Method for preparing plant single cell

Abstract

According to the method, a plant explant callus culture mode is adopted, a carbon source containing a fluorophore is added into a culture medium, the callus which is provided with the fluorophore, thin in cell wall, easy to dissociate by a single enzyme and uniform in cell shape and size is generated through induction, and the cell dissociated by the single enzyme is detected by a flow cytometry, so that the cell quality is improved. The single cell purity and activity of the prepared leaf are 98.5% and 99.95% respectively, the clotting rate is only 1.5%, the cell purity and activity are improved by 32.6% and 21.05% respectively compared with those of a traditional technical scheme, and the clotting rate is reduced by 31.0%; according to the method, rice seeds are used as explants, seed single cells with the cell purity and activity being up to 99.5% and 100% respectively are obtained, and the limitation that the seed single cells cannot be prepared by a traditional enzymolysis method is broken through; besides, through co-induction of the safflower leaves and the rice seeds, the purity and activity of the prepared cells are as high as 99.98% and 100.0 respectively, and the limitation of single cell preparation caused by heterogeneity among species is overcome.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and specifically relates to a method for preparing plant single cells. Background Art

[0002] Living organisms such as animals and plants are advanced life forms composed of many single cells and can carry out complex life activities. Plant roots, stems, leaves, and animal internal organs are common samples for single cell separation. Separating single cells from animal and plant tissues and culturing them is of great significance for studying the functions of animal and plant organs, gene genetic mutations, etc. However, multiple cells and proteins in animal and plant tissues are adhered together, making it difficult to separate single cells; especially in the field of botany, plant cells have a cell wall structure, and the connection between cells mainly depends on the middle lamella in the cell wall. The interaction of cellulose, hemicellulose, and lignin in the cell wall determines the degree of cell adhesion.

[0003] The preparation of plant single cell suspensions usually uses mechanical methods and enzymatic methods. The mechanical method often uses a blade, etc. to chop the plant material immersed in the enzyme extraction buffer, incubate for several minutes to release the cells, and then obtain the cell suspension through filtration and centrifugation. However, the cell structures of each plant are highly heterogeneous, and there are obvious differences even in different tissue types of the same species and different tissue parts of the same tissue type. For example, cell size (at different developmental stages, different tissue parts, etc.), cell structure (spongy tissue, palisade tissue, stomatal cells, endosperm cells, mesophyll cells, vascular bundle cells, etc.), cell morphology (elongated, round, oval, fusiform, rhomboid, etc.) ( Figures 1 - 6 ), therefore, the cells obtained by centrifugation and resuspension are a mixture of all cell types of the material taken, and each cell type cannot be separately distinguished. At the same time, the obtained cell suspension cannot accurately distinguish single cells from cell clusters, which is not conducive to subsequent single cell research.

[0004] Although the enzymatic method mainly uses a mixed enzyme solution of cellulase, pectinase, and macerozyme to treat young tissue parts of plants. Since the cell wall compositions of different species, tissues, and organs are different ( Figures 1 - 7 ), not all cell walls can be digested with the same efficiency. Therefore, it is necessary to optimize the enzyme solution and reaction conditions for different species and different tissue types, which greatly increases the application difficulty of plant single cells; at the same time, it also has great limitations for relatively old and difficult-to-obtain intact and active protoplast parts such as plant flowers, stems, fruits, and seeds; in addition, the long-term enzymatic hydrolysis of multiple complex enzyme solutions and the influence of osmotic pressure will cause changes in cell gene expression and also cause the fragmentation of easily digested cells.

[0005] Plant callus refers to the morphogenetic process in which somatic cells, under specific conditions, form new individuals through a developmental pathway similar to that of zygotic embryos without sexual cell fusion. Callus cells are mostly isodiametric and thin-walled meristematic cells, and are more likely to obtain single cells with high activity, good integrity, and consistent size through enzymatic digestion solution compared to mature plant tissue cells.

[0006] Flow cytometry, on the other hand, is a fluorescence-based cell biology analysis method that rapidly detects the properties of individual cells in a cell population. Multiple cell parameters can be collected in a single detection, such as cell number, cell volume, and the response to fluorescent probes. The detection and classification of cells by flow cytometry are based on cell morphology and fluorescent substances. By adjusting various parameters of the cells in the sample and the particle size of the sorted cells, active cells can be captured. With the help of an imaging system, the droplet state is observed. By adjustment, parameters such as the diameter and roundness of the cells in the sample are obtained, and the particle size of the sorted cells (10 - 40 μm) is adjusted to precisely capture active cells.

[0007] Therefore, developing an efficient method for preparing plant single cells, especially a method for preparing single cells and separation and sorting techniques applicable to different plant types, tissue parts, and cell morphologies with large cell structure heterogeneity, is the key to solving the above technical problems. Summary of the Invention

[0008] One of the objectives of the present invention is to provide a culture method for obtaining plant callus containing fluorescent labels, mainly achieved by adding a carbon source containing fluorescent labels during callus culture.

[0009] Another objective of the present invention is to provide a callus enzymatic digestion solution, which is one of cellulase, hemicellulase, macerozyme, pectinase, lysing enzyme, or snail enzyme, simplifying the composition of the enzymatic digestion solution.

[0010] Another objective of the present invention is to provide a method for obtaining callus cell clusters with fluorescent groups, thin cell walls, easily dissociated by a single enzymatic digestion solution, and uniform cell morphology and size.

[0011] Another objective of the present invention is to provide a method for separating single cells from fluorescently labeled plant callus, especially for tissue parts such as hard seeds, fruits, and plant roots, stems, and leaves with a high degree of fibrosis that have poor enzymatic digestion effects. The fluorescent groups on the callus are identified by a flow cytometer to capture and separate single cells.

[0012] Another objective of the present invention is to provide an application of plant single cells obtained by the method for preparing plant single cells.

[0013] The present invention adopts the method of culturing callus from plant explants. Especially for hard seeds, fruits, and tissue parts such as roots, stems, and leaves of plants with a high degree of fibrosis that are poorly enzymolyzed, callus culture of explants is first carried out. By adding a carbon source containing a fluorescent group to the culture medium, callus cell clusters with a fluorescent group, thin cell walls, easily dissociated by a single enzyme solution, and uniform cell morphology and size are generated. The cell suspension formed after dissociating the cell clusters with a single enzyme solution is used to separate and sort single cells with fluorescent labels through a flow cytometer to obtain plant single cells with high purity, strong vitality, and low aggregation rate.

[0014] The present invention provides a method for preparing plant single cells, mainly including the following steps.

[0015] S1. Disinfection of plant explants:

[0016] Select suitable plant tissue parts, cut, slice, or peel the parts as explants for callus culture according to needs, soak the explants in 75% ethanol for 2 min - 10 min, then soak them in sodium hypochlorite solution with an active chlorine content of 8% - 15% for 15 min - 20 min, and rinse them with sterile water 3 - 6 times for surface disinfection of the explants.

[0017] Furthermore, the explants are derived from one or more of algae plants and / or bryophyte plants and / or pteridophyte plants and / or spermatophyte plants and / or gramineous plants and / or ligneous plants and / or gymnosperm plants and / or angiosperm plants and / or arbors and / or shrubs and / or wild plants and / or artificially cultivated plants and / or monocotyledonous plants and / or dicotyledonous plants.

[0018] Furthermore, the explants are derived from one or more of food crops and / or fiber crops and / or oil crops and / or sugar crops and / or medicinal crops and / or spice crops and / or beverage crops and / or addictive crops and / or industrial crops and / or tropical cash crops and / or subtropical cash crops and / or temperate cash crops.

[0019] Preferably, the explants are derived from one or more of wheat and / or rice and / or corn and / or oats and / or rye and / or barley and / or millet and / or sorghum and / or highland barley and / or soybean and / or broad bean and / or pea and / or mung bean and / or adzuki bean and / or sweet potato and / or potato and / or cassava and / or cotton and / or hemp and / or flax and / or jute and / or rape and / or peanut and / or sesame and / or beet and / or sugarcane and / or rubber and / or mulberry and / or wolfberry and / or licorice and / or astragalus and / or ginseng and / or notoginseng and / or mint and / or ginkgo and / or lavender and / or fennel and / or clove and / or tea and / or coffee and / or cocoa and / or tobacco leaf and / or tomato and / or hops.

[0020] Preferably, the explant is derived from one or more of roots, stems, leaves, flowers, fruits, and seeds of a plant.

[0021] Preferably, the size of the explant is one or more of roots (2.0 mm - 10.0 mm), stems (1.5 mm - 15.0 mm), leaves (1.0 mm - 5.0 mm), flowers (2.0 mm - 8.0 mm), fruits (2.0 mm - 8.0 mm), and seeds (2.0 mm - 10.0 mm).

[0022] Preferably, the seed includes a seed embryo and / or endosperm.

[0023] S2. Obtaining plant callus:

[0024] Place the disinfected explant in step S1 on the sterilized absorbent paper in a laminar flow hood, gently press the absorbent paper with forceps to absorb the surface moisture, and inoculate it onto the solid callus induction medium, and culture it at 24°C - 37°C, in the dark, and under light avoidance for 24 hr - 72 hr.

[0025] Preferably, the solid induction medium includes MS solid medium and / or B5 solid medium and / or White solid medium and / or N6 solid medium, and is mainly composed of inorganic salts, hormones, carbon sources, and agar, with a pH of 5.5 - 7.5.

[0026] Preferably, the inorganic salts include ammonium nitrate and / or calcium chloride and / or magnesium sulfate and / or potassium hydrogen phosphate and / or potassium dihydrogen phosphate and / or potassium nitrate and / or calcium nitrate and / or boric acid and / or cobalt chloride and / or ferrous sulfate and / or manganese(II) sulfate and / or potassium iodide and / or sodium molybdate and / or zinc sulfate and / or sodium ferric ethylenediaminetetraacetate and / or copper sulfate, and can be added individually and / or in combination of two or more.

[0027] Furthermore, the inorganic salts include ammonium nitrate at 0.1 g / L - 1.0 g / L and / or calcium chloride at 0.5 g / L - 1.5 g / L and / or magnesium sulfate at 0.01 g / L - 0.1 g / L and / or potassium hydrogen phosphate at 0.1 g / L - 1.0 g / L and / or potassium dihydrogen phosphate at 1.5 g / L - 2.0 g / L and / or potassium nitrate at 0.5 g / L - 1.0 g / L and / or calcium nitrate at 0.25 g / L - 1.58 g / L and / or boric acid at 1.1 g / L - 1.96 g / L and / or cobalt chloride at 0.001 g / L - 0.01 g / L and / or ferrous sulfate at 0.01 g / L - 0.09 g / L and / or manganese(II) sulfate at 0.01 g / L - 0.1 g / L and / or potassium iodide at 0.025 g / L - 0.055 g / L and / or sodium molybdate at 0.002 g / L - 0.005 g / L and / or zinc sulfate at 0.025 g / L - 0.25 g / L and / or sodium ferric ethylenediaminetetraacetate at 1.12 g / L - 1.59 g / L and / or copper sulfate at 0.001 g / L - 0.02 g / L, and can be added individually and / or in combination of two or more.

[0028] Preferably, the hormones include auxin and / or synthetic auxin and / or auxin analogues and / or 2,4-dichlorophenoxyacetic acid and / or 3,5-dimethylphenoxyacetic acid and / or phenoxyacetic acid and / or phenylacetic acid and / or 4-chlorophenoxyacetic acid and / or 3,6-dichloro-o-anisic acid and / or naphthaleneacetic acid and / or indoleacetic acid and / or indole-3-butyric acid and / or 6-benzylaminopurine and / or 6-(γ,γ-dimethylallylamino)purine and / or thidiazuron and / or gibberellin and / or abscisic acid, and can be added individually and / or in combination of two or more.

[0029] Furthermore, the hormones include auxin at 0.001 g / L - 0.005 g / L and / or synthetic auxin at 0.001 g / L - 0.005 g / L and / or auxin analog at 0.002 g / L - 0.005 g / L and / or 2,4-dichlorophenoxyacetic acid at 0.02 g / L - 0.05 g / L and / or 3,5-dimethylphenoxyacetic acid at 0.005 g / L - 0.010 g / L and / or phenoxyacetic acid at 0.005 g / L - 0.015 g / L and / or phenylacetic acid at 0.001 g / L - 0.010 g / L and / or 4-chlorophenoxyacetic acid at 0.002 g / L - 0.008 g / L and / or 3,6-dichloro-o-anisic acid at 0.0025 g / L - 0.0095 g / L and / or naphthylacetic acid at 0.002 g / L - 0.009 g / L and / or indoleacetic acid at 0.005 g / L - 0.009 g / L and / or indole-3-butyric acid at 0.001 g / L - 0.009 g / L and / or 6-benzylaminopurine at 0.002 g / L - 0.01 g / L and / or 6-(γ,γ-dimethylallylamino)purine at 0.001 g / L - 0.02 g / L and / or thidiazuron at 0.001 g / L - 0.005 g / L and / or gibberellin at 0.001 g / L - 0.01 g / L and / or abscisic acid at 0.001 g / L - 0.02 g / L, which can be added alone and / or in combination of two or more.

[0030] Preferably, the carbon sources include glucose and / or fructose and / or sucrose and / or mannose and / or galactose and / or lactose and / or maltodextrin and / or honey, etc., which are sugars and their derivatives that can be used by the explant to induce callus, and can be added alone or in combination of two or more.

[0031] Furthermore, the carbon sources include glucose at 15.0 g / L - 50.0 g / L and / or fructose at 30.0 g / L - 60.0 g / L and / or sucrose at 10.0 g / L - 30.0 g / L and / or mannose at 15.0 g / L - 45.0 g / L and / or galactose at 5.0 g / L - 20.0 g / L and / or lactose at 30.0 g / L - 45.0 g / L and / or maltodextrin at 30.0 g / L - 60.0 g / L and / or honey at 50.0 g / L - 65.0 g / L, etc., which are sugars and their derivatives that can be used by the explant to induce callus, and can be added alone or in combination of two or more.

[0032] Furthermore, the carbon source includes glucose at 15.0 g / L - 50.0 g / L and / or fructose at 30.0 g / L - 60.0 g / L and / or sucrose at 10.0 g / L - 30.0 g / L and / or mannose at 15.0 g / L - 45.0 g / L and / or galactose at 5.0 g / L - 20.0 g / L and / or lactose at 30.0 g / L - 45.0 g / L and / or maltodextrin at 30.0 g / L - 60.0 g / L and / or honey at 50.0 g / L - 65.0 g / L, etc., which are labeled with fluorescent groups such as FAM and / or TET and / or CY3 and / or Texas Red and / or LC Red and / or LC Green and / or CY5, etc., and can be labeled with a single type or a combination of multiple types.

[0033] Preferably, the agar content is 2.0 g / L - 5.0 g / L, including agarose and / or agar powder and / or agar strips, and can be added alone or in combination of two or more.

[0034] Preferably, the callus induction culture temperature is 26°C - 34°C.

[0035] Preferably, the induction culture time is 48 hr - 60 hr.

[0036] S3. Preparation of plant cell suspension:

[0037] Place the callus obtained in step S2 into 5.0 ml - 20.0 ml of a single enzymatic hydrolysis solution, at 25.0°C - 37.0°C, with a rotation speed of 20.0 rpm / min - 80.0 rpm / min, and perform constant temperature, dark, and oscillating culture for 15 min - 180 min to obtain a callus cell suspension.

[0038] Furthermore, the single enzymatic hydrolysis solution is one of cellulase or hemicellulase or macerozyme or pectinase or lytic enzyme or snail enzyme.

[0039] Furthermore, in the single enzymatic hydrolysis solution, the cellulase content is 0.005 g / L - 0.1 g / L, or the hemicellulase content is 0.001 g / L - 0.5 g / L, or the macerozyme content is 0.001 g / L - 0.01 g / L, or the pectinase content is 0.001 g / L - 0.02 g / L, or the lytic enzyme content is 0.01 g / L - 0.02 g / L, or the snail enzyme content is 0.001 g / L - 0.002 g / L.

[0040] Further, in the single enzymatic hydrolysate, the solvent components include potassium dihydrogen phosphate at 0.027 g / L - 0.5 g / L and / or sodium dihydrogen phosphate at 0.02 g / L - 0.8 g / L and / or potassium nitrate at 0.005 g / L - 0.05 g / L and / or potassium chloride at 0.5 g / L - 2.5 g / L and / or sodium chloride at 0.5 g / L - 1.5 g / L and / or calcium chloride at 0.05 g / L - 0.25 g / L and / or magnesium sulfate at 0.005 g / L - 0.05 g / L and / or copper sulfate at 0.02 g / L - 0.03 g / L, with pH = 6.0 - 7.5, and it is sterilized by filtration. It can be added individually or in combination of two or more.

[0041] Further, during the single enzymatic hydrolysis of callus, the volume of the enzymatic hydrolysate is 1 - 5 times the volume of the callus.

[0042] Further, the volume of the enzymatic hydrolysate is 2 times the volume of the callus.

[0043] Further, the reaction temperature of the enzymatic hydrolysate is 25.0°C - 30.0°C, the oscillation speed is 50.0 rpm / min - 60.0 rpm / min, and it is cultured under constant temperature and dark oscillation for 20 min - 120 min.

[0044] S4. Obtaining single cells:

[0045] The cell suspension obtained by single enzymatic hydrolysis of the callus in step S3 is separated, sorted, and counted for fluorescently labeled cells using a FACS flow cytometer; the parameters of the FACS flow cytometer are set as nozzle diameter 30 µm - 200 µm, sheath fluid pressure 10 - 75 psi, excitation light wavelength 375 nm - 700 nm, and emission light wavelength 518 nm - 780 nm.

[0046] Preferably, the parameters of the FACS flow cytometer are set as nozzle diameter 30 µm - 100 µm, sheath fluid pressure 20 - 50 psi, excitation light wavelength 488 nm - 650 nm, and emission light wavelength 518 nm - 670 nm.

[0047] S5. Determination of single cell viability:

[0048] The single cell suspension obtained in step S4 is used with a flow cytometry cell viability kit, Molecular Probe LIVE / DEAD Fixable viability dye and eBioscience FVD eFluor viability dye, to detect the viability of the prepared single cells.

[0049] S6. Determination of single cell purity:

[0050] The single-cell suspension obtained in step S4 is used to determine the purity of single cells in accordance with the national standard GB / T 39729-2020, "General Requirements for Cell Purity Determination - Flow Cytometry".

[0051] S7. Application of single cells:

[0052] The single cells obtained in step S4 can be applied to transcriptome sequencing of single cells, single cell nuclei, and single microorganisms in single-cell or single-cell nucleus samples.

[0053] Preferably, it is applied to single-cell sorting and / or single-cell sequencing and / or single-cell transcriptome library construction.

[0054] Preferably, it is applied to subcellular localization and / or promoter activity research and / or cell fusion transformation and / or transient expression detection and / or organelle implantation and / or cell mutant screening and / or genome editing, etc.

[0055] Preferably, it is applied to microbiology and / or basic medicine and / or clinical medicine and / or agronomy and / or cell biology and / or immunology and / or developmental biology and / or pathology and / or neurobiology and / or development and / or genetics and / or stem cells and / or tumors and / or reproductive health and / or metagenomics and / or microecology and / or new drug research and development fields.

[0056] Definition

[0057] The words used in this specification to describe the present invention and its various embodiments should be understood not only in their ordinary defined meanings, but also to include structures, materials, or actions that exceed the scope of the ordinary defined meanings through special definitions in this specification. Therefore, if an element can be understood to include more than one meaning in the context of this specification, its use in the claims must be understood to be generic to all possible meanings supported by this specification and the word itself.

[0058] The various embodiments and aspects of the embodiments disclosed in this specification should be understood not only in the order and context specifically described in this specification, but also to include any order and any combination. Whenever the context requires, all words used in the singular should be considered to include the plural, and vice versa.

[0059] Unless otherwise defined, all technical and scientific terms used in this specification generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used in this specification and the laboratory procedures described in this specification in explants, callus culture, carbon source, fluorophore, culture medium, flow cytometer, cell sorting, cell counting, and cell separation are those well known and commonly employed in the art.

[0060] In the drawings and the specification, the disclosed embodiments of the invention have been set forth, and although specific terms are employed, they are used in a descriptive sense only and not for purposes of limitation. The scope of the invention is set forth in the following claims. It must be understood that the illustrative embodiments have been set forth only by way of example and that it should not be regarded as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, improvements, details, and uses can be made in accordance with the letter and spirit of the disclosure herein and within the scope of the present disclosure, the scope of which is limited only by the claims, interpreted in accordance with the patent laws, including the doctrine of equivalents. In the following claims, the reference characters used to designate claim steps are provided for convenience of description only and are not intended to imply any particular order of performing the steps.

[0061] In the present invention, in addition to its literal meaning, as used herein, the term "comprising" also includes and specifically refers to the terms "consisting essentially of" and "consisting of". Thus, the term "comprising" refers to embodiments in which the subject matter "comprising" the specifically listed elements does not contain additional elements, as well as embodiments in which the subject matter "comprising" the specifically listed elements may and / or does cover additional elements. Similarly, the term "having" should be understood as the term "comprising", and also includes and specifically refers to the terms "consisting essentially of" and "consisting of".

[0062] In the present invention, the terms "specifically", "preferably", "typically", "generally", and "frequently" are not used herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Instead, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in particular embodiments of the present invention. It should also be noted that the terms such as "substantially" and "about" are used herein to represent the inherent uncertainty attributable to any quantitative comparative value, measurement, or other representation.

[0063] The term "single cell" refers to one cell. The term "isolated single cell" refers to a single cell that has been completely, substantially, or partially separated, isolated, excluded, or purified from other components (e.g., cells or cell debris, including but not limited to membrane, protein, or nucleic acid molecules).

[0064] The term "cell" refers to the basic functional unit of a living organism. Suspended cells from any population can be used in the described methods and / or systems; examples include but are not limited to plant cells, eukaryotic single cells, mammalian cells, mammalian mononuclear blood cells, prokaryotic cells, and organisms including animals, plants, microorganisms, algae, or combinations thereof. Suspended cells can be obtained from tissues in several ways well known in the art. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase that break down the extracellular matrix. Optionally, pieces of tissue can be placed in growth medium, and the outgrowing cells can be used for culture.

[0065] The term "isolating" is intended to mean that a substance has been completely, substantially, or partially separated, isolated, excluded, or purified from other components (e.g., cells or cell debris including but not limited to membrane, protein, or nucleic acid molecules).

[0066] The term "fluorophore" refers to a moiety that emits a fluorescent signal in response to a light-stimulating event. A fluorophore can be a small molecule compound or a fluorescent protein. In some embodiments, the fluorophore can be selected from chemiluminescent fluorophores or fluorescent fluorophores. In some embodiments, small molecule fluorophores can include, or not include: FAM, Cy dyes, AlexaFluors, Pacific Blue, coumarin, BODIPY, dansyl, fluorescein, rhodamine, Texas Red-X, Pacific Green, Oregon Green, Texas Red, tetramethylrhodamine, Pacific Orange, eFlours, PE-eFlours, Super Bright Fluors, DyLight Fluors, StarBright Fluors, DRAQ and CyTRAK probes, EverFluor fluors, BellaFluors, Atto tags, Abberior dyes, MegaSTOKES dyes, DYfluors, HiLyte Fluors, SeTauDytes, Quasar and Cal Fluors, SureLight dyes, APC (allophycocyanin), APCXL, RPE, BPE, YOYO-1, and other fluorophores described in the following literature: The Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition, Iain Johnson and Michelle T.Z. Spence, published by Life Technologies in 2010, and InvitrogenTM Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition (Catalog Number InvitrogenTM H37126), both of which are hereby incorporated by reference in their entirety. A fluorescent fluorophore can be any fluorophore understood in the art and can be attached to an antibody by methods well known in the art. In some embodiments, the functionalization of the antibody with a fluorophore can be carried out by NHS chemistry or maleimide chemistry such that the fluorophore is covalently conjugated to the antibody via an amine or a cysteine residue, respectively.In some embodiments, the fluorescent protein is co-expressed independently with the bait biomolecule, the prey biomolecule, or both. In some embodiments, the fluorescent protein may include or not include: GFP (green fluorescent protein), YFP (yellow fluorescent protein), CFP (cyan fluorescent protein), RFP (red fluorescent protein), mCherry, mNeon Green, Sirius, Sandercyanin, shBFP-N158S, Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, shBFP, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquise, mTFP1, monomeric Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, monomeric AzamiGreen, mWasabi, Clover, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, IanRFP-deltaS83, mPapaya1, mCyRFP1, monomeric Kusabira-Orange, mOrange, mOrange2, MOKk, MKO2, TagRFP, TagRFP-T, RRvT, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mNectarine, mRuby3, mScarlet, mScarlet-I, mKate2, HcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, mCardinal, mStable, mMaroon1, mGarnet2, iFP1.4, iFP713, iFP670, iFP682, iFP702, iFP720, iFP2.0, mIFP, TDsmURFP, miRFP670, Sapphire, T-Sapphire, mAmetrine, mKeima, mBeRFP, LSS[1]mKate2, LSS-mKate1, LSSmOrange or CyOFP1.

[0067] The term "callus" refers to a dedifferentiated cell mass that forms after an explant (such as roots, stems, leaves, etc.) detaches from the parent body and proliferates on a culture medium containing plant hormones. Such cell masses are totipotent and can be induced to regenerate into complete plants, which are widely used in plant breeding and rapid propagation. It can also refer to microcallus or structures, or parts thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] 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 embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0069] Figure 1 Results of image acquisition of a longitudinal section of a plant flower bud stained with safranin-fast green at 100 times magnification, used to show differences in cell structure, cell type, cell size, and cell morphology of the longitudinal section of the flower bud.

[0070] Figure 2 Results of image acquisition of a longitudinal section of a plant flower bud stained with safranin-fast green at 200 times magnification, used to show differences in cell structure, cell type, cell size, and cell morphology of the longitudinal section of the flower bud. The longitudinal section of the same flower bud tissue contains various cell structures, cell types, and cell morphologies, and the cell sizes are not uniform.

[0071] Figure 3 Results of image acquisition of a cross-section of a cucumber fruit stained with toluidine blue at 100 times magnification, used to show differences in cell structure, cell type, cell size, and cell morphology of the cross-section of the cucumber fruit.

[0072] Figure 4 Results of image acquisition of a cross-section of a cucumber fruit stained with toluidine blue at 200 times magnification, used to show differences in cell structure, cell type, cell size, and cell morphology of the cross-section of the cucumber fruit. The cross-section of the same cucumber fruit tissue contains various cell structures, cell types, and cell morphologies, and the cell sizes are not uniform.

[0073] Figure 5 Results of image acquisition of a cross-section of a mango fruit stalk stained with safranin-fast green at 100 times magnification, used to show differences in cell structure, cell type, cell size, and cell morphology of the longitudinal section of the flower bud.

[0074] Figure 6 Results of image acquisition of a cross-section of a mango fruit stalk stained with safranin-fast green at 200 times magnification, used to show differences in cell structure, cell type, cell size, and cell morphology of the longitudinal section of the flower bud. The cross-section of the same mango fruit stalk contains various cell structures, cell types, and cell morphologies, and the cell sizes are not uniform.

[0075] Figure 7 The induction process of rice seeds in the callus induction medium. A is the initial stage of induction, where the radicle and callus appear simultaneously. B and C are the calli in the middle stage of induction, and D is the callus in the final stage of induction. Some calli begin to differentiate into seedlings. Specific implementation manners

[0076] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0077] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention, and the upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0078] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described in the present invention can also be used in the implementation or testing of the present invention.

[0079] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention's specification, which are obvious to those of ordinary skill in the art. The specification and examples of the present invention are only exemplary.

[0080] Regarding the use of "comprising", "including", "having", "containing", etc. in the present invention, they are all open-ended terms, meaning including but not limited to.

[0081] The raw materials used in the present invention are all conventional commercially available products unless otherwise specified. The methods used in the present invention are all conventional methods in the art unless otherwise specified. The masses of the various substances used in the present invention are all conventional usage masses and are purchased from commercial channels or are publicly available unless otherwise specifically stated.

[0082] The technical solutions of the present invention will be further described in detail below in combination with specific implementation manners. Example 1

[0083] Take 3 to 4 aseptic tobacco seedlings about 8 weeks old, remove the main leaf veins and leaf tip tissues, and cut them into uniform filaments of 0.5 mm - 1.5 mm with scissors in a laminar flow hood.

[0084] Put the filaments into 10 ml of enzyme solution with a cellulase content of 0.1 g / L, mix gently to make the tobacco leaf filaments fully contact with the enzyme solution, place them on a horizontal constant temperature shaker at 45 rpm / min and a temperature of 26 °C, and enzymatically hydrolyze in the dark for 150 min - 180 min.

[0085] Take a 50 ml centrifuge tube, place a 200-mesh filter screen at the opening, slowly inject the enzymatically hydrolyzed mesophyll solution into the central area of the filter screen for filtration, remove the filter screen, collect the cell solution filtered through the screen, centrifuge at 100 rpm / min for 7 min, and discard the supernatant of the centrifuged solution.

[0086] Resuspend the precipitate with 1X PBS buffer, and determine the single-cell purity according to the national standard GB / T 39729-2020 "General Requirements for Cell Purity Determination - Flow Cytometry". The obtained cell purity is 65.9%, and the cell aggregation rate is 32.5%.

[0087] Use a flow cytometry cell viability kit and Molecular Probe LIVE / DEAD Fixable viability dye to detect the viability of the prepared single cells, which is 78.9. Example 2

[0088] Take 3 to 4 aseptic tobacco seedlings about 8 weeks old, remove the main leaf veins and leaf tip tissues, and cut them into uniform filaments of 0.5 mm - 1.5 mm with scissors in a laminar flow hood.

[0089] Soak the filaments in 75% ethanol for 5 min, then soak them in sodium hypochlorite solution with an active chlorine content of 8% for 15 min, and rinse with sterile water 3 times.

[0090] The rinsed tobacco leaf filaments are inoculated on a solid medium of MS + 6-benzylaminopurine (0.0025 g / L) + naphthaleneacetic acid (0.005 g / L) + agar powder (5.0 g / L) + sucrose (CY3 fluorescent label) (30.0 g / L), pH = 6.5, and induced to culture at 30 °C in the dark and away from light for 48 hr.

[0091] Put the callus induced to culture in the dark for 48 hr into 20.0 ml of enzyme solution with a cellulase content of 0.1 g / L. The enzyme solution is 3 times the volume of the callus, and culture at 26.0 °C, 50.0 rpm / min, constant temperature, in the dark, and with shaking for 30 min to obtain a callus cell suspension.

[0092] The obtained callus cell suspension was separated, sorted, and counted for fluorescently labeled cells using a FACS flow cytometer.

[0093] The parameters of the FACS flow cytometer were set as follows: nozzle diameter 50 µm, sheath fluid pressure 25 psi, excitation light wavelength 550 nm, and emission light wavelength 570 nm.

[0094] According to the national standard GB / T 39729-2020 "General Requirements for Cell Purity Determination - Flow Cytometry", the purity of single cells was determined. The number of effective cells obtained was 10,515, the cell purity was 98.5%, and the cell aggregation rate was 1.5%.

[0095] Using a flow cytometry cell viability kit and Molecular Probe LIVE / DEAD Fixable viability dye, the viability of the prepared single cells was detected to be as high as 99.95.

[0096] Beneficial effects: In Example 1, the traditional method for preparing single cells from plant leaves was used. The cell purity of the prepared single cells was 65.9%, the cell aggregation rate was 32.5%, and the viability was 78.9. In Example 2, the technical solution of the present invention was adopted. The cell purity of the prepared single cells was as high as 98.5%, the cell aggregation rate was only 1.5%, and the viability of single cells was as high as 99.95. The cell purity and the viability of single cells were increased by 32.6% and 21.05% respectively compared with the traditional technical solution, and the cell aggregation rate was decreased by 31.0% compared with the traditional technical solution. Example 3

[0097] The brown rice obtained after removing the husk from mature rice seeds was placed in a 50 ml centrifuge tube, disinfected with 75% ethanol for 10 min, and then soaked in a 30.0%-50.0% sodium hypochlorite solution (active chlorine content 8.25%) for 20 min. During this period, the centrifuge tube was gently shaken, and after rinsing 4 times with sterile water in a laminar flow hood, the disinfected explants were obtained.

[0098] The explants were inoculated onto a solid medium of N6 + 6-benzylaminopurine (0.005 g / L) + naphthaleneacetic acid (0.005 g / L) + agar powder (5.0 g / L) + glucose (‌FAM fluorescent label) (50.0 g / L), pH = 6.0, and cultured under dark and light-free conditions at 30 °C for 72 h ( Figure 7 )

[0099] The callus induced in the dark for 72 h ( Figure 7), placed in 15.0 ml of an enzyme solution containing 0.01 g / L of macerozyme. The enzyme solution is 1.5 times the volume of the callus, and cultured at 28.0 °C, 60.0 rpm / min, under constant temperature, darkness, and shaking for 60 min to obtain a callus cell suspension.

[0100] The obtained callus cell suspension was separated, sorted, and counted for fluorescently labeled cells using a FACS flow cytometer.

[0101] The parameters of the FACS flow cytometer were set as follows: nozzle diameter 60 µm, sheath fluid pressure 30 psi, excitation light wavelength 495 nm, and emission light wavelength 521 nm.

[0102] According to the national standard GB / T 39729-2020 "General requirements for cell purity determination - Flow cytometry", the purity of single cells was determined. A total of 11,189 effective cells were detected, with a cell purity of 99.5% and a cell aggregation rate of 0.58%.

[0103] Using a flow cytometry cell viability kit and Molecular Probe LIVE / DEAD Fixable viability dye, the viability of the prepared single cells was detected to be as high as 100.0.

[0104] Beneficial effects: The enzyme digestion method mainly uses a mixed enzyme solution of cellulase, pectinase, and macerozyme to treat the tender tissue parts of plants. It has great limitations for plant parts such as flowers, stems, fruits, and seeds, which are older and more difficult to obtain intact and active protoplasts. In Example 3, mature rice seeds were used as explants, and through callus induction, single cells of mature rice seeds with cell purity and cell viability as high as 99.5% and 100% were successfully obtained, breaking through the limitation that the traditional enzyme digestion method cannot separate single cells of plant seeds, especially the embryo and endosperm single cells inside mature plant seeds. Example 4

[0105] The outer scales of the peony bud were removed, and the bud was placed in 75% ethanol for disinfection for 5 min on a super clean bench, then soaked in 30.0% - 50.0% sodium hypochlorite solution (active chlorine content 8.25%) for 15 min, and rinsed 3 times with sterile water in the super clean bench to obtain a disinfected peony bud explant.

[0106] The disinfected peony bud explant was inoculated onto a solid medium of B5 + auxin (0.005 g / L) + 2,4-dichlorophenoxyacetic acid (0.02 g / L) + gibberellin (0.001 g / L) + agar (3.5 g / L) + fructose (40 g / L) (LC Red fluorescence labeling), pH = 6.2, and induced to culture at 30 °C, in darkness, and avoiding light for 48 hr.

[0107] The callus induced in the dark for 48 h was placed in 10.0 ml of an enzymatic hydrolysis solution containing 0.02 g / L of pectinase. The enzymatic hydrolysis solution was 2.0 times the volume of the callus. It was cultured at 28.0 °C, 50.0 rpm / min, under constant temperature, in the dark, with shaking for 40 min to obtain a callus cell suspension.

[0108] The obtained callus cell suspension was separated, sorted, and counted for fluorescently labeled cells using a FACS flow cytometer.

[0109] The parameters of the FACS flow cytometer were set as follows: nozzle diameter 40 µm, sheath fluid pressure 20 psi, excitation light wavelength 625 nm, and emission light wavelength 640 nm.

[0110] The determination of single-cell purity was carried out in accordance with the national standard GB / T 39729-2020 "General Requirements for Cell Purity Determination - Flow Cytometry". 9892 effective cells were detected, the cell purity was 99.9%, and the cell aggregation rate was 0.10%.

[0111] The viability of the prepared single cells was detected using a flow cytometry cell viability detection kit and the eBioscience FVD eFluor viability dye, and the viability was as high as 99.98%. Example 5

[0112] Take Loropetalum chinense var. rubrum leaf material, remove the main vein and leaf tip tissue, cut it into uniform leaf segments of 1.5 mm - 5.0 mm, and make two transverse cuts perpendicular to the vein with scissors in a laminar flow hood without disconnecting the leaf. Then place the leaf in 75% ethanol for disinfection for 10 min in the laminar flow hood, and then soak it in 30.0% - 50.0% sodium hypochlorite solution (active chlorine content 8.25%) for 15 min, and rinse it 3 times with sterile water in the laminar flow hood for later use; the brown rice obtained after removing the husk from mature rice seeds was placed in a 50 ml centrifuge tube, disinfected with 75% ethanol for 10 min, and then soaked in 30.0% - 50.0% sodium hypochlorite solution (active chlorine content 8.25%) for 20 min, gently shaking the centrifuge tube during this period, and after rinsing 4 times with sterile water in the laminar flow hood, disinfected explants were obtained. The disinfected rice seed explants and Loropetalum chinense var. rubrum leaves were inoculated together on the callus medium with the back facing up. The induction medium used was White solid medium + auxin (0.005 g / L) + 2,4-dichlorophenoxyacetic acid (0.02 g / L) + gibberellin (0.001 g / L) + agar (4.0 g / L) + glucose (30 g / L) (Texas Red fluorescence labeling), pH = 6.0, and co-induced culture was carried out at 28 °C, in the dark, avoiding light for 60 h.

[0113] The callus induced by common darkness for 60 hr was placed in 12.0 ml of an enzyme solution containing 0.02 g / L of pectinase. The volume of the enzyme solution was 1.8 times that of the callus. The mixture was cultured at 30.0 °C, 60.0 rpm / min, under constant temperature, in the dark, and with shaking for 60 min to obtain a callus cell suspension.

[0114] The obtained callus cell suspension was separated, sorted, and counted for fluorescently labeled cells using a FACS flow cytometer.

[0115] The parameters of the FACS flow cytometer were set as follows: nozzle diameter 60 µm, sheath fluid pressure 20 psi, excitation light wavelength 589 nm, and emission light wavelength 610 nm.

[0116] The determination of single-cell purity was carried out in accordance with the national standard GB / T 39729-2020 "General Requirements for Cell Purity Determination - Flow Cytometry". A total of 11,269 effective cells were detected, with a cell purity of 99.98% and a cell aggregation rate of 0.5%.

[0117] The viability of the prepared single cells was detected using a flow cytometry cell viability kit, eBioscience FVD eFluor viability dye, and the viability was as high as 100.0.

[0118] The present invention adopts the method of culturing plant explant callus. Especially for tissue parts with poor enzymatic hydrolysis effects, such as hard seeds, fruits, and plant roots, stems, and leaves with a high degree of fibrosis, the explant callus is first cultured. By adding a carbon source containing a fluorescent group to the culture medium, callus cell clusters with fluorescent groups, thin cell walls, easily dissociated by a single enzyme solution, and uniform cell morphology and size are generated. The cell suspension formed after dissociating the cell clusters with a single enzyme solution is separated and sorted for single cells with fluorescent labels using a flow cytometer. Finally, the purity of the prepared single plant leaf cells is as high as 98.5%, the cell aggregation rate is only 1.5%, and the viability of single cells is as high as 99.95%. The cell purity and the viability of single cells are increased by 32.6% and 21.05% respectively compared with the traditional technical scheme, and the cell aggregation rate is reduced by 31.0% compared with the traditional technical scheme. Using mature rice seeds as explants, single seeds with cell purity and cell viability as high as 99.5% and 100% respectively were successfully obtained through callus induction, breaking through the limitation that traditional enzymatic hydrolysis methods cannot prepare single seed cells, especially cannot prepare single embryo and endosperm cells of seeds. In addition, through the co-induction of Loropetalum chinense var. rubrum leaves and mature rice seeds, the prepared cell purity is 99.98%, the cell aggregation rate is only 0.5%, and the viability of single cells is as high as 100.0, overcoming the limitation of single-cell preparation caused by species differences.

Claims

1. A method for preparing a plant single cell, characterized in that: It consists of the following steps: S1. isolating and obtaining explants of plant roots and / or stems and / or leaves and / or flowers and / or fruits and / or seeds and / or seed embryos and / or seed endosperms for inducing callus; S2. Placing the explant in a callus culture medium containing a fluorescently labeled carbon source to induce callus tissue, wherein the fluorescent markers include FAM and / or TET and / or CY3 and / or Texas Red and / or LC Red and / or LC Green and / or CY5, etc., and may be a single marker or a combination of multiple markers; the carbon source includes glucose and / or fructose and / or sucrose and / or mannose and / or galactose and / or lactose and / or maltodextrin and / or honey, etc., which can be used to induce explant callus tissue and their derivatives, and may be added alone or in combination of two or more; S3, using one of cellulase, hemicellulase, macerate, pectinase, cleavase or snailase to dissociate the callus induced in step S2, to prepare a callus cell suspension with thin cell walls and uniform cell morphology and size; S4. For the cell suspension obtained in step S3, single cells are separated, sorted and counted using a flow cytometer.

2. A method for preparing a plant single cell according to claim 1, characterized in that: The plants are derived from algae and / or bryophytes and / or ferns and / or seed plants and / or gramineous plants and / or woody plants and / or gymnosperms and / or angiosperms and / or trees and / or shrubs and / or wild plants and / or artificially cultivated plants and / or monocotyledons and / or dicotyledons.

3. A method for preparing a plant single cell according to claim 1, characterized in that: The explant is derived from one or more of wheat and / or rice and / or corn and / or oats and / or rye and / or barley and / or millet and / or sorghum and / or highland barley and / or soybean and / or broad bean and / or pea and / or mung bean and / or adzuki bean and / or sweet potato and / or potato and / or cassava and / or cotton and / or hemp and / or flax and / or jute and / or rape and / or peanut and / or sesame and / or sugar beet and / or sugar cane and / or rubber and / or mulberry and / or wolfberry and / or liquorice and / or astragalus and / or ginseng and / or notoginseng and / or mint and / or ginkgo and / or lavender and / or fennel and / or clove and / or tea and / or coffee and / or cocoa and / or tobacco and / or tomato and / or hops.

4. The method for preparing a plant single cell according to claim 1, characterized in that: The callus tissue culture medium is composed of inorganic salts, hormones, carbon sources and agar, and includes MS solid culture medium and / or B5 solid culture medium and / or White solid culture medium and / or N6 solid culture medium.

5. The method for preparing a plant single cell according to claim 4, characterized in that: The inorganic salts include ammonium nitrate and / or calcium chloride and / or magnesium sulfate and / or potassium hydrogen phosphate and / or potassium dihydrogen phosphate and / or potassium nitrate and / or calcium nitrate and / or boric acid and / or cobalt chloride and / or ferrous sulfate and / or manganese (II) sulfate. and / or potassium iodide and / or sodium molybdate and / or zinc sulfate and / or sodium ferric ethylenediaminetetraacetate and / or copper sulfate, which can be added alone and / or in combination of two or more; the content is ammonium nitrate 0.1g / L-1.0g / L and / or calcium chloride 0.5g / L-1.5g / L and / or magnesium sulfate 0.01g / L-0.1g / L and / or potassium hydrogen phosphate 0.1g / L-1.0g / L and / or potassium dihydrogen phosphate 1.5g / L-2.0g / L and / or potassium nitrate 0.5g / L-1.0g / L and / or calcium nitrate 0.25g / L-1.58g / L and / or boric acid 1.1g / L-1. 96g / L and / or cobalt chloride 0.001g / L-0.01g / L and / or ferrous sulfate 0.01g / L-0.09g / L and / or manganese (II) sulfate 0.01g / L-0.1g / L and / or potassium iodide 0.025g / L-0.055g / L and / or sodium molybdate 0.002g / L-0.005g / L and / or zinc sulfate 0.025g / L-0.25g / L and / or sodium ferric ethylenediaminetetraacetate 1.12g / L-1.59g / L and / or copper sulfate 0.001g / L-0.02g / L, which can be added alone or in combination of two or more.

6. A method for preparing a plant single cell according to claim 4, characterized in that: The hormones include auxin and / or synthetic auxin and / or auxin analogs and / or 2,4-dichlorophenoxyacetic acid and / or 3,5-dimethylphenoxyacetic acid and / or phenoxyacetic acid and / or phenylacetic acid and / or p-chlorophenoxyacetic acid and / or 3,6-dichloro-o-anisic acid and / or naphthylacetic acid and / or indoleacetic acid and / or indole-3-butyric acid and / or 6-benzylaminopurine and / or 6-( γ,γ-dimethylallylamino) purine and / or thidiazuron and / or gibberellin and / or abscisic acid, which can be added alone and / or in combination of two or more; the content is 0.001g / L-0.005g / L of auxin and / or 0.001g / L-0.005g / L of synthetic auxin and / or 0.002g / L-0.005g / L of auxin analogs and / or 0.02g / L-0.05g / L of 2,4-dichlorophenoxyacetic acid and / or 0.005g / L-0.010g / L of 3,5-dimethylphenoxyacetic acid and / or 0.005g / L-0.010g / L of phenoxyacetic acid. L-0.015g / L and / or phenylacetic acid 0.001g / L-0.010g / L and / or p-chlorophenoxyacetic acid 0.002g / L-0.008g / L and / or 3,6-dichloro-o-anisic acid 0.0025g / L-0.0095g / L and / or naphthylacetic acid 0.002g / L-0.009g / L and / or indoleacetic acid 0.005g / L-0.009g / L and / or indole-3-butyric acid 0.001g / L-0.009g / L and / or 6-benzylaminopurine 0.002g / L-0.01g / L and / or 6-( γ,γ-dimethylallylamino)purine 0.001g / L-0.02g / L and / or thidiazuron 0.001g / L-0.005g / L and / or gibberellin 0.001g / L-0.01g / L and / or abscisic acid 0.001g / L-0.02g / L, which can be added alone or in combination of two or more.

7. The method for preparing a plant single cell according to claim 1, characterized in that: The glucose and / or fructose and / or sucrose and / or mannose and / or galactose and / or lactose and / or maltodextrin and / or honey and the like can be used to induce explant callus tissue and their derivatives, including glucose 15.0g / L-50.0g / L and / or fructose 30.0g / L-60.0g / L and / or sucrose 10.0g / L-30.0g / L and / or mannose 15.0g / L-45.0g / L and / or galactose 5.0g / L-20.0g / L and / or lactose 30.0g / L-45.0g / L and / or maltodextrin 30.0g / L-60.0g / L and / or honey 50.0g / L-65.0g / L and the like can be used to induce explant callus tissue and their derivatives, which can be added alone or in combination of two or more.

8. The method for preparing a plant single cell according to claim 7, characterized in that: The glucose and / or fructose and / or sucrose and / or mannose and / or galactose and / or lactose and / or maltodextrin and / or honey and the like can be used to induce explant callus and their derivatives, including glucose 15.0 g / L-50.0 g / L and / or fructose 30.0 g / L-60.0 g / L and / or sucrose 10.0 g / L-30.0 g / L and / or mannose 15.0 g / L-45.0 g / L and / or galactose 5.0 g / L-20.0 g / L and / or lactose 30.0 g / L-45.0 g / L and / or maltodextrin 30.0 g / L-60.0 g / L and / or honey 50.0 g / L-65.0 g / L, etc., which are induced by FAM and / or TET and / or CY3 and / or Texas Red and / or LC Fluorescent markers such as Red and / or LC Green and / or CY5 can be used alone or in combination.

9. The method for preparing a plant single cell according to claim 1, characterized in that: The single enzyme is one of the six enzymes with a cellulase content of 0.005g / L-0.1g / L, a hemicellulase content of 0.001g / L-0.5g / L, a cleavage enzyme content of 0.001g / L-0.01g / L, a pectinase content of 0.001g / L-0.02g / L, a colaptase content of 0.01g / L-0.02g / L, or a snailase content of 0.001g / L-0.002g / L.

10. The method for preparing a plant single cell according to claim 1, characterized in that: The single cell separation, sorting and counting are prepared by flow cytometry and / or fluorescence activated flow cytometry sorter.

Images