociation buffer comprising mannitol and antibiotics.

[0048] In one embodiment, the concentration of mannitol is 600 mM, which can increase the osmotic pressure level and significantly improve the integrity of protoplasts. In one embodiment, the concentration of antibiotics is 1%, wherein the percentage refers to the final concentration volume percentage.

[0049] In a third aspect, the present invention provides the use of the method described in the first aspect or the kit described in the second aspect in protoplast culture, transient transformation, cell fusion, and single-cell sequencing.

[0050] Example

[0051] It should be noted that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The above summary of the invention and the detailed description below are only for the purpose of specifically explaining the present invention, and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the attached claims.

[0052] Example 1

[0053] In this example, single-cell omics research was conducted on cucumber callus tissue. The method provided by the present invention was used to prepare cucumber callus protoplasts, and library sequencing was performed on the protoplasts. After analysis, the data quality was found to be qualified, confirming the feasibility of the method provided by the present invention.

[0054] The protoplast preparation process is as follows:

[0055] (1) Sample preparation: Use a blade to remove callus tissues from 10 cultured cucumber plants (note the division between leaf tissue and callus tissue), cut the callus tissue into thin slices, take an appropriate amount of callus tissue (about 5 g) in a 10 cm culture dish, and wash the tissue once with 5 mL of plant lysis buffer (Table 1). Five callus tissues were used as one sample for subsequent experiments.

[0056] Table 1: Plant lysis buffer composition

[0057] Reagent name Final concentration Mannitol 600mM Morpholineethanesulfonic acid solution 20mM Potassium chloride 20mM Calcium chloride 10mM Dithiothreitol 1mM BSA 0.1％ antibiotic 1％

[0058] The preparation method of the morpholineethanesulfonic acid solution is as follows: weigh 0.975 g of morpholineethanesulfonic acid and dissolve it in 25 mL of nuclease-free water; adjust the pH of the solution to 6.0 with 1N sodium hydroxide solution, and dilute to 50 mL for use.

[0059] (2) Sampling: Transfer the cleaned callus tissue to a new 10 cm culture dish and cut the tissue into 0.5 mm slices using a clean surgical blade. Add 10-20 mL of plant enzymatic hydrolysis buffer (Table 2), mix gently, and cover the dish for later use.

[0060] Table 2: Plant enzymatic digestion buffer composition

[0061] Reagent name Final concentration Mannitol 600mM Morpholineethanesulfonic acid solution 20mM Potassium chloride 20mM Calcium chloride 10mM Dithiothreitol 1mM BSA 0.1％ antibiotic 1％ Cellulase Onozuka R-10 2％ Macerozyme R-10 (Macerozyme) 1％

[0062] (3) Enzymatic hydrolysis: Adjust the speed of the horizontal shaker to 40 rpm. After the speed stabilizes, fix the culture dish containing the tissue with sealing film and place it on the shaker in the dark for 2 hours for enzymatic hydrolysis. During this period, remove the dish every 30 minutes and gently shake to disperse the tissue clumps gathered in the middle area.

[0063] (4) Microscopic examination: When the enzymatic digestion solution becomes slightly turbid and the tissue edge is diffuse, aspirate 10 μL of the cell suspension, add 10 μL of trypan blue, mix well, and observe under a microscope; when it is confirmed that a single, round protoplast appears in the field of view and the amount of protoplasts is sufficient, the enzymatic digestion can be terminated.

[0064] (5) Sieving: After enzymatic hydrolysis, filter the cell suspension through a 70 μm cell sieve into a 50 mL round-bottom centrifuge tube. Add 10 mL of buffer to wash the plate and filter it through a 70 μm cell sieve into the tube.

[0065] (6) Centrifugation: Set the centrifugal parameters of the horizontal rotor centrifuge to: 100 g centrifugal force, speed increase 1, speed decrease 3, centrifugation at room temperature for 5 minutes. After centrifugation, discard the supernatant.

[0066] (7) Washing: Add 10 mL of plant lysis buffer to the precipitate and gently shake the precipitate.

[0067] (8) Recentrifugation: Set the centrifugal parameters of the horizontal rotor centrifuge to: 100 g centrifugal force, speed increase 1, speed decrease 3, centrifuge at room temperature for 5 minutes. After the centrifugation is completed, discard the supernatant.

[0068] (9) Resuspension: Add an appropriate volume of plant dissociation buffer (Table 3) to resuspend the cell pellet according to the amount of pellet (to ensure that the cell concentration is 1000-10000 cells / μL). Transfer the cell suspension to a 1.5 mL centrifuge tube and mix gently with a pipette (be careful not to create bubbles when pipetting).

[0069] Table 3: Plant dissociation buffer composition

[0070] Reagent name Final concentration Mannitol 600mM Morpholineethanesulfonic acid solution 20mM Potassium chloride 20mM Calcium chloride 10mM Dithiothreitol 1mM BSA 0.1％ antibiotic 1％ Nuclease-free water /

[0071] (10) Microscopic examination: Pipette 1 μL of cell suspension, add 1 μL of trypan blue, mix well, and observe under a microscope to confirm cell viability and estimate cell concentration. The qualified standards for microscopic examination include: a) the total number of cells obtained by dissociation exceeds 100,000; b) the protoplast clumping rate does not exceed 10%; c) the cell viability identified by trypan blue staining is higher than 70%.

[0072] The dissociation results are shown in Table 4. Figure 1 As shown in the figure, both callus samples were of qualified quality and could continue to be used for library construction.

[0073] Table 4: Dissociation experiment results

[0074]

[0075] The library construction and sequencing process are as follows:

[0076] (1) Each sample was fed with approximately 46,000 cells for subsequent library construction, and the machine library was constructed using the DNBelab C series high-throughput single-cell RNA library preparation kit.

[0077] (2) The prepared library was sequenced using the DIPSEQ T1 sequencing platform, and the final data results were obtained (Table 5).

[0078] Table 5: Sequencing data results

[0079]

[0080] The reagents and consumables used in this example are shown in Table 6.

[0081] Table 6: Reagents and consumables

[0082]

[0083]

[0084] Example 2

[0085] This example uses tomato leaf callus as the object and uses the method provided by the present invention to prepare tomato leaf callus protoplasts.

[0086] In this embodiment, two experimental groups were set up, each group had 4 young tomato leaves, which were cut into thin filaments by a blade for dissociation, and protoplasts were prepared using the same method as in Example 1. The components of the dissociation buffer used in the two experimental groups (the difference was the concentration of mannitol) were shown in Tables 7 and 8.

[0087] Table 7: Dissociation buffer composition for experimental group 1

[0088]

[0089]

[0090] Table 8: Dissociation buffer composition for experimental group 2

[0091] Reagent name Final concentration Mannitol 400mM Morpholineethanesulfonic acid solution 20mM Potassium chloride 20mM Calcium chloride 10mM Dithiothreitol 1mM BSA 0.1％ antibiotic 1％

[0092] In experimental group 1, the total number of protoplasts was 114,000, the viability was 96.03%, and the aggregation rate was 5.56% ( Figure 2 In experimental group 2, the number of intact protoplasts was too small to be counted, and there were many broken protoplasts and cell contents in the field of view ( Figure 3 ).

[0093] It can be seen that the concentration of mannitol is crucial for the dissociation of protoplasts. Low concentrations of mannitol will cause cells to break, making it difficult to obtain sufficient and complete protoplasts. In addition, although high concentrations of mannitol will not break a large number of protoplasts, they will cause the protoplasts to lose water and shrink, making it difficult to maintain their original state, thus affecting the experimental results. Figure 4 The results of using high concentration of mannitol (800mM) to prepare soybean cotyledon protoplasts showed that in addition to regular round protoplasts, there were also some irregular cells, which may be caused by high osmotic pressure and water loss.

[0094] Example 3

[0095] This example uses watermelon fruit callus as the object and uses the method provided by the present invention to prepare watermelon fruit callus protoplasts.

[0096] In this embodiment, two experimental groups were set up, each group had three young watermelon fruits, which were sliced ​​for dissociation, and protoplasts were prepared using the same method as in Example 1. The components of the dissociation buffer used in the two experimental groups (the difference was whether antibiotics were added) were shown in Tables 9 and 10.

[0097] Table 9: Dissociation buffer composition for experimental group 1

[0098] Reagent name Final concentration Mannitol 600mM Morpholineethanesulfonic acid solution 20mM Potassium chloride 20mM Calcium chloride 10mM Dithiothreitol 1mM BSA 0.1％

[0099] Table 10: Dissociation buffer composition for experimental group 2

[0100] Reagent name Final concentration Mannitol 600mM Morpholineethanesulfonic acid solution 20mM Potassium chloride 20mM Calcium chloride 10mM Dithiothreitol 1mM BSA 0.1％ antibiotic 1％

[0101] In experimental group 1, the total number of protoplasts was 980,000, the viability was 82.4%, and the aggregation rate was 21% ( Figure 5 In experimental group 2, the total number of protoplasts was 755,000, the viability was 86.85%, and the aggregation rate was 15.96% ( Figure 6 ). It can be seen that the protoplast viability in experimental group 2 with antibiotics added was slightly higher and the agglomeration rate was slightly lower.

[0102] The prepared protoplasts were sequenced and the results are shown in Table 11. It can be seen that the data of experimental group 2 performed better, such as Mean UMIs / cell, Mean genes / cell, mapping rate to Genome, mapping rate to Exon, mapping rate to Inter and other indicators.

[0103] Table 11: Sequencing data results

[0104]

[0105] Note: LibName: library name; cell_number: cell number; mean_reads / cell: average reads / cell; Mean UMIs / cell: average UMI / cell; Mean genes / cell: average genes / cell; Raw_Data(M_reads)_cDNA: raw data (M_reads) cDNA; Raw_Data(M_reads)_oligo: raw data (M_reads) oligo; PassQC_reads(M): reads passed quality control(M); mapping rate to Genome: genome alignment rate; mapping rate to Exon: exon alignment rate; mapping rate to Inter: intergenic region alignment rate.

[0106] The preparation method of plant protoplasts provided by the present invention is introduced in detail above. Specific embodiments are used herein to illustrate the principle and implementation mode of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core concept. Meanwhile, for those skilled in the art, according to the concept of the present invention, there will be changes in the specific implementation mode and the scope of application. In summary, the contents of this specification should not be construed as limitation of the present invention.

Claims

1. A method for preparing plant protoplasts, characterized in that: The method comprises the following steps: S1, sample preparation: wash plant tissues with plant lysis buffer; S2, protoplast enzymatic hydrolysis: cutting the plant tissue into thin slices and performing enzymatic hydrolysis using a plant enzymatic hydrolysis buffer; S3, centrifugation purification: after the first centrifugation, discard the supernatant; add plant lysis buffer to the precipitate and shake the precipitate; after the second centrifugation, discard the supernatant; add plant dissociation buffer to the precipitate, resuspend the cell precipitate and obtain protoplasts; The plant lysis buffer, the plant enzymatic hydrolysis buffer and the plant dissociation buffer contain mannitol and antibiotics.

2. The method according to claim 1, characterized in that The concentration of mannitol is 600 mM.

3. The method according to claim 1 or 2, characterized in that: The concentration of the antibiotic is 1%, wherein the percentage refers to the final concentration volume percentage.

4. The method according to any one of claims 1 to 3, characterized in that The plant lysis buffer further comprises morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol and bovine serum albumin.

5. The method according to any one of claims 1 to 4, characterized in that The plant enzymolysis buffer further comprises morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, bovine serum albumin, cellulase and cleavage enzyme.

6. The method according to any one of claims 1 to 5, characterized in that The plant dissociation buffer further comprises morpholineethanesulfonic acid, potassium chloride, calcium chloride, dithiothreitol, bovine serum albumin, and nuclease-free water.

7. The method according to any one of claims 4 to 6, characterized in that: The concentration of the morpholineethanesulfonic acid is 20 mM, the concentration of the potassium chloride is 20 mM, the concentration of the calcium chloride is 10 mM, the concentration of the dithiothreitol is 1 mM, and the concentration of the bovine serum albumin is 0.1%, wherein the percentage refers to the final concentration volume percentage.

8. A kit for preparing plant protoplasts, characterized in that, The kit comprises reagents for performing the method according to any one of claims 1 to 7, and the plant lysis buffer, the plant enzymatic hydrolysis buffer, and the plant dissociation buffer comprise mannitol and antibiotics.

9. The kit according to claim 8, characterized in that The concentration of mannitol is 600 mM.

10. The kit according to claim 8 or 9, characterized in that The concentration of the antibiotic is 1%, wherein the percentage refers to the final concentration volume percentage.

11. Use of the method according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 10 in protoplast culture, transient transformation, cell fusion, and single cell sequencing.

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