A method for preparing plant single cells

Abstract

The present application adopts the way of plant explant callus culture, adds carbon source containing fluorescent group in the culture medium, induces callus with fluorescent group, thin cell wall, easy to be single enzymolysis, uniform cell morphology and size, the cell after single enzymolysis is detected by flow cytometry, the purity and activity of the prepared leaf single cell are 98.5% and 99.95 respectively, the agglomeration rate is only 1.5%, the cell purity and activity are increased by 32.6% and 21.05 respectively compared with the traditional technical scheme, and the agglomeration rate is reduced by 31.0%; the rice seed is used as the explant, and the seed single cell with cell purity and activity of 99.5% and 100% is obtained, which breaks through the limitation of traditional enzymolysis method that cannot prepare seed single cell; in addition, through the common induction of safflower leaf and rice seed, the cell purity and activity prepared are as high as 99.98% and 100.0, which overcomes the limitation of single cell preparation caused by interspecific heterogeneity.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically, it relates to a method for preparing plant single cells. Background Technology

[0002] Plants and animals are highly evolved life forms composed of many single cells, capable of complex life activities. Plant roots, stems, leaves, and animal viscera are common samples for single-cell isolation. Isolating and culturing single cells from plant and animal tissues is of great significance for studying organ function and genetic mutation. However, in plant and animal tissues, multiple cells and proteins adhere together, making it difficult to isolate individual cells. Especially in botany, plant cells have cell walls, and intercellular connections mainly rely on the mucilage layer within the cell wall. The interaction of cellulose, hemicellulose, and lignin in the cell wall determines the degree of cell adhesion.

[0003] Plant single-cell suspensions are typically prepared using mechanical and enzymatic methods. Mechanical methods often involve chopping plant material immersed in enzyme extraction buffer using a blade, incubating for several minutes to release cells, and then filtering and centrifuging to obtain the cell suspension. However, the cell structure of each plant species exhibits significant heterogeneity, and even within the same species, significant differences exist between different tissue types and within the same tissue type. These differences include cell size (different developmental stages, different tissue locations, etc.), cell structure (spongy tissue, palisade tissue, stomatal cells, endosperm cells, mesophyll cells, vascular bundle cells, etc.), and cell morphology (elongated, round, elliptical, spindle-shaped, rhomboid, etc.). Figures 1-6 Therefore, the cells obtained by centrifugation and resuspension are a mixture of all cell types in the material taken, and each cell type cannot be distinguished individually. At the same time, the cell suspension obtained by separation cannot accurately distinguish between individual cells and cell clusters, which is not conducive to subsequent single-cell research.

[0004] Although enzymatic hydrolysis mainly utilizes a mixed enzymatic hydrolysate of cellulase, pectinase, and dissociative enzymes to treat young plant tissues, the cell wall composition varies among different species, tissues, and organs. Figures 1-7 Therefore, not all cell walls can be digested with the same efficiency. Thus, it is necessary to optimize the enzymatic hydrolysate and reaction conditions for different species and tissue types, which greatly increases the difficulty of applying it to single-cell plants. At the same time, it also has great limitations for older parts of plants such as flowers, stems, fruits, and seeds, where it is difficult to obtain intact protoplasts. In addition, the effects of long-term enzymatic hydrolysis of multiple complex enzyme solutions and osmotic pressure can lead to changes in cell gene expression and also cause the breakage of easily digestible cells.

[0005] Plant callus refers to the morphogenesis process in which somatic cells, under specific conditions, form new individuals through a developmental pathway similar to that of a zygote without the fusion of sex cells. Callus cells are mostly isodiametric, thin-walled meristematic cells, which, compared to mature plant tissue cells, are more easily obtained as highly active, intact, and uniformly sized single cells through enzymatic hydrolysis.

[0006] Flow cytometry is a fluorescence-based cell biology analysis technique that rapidly detects the properties of individual cells within a cell population. It can collect multiple cellular parameters in a single analysis, such as cell number, cell volume, and response to fluorescent probes. Flow cytometry detects and classifies cells based on cell morphology and fluorescent substances. By adjusting various parameters of the cells in the sample and the sorted cell size, viable cells are captured. With the aid of an imaging system, the droplet state is observed, and adjustments are made to obtain parameters such as cell diameter and roundness in the sample. Adjusting the sorted cell size (10-40 μm) allows for precise capture of viable cells.

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

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

[0009] Another object of the present invention is to provide a callus enzymatic hydrolysate, wherein the hydrolysate is one of cellulase, hemicellulase, dissociative enzyme, pectinase, breakdown enzyme, or snail enzyme, thereby simplifying the composition of the hydrolysate.

[0010] Another object of the present invention is to provide a method for obtaining callus cell clusters with fluorescent groups, thin cell walls, easy dissociation by a single enzymatic hydrolysate, and uniform cell morphology and size.

[0011] Another objective of this invention is to provide a method for isolating single cells from fluorescently labeled plant callus, particularly for hard seeds, fruits, and plant roots, stems, and leaves with high fibrosis that are difficult to digest with enzymatic methods. The method uses flow cytometry to identify fluorescent groups on the callus and capture and separate single cells.

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

[0013] This invention employs plant explant callus culture, particularly suitable for hard seeds, fruits, and plant roots, stems, and leaves with high fibrosis that are difficult to hydrolyze with enzymes. First, explant callus culture is performed. By adding a carbon source containing a fluorescent group to the culture medium, callus cell clusters with fluorescent groups, thin cell walls, and uniform cell morphology and size are generated, which are easily dissociated by a single enzyme hydrolysate. The cell suspension formed after dissociation by the single enzyme hydrolysate is then separated and sorted using flow cytometry to obtain plant single cells with high purity, high viability, and low aggregation rate.

[0014] This invention provides a method for preparing plant single cells, which mainly includes the following steps.

[0015] S1. Disinfection of plant explants:

[0016] Select suitable plant tissue parts, and cut, slice or peel the parts as needed to use them as explants for callus culture. 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 to disinfect the surface of the explants.

[0017] Furthermore, the explants are derived from one or more of the following: algae and / or bryophytes and / or ferns and / or seed plants and / or grasses and / or woody plants and / or gymnosperms and / or angiosperms and / or trees and / or shrubs and / or wild plants and / or cultivated plants and / or monocots and / or dicots.

[0018] Furthermore, the explants are derived from one or more of the following: 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 hobby crops and / or industrial crops and / or tropical economic crops and / or subtropical economic crops and / or temperate economic crops.

[0019] Preferably, the explant is derived from one or more of the following: 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 red 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 rapeseed 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 pepper 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.

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

[0021] Preferably, the size of the explant is one or more of the following: root (2.0mm-10.0mm) and / or stem (1.5mm-15.0mm) and / or leaf (1.0mm-5.0mm) and / or flower (2.0mm-8.0mm) and / or fruit (2.0mm-8.0mm) and / or seed (2.0mm-10.0mm).

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

[0023] S2. Obtaining plant callus:

[0024] Place the sterilized explants from step S1 onto sterilized absorbent paper in a clean bench, gently press the absorbent paper with tweezers to absorb the surface moisture, and inoculate them onto callus solid induction medium. Induce culture at 24℃-37℃ in the dark for 24-72 hours.

[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, mainly composed of inorganic salts, hormones, carbon sources and agar, with a pH of 5.5-7.5.

[0026] Preferably, the inorganic salts, including 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 ferric sodium ethylenediaminetetraacetate and / or copper sulfate, can be added alone and / or in mixtures of two or more.

[0027] Furthermore, the inorganic salts include ammonium nitrate 0.1 g / L-1.0 g / L and / or calcium chloride 0.5 g / L-1.5 g / L and / or magnesium sulfate 0.01 g / L-0.1 g / L and / or potassium hydrogen phosphate 0.1 g / L-1.0 g / L and / or potassium dihydrogen phosphate 1.5 g / L-2.0 g / L and / or potassium nitrate 0.5 g / L-1.0 g / L and / or calcium nitrate 0.25 g / L-1.58 g / L and / or boric acid 1.1 g / L-1.96 g / L and / or cobalt chloride 0.001 g / L-0. 0.01 g / L and / or ferrous sulfate 0.01 g / L-0.09 g / L and / or manganese(II) sulfate 0.01 g / L-0.1 g / L and / or potassium iodide 0.025 g / L-0.055 g / L and / or sodium molybdate 0.002 g / L-0.005 g / L and / or zinc sulfate 0.025 g / L-0.25 g / L and / or ferric sodium EDTA 1.12 g / L-1.59 g / L and / or copper sulfate 0.001 g / L-0.02 g / L, which can be added alone and / or in combination with two or more.

[0028] Preferably, the hormones, including 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 p-chlorophenoxyacetic acid and / or 3,6-dichloro-o-anisolic 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, can be added alone and / or in mixtures of two or more.

[0029] Furthermore, the hormones include auxin 0.001 g / L-0.005 g / L and / or synthetic auxin 0.001 g / L-0.005 g / L and / or auxin analogue 0.002 g / L-0.005 g / L and / or 2,4-dichlorophenoxyacetic acid 0.02 g / L-0.05 g / L and / or 3,5-dimethylphenoxyacetic acid 0.005 g / L-0.010 g / L and / or phenoxyacetic acid 0.005 g / L-0.015 g / L and / or phenylacetic acid 0.001 g / L. 0.010 g / L and / or p-chlorophenoxyacetic acid 0.002 g / L-0.008 g / L and / or 3,6-dichloro-o-anisolic acid 0.0025 g / L-0.0095 g / L and / or naphthaleneacetic acid 0.002 g / L-0.009 g / L and / or indoleacetic acid 0.005 g / L-0.009 g / L and / or indole-3-butyric acid 0.001 g / L-0.009 g / L and / or 6-benzylaminopurine 0.002 g / L-0.01 g / 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, may be added alone and / or in combination with two or more.

[0030] Preferably, 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 are sugars and their derivatives that can be used by explants to induce callus, and can be added alone or in combination of two or more.

[0031] Furthermore, the carbon source includes 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 can be used by explants to induce callus, and can be added alone or in combination of two or more.

[0032] Furthermore, the carbon source, 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, is 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, and can be labeled with one or multiple combinations.

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

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

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

[0036] S3. Preparation of plant cell suspension:

[0037] The callus obtained in step S2 was placed in 5.0 ml to 20.0 ml of a single enzyme digest and cultured at 25.0 °C to 37.0 °C and at a rotation speed of 20.0 rpm / min to 80.0 rpm / min for 15 min to 180 min in the dark with shaking to obtain a callus cell suspension.

[0038] Furthermore, the single enzymatic hydrolysate is one of cellulase, hemicellulase, dissociative enzyme, pectinase, breakdown enzyme, or snail enzyme.

[0039] Furthermore, in the single enzymatic hydrolysate, the content of cellulase is 0.005 g / L-0.1 g / L, or the content of hemicellulase is 0.001 g / L-0.5 g / L, or the content of dissociative enzyme is 0.001 g / L-0.01 g / L, or the content of pectinase is 0.001 g / L-0.02 g / L, or the content of catabolase is 0.01 g / L-0.02 g / L, or the content of snailase is 0.001 g / L-0.002 g / L.

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

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

[0042] Furthermore, the volume of the enzymatic hydrolysate is twice the volume of the callus tissue.

[0043] Furthermore, the reaction temperature of the enzymatic hydrolysate was 25.0℃-30.0℃, the shaking speed was 50.0 rpm / min-60.0 rpm / min, and the culture was carried out at a constant temperature in the dark for 20min-120min.

[0044] S4. Obtaining single cells:

[0045] The cell suspension obtained from the callus tissue after single enzymatic digestion in step S3 was used to separate, sort, and count fluorescently labeled cells using a FACS flow cytometer. The parameters of the FACS flow cytometer were set as follows: nozzle diameter 30µm-200µm, sheath fluid pressure 10-75 psi, excitation wavelength 375nm-700nm, and emission wavelength 518nm-780nm.

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

[0047] S5. Determination of single-cell viability:

[0048] The single-cell suspension obtained in step S4 was used to detect the activity of the prepared single cells using a flow cytometry kit containing Molecular ProbeLIVE / DEAD Fixable activity dye and eBioscience FVD eFluor activity dye.

[0049] S6. Determination of single-cell purity:

[0050] The single-cell suspension obtained in step S4 was subjected to single-cell purity determination in accordance with the national standard GB / T 39729-2020 "General requirements for cell purity determination - flow cytometry".

[0051] S7. Applications of single cells:

[0052] The single cells obtained in step S4 can be used in transcriptome sequencing of single cells or single cell nuclei samples, including single cells, single cell nuclei, and single microorganisms.

[0053] Preferably, it is used in 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 studies 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 in the fields of 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 oncology and / or reproductive health and / or metagenomics and / or microecology and / or new drug development.

[0056] definition

[0057] The terms used in this specification to describe the invention and its various embodiments should be understood not only in their commonly defined meanings, but also, through the specific definitions herein, to include structures, materials, or actions that extend beyond their commonly defined meanings. Therefore, if an element can be understood to include more than one meaning within the context of this specification, its use in the claims must be understood to be superior to all possible meanings supported by this specification and by the term itself.

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

[0059] Unless otherwise defined, all technical and scientific terms used in this description generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used in this description and the laboratory procedures described herein for explants, callus culture, carbon sources, fluorescent groups, culture media, flow cytometry, cell sorting, cell counting, and cell isolation are those well-known and commonly used in the art.

[0060] The disclosed embodiments of the invention have been shown in the accompanying drawings and description. While specific terminology has been used, it is for descriptive purposes only and not for limiting purposes. The scope of the invention is set forth in the following claims. It must be understood that illustrative embodiments have been described for purposes of example only and should not be construed as limiting the invention. It will be apparent to those skilled in the art that changes, other embodiments, modifications, details, and uses can be made consistent with the text and spirit of the disclosure herein and within the scope of this disclosure, the scope of which is limited only by the claims and interpreted in accordance with patent law, including the doctrine of equivalents. In the following claims, reference characters used to designate claim steps are provided for ease of description only and are not intended to imply any particular order of performing the steps.

[0061] In this invention, in addition to its literal meaning, the expression "comprising" as used herein also includes and specifically refers to the expressions "substantially constitutes" and "consisting of". Therefore, the expression "comprising" means an embodiment in which the subject matter of the specifically listed elements "comprising" does not contain other elements, and an embodiment in which the subject matter of the specifically listed elements "comprising" may and / or does cover other elements. Similarly, the expression "having" should be understood to mean that the expression "comprising" also includes and specifically refers to the expressions "substantially constitutes" and "consisting of".

[0062] In this invention, the terms “specifically,” “preferably,” “typically,” “generally,” and “often” are not used herein to limit the scope of the claimed invention or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed invention. Rather, these terms are intended only to highlight alternative or additional features that may or may not be used in a particular embodiment of the invention. It should also be noted that terms such as “substantially” and “about” are used herein to represent inherent uncertainty attributable to any quantitative comparison, measurement, or other representation.

[0063] The term "single cell" refers to a single cell. The term "isolated single cell" refers to a single cell that is completely, substantially, or partially separated, isolated, excluded, or purified from other components (e.g., cells or cell debris, including but not limited to membranes, proteins, 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 monocytes, mammalian cells, mammalian monocytes, 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. Monocytes can be released from soft tissues by enzymatic digestion with enzymes that break down the extracellular matrix, such as collagenase, trypsin, or streptomycin. Alternatively, pieces of tissue can be placed in a growth medium, and the resulting cells can be cultured.

[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 membranes, proteins or nucleic acid molecules).

[0066] The term "fluorophore" refers to the portion that emits a fluorescent signal in response to a photostimulatory event. A fluorophore can be a small molecule compound or a fluorescent protein. In some embodiments, the fluorophore may be selected from a chemiluminescent fluorophore or a fluorescent fluorophore. In some embodiments, the small molecule fluorophore may or may not include: FAM, Cy dyes, AlexaFluors, Pacific Blue, coumarin, BODIPY, dansyl sulfonyl, 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, Abberio dyes, MegaSTOKES dyes, DYfluors, HiLyte Fluors, SeTauDytes, Quasar and Cal Fluors, SureLight dye, 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, by Iain Johnson and Michelle T.Z. Spence, published by Life Technologies, 2010; and *Invitrogen™ Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies*, 11th edition (manufacturer number Invitrogen™ H37126), both of which are incorporated herein by reference in their entirety. Fluorescent fluorophores can be any fluorophore understood in the art and can be attached to antibodies using methods well known in the art. In some embodiments, the functionalization of the antibody with a fluorophore can be performed by NHS chemistry or maleimide chemistry, such that the fluorophore is covalently conjugated to the antibody via an amine or cysteine ​​residue, respectively.In some embodiments, the fluorescent protein is independently co-expressed with a bait biomolecule, a prey biomolecule, or both. In some embodiments, the fluorescent protein may or may 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, monomer Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder. GFP, Emerald, EGFP, Monomeric AzamiGreen, mWasabi, Clover, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Yp et, IanRFP-deltaS83, mPapaya1, mCyRFP1, monomer Kusabira-Orange, mOrange, mOrange2, MOKk, MKO2, TagRFP, TagRFP-T, RRvT, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mNectarine, mRuby3, mScarlet, mScarlet-I, mKate2, HcR ed-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 the dedifferentiated cell mass formed after an explant (such as a root, stem, or leaf) detaches from the parent plant and proliferates on a culture medium containing plant hormones. These cell masses are totipotent and can be induced to regenerate into complete plants, making them widely used in plant breeding and rapid propagation. It can also refer to microcallus tissue or structures, or portions thereof. Attached Figure Description

[0068] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0069] Figure 1 Images of plant flower bud longitudinal sections stained with safranin and fast green at 100x magnification were acquired to demonstrate differences in cell structure, cell type, cell size, and cell morphology in the longitudinal sections of flower buds.

[0070] Figure 2 Images of longitudinal sections of plant flower buds stained with safranin and fast green at 200x magnification were acquired to demonstrate differences in cell structure, cell type, cell size, and cell morphology within the longitudinal sections. The same flower bud tissue contains various cell structures, cell types, and cell morphologies, and the cells are not uniform in size.

[0071] Figure 3 The images of cucumber fruit cross sections stained with toluidine blue at 100x magnification were used to demonstrate the differences in cell structure, cell type, cell size, and cell morphology in cucumber fruit cross sections.

[0072] Figure 4 Images of cucumber fruit cross-sections stained with toluidine blue at 200x magnification were acquired to demonstrate the differences in cell structure, cell type, cell size, and cell morphology within the cross-sections. A cross-section of the same cucumber fruit tissue contains various cell structures, cell types, and cell morphologies, and the cells are not uniform in size.

[0073] Figure 5 Images of mango peduncle cross sections stained with safranin and fast green at 100x magnification were acquired to demonstrate differences in cell structure, cell type, cell size, and cell morphology in longitudinal sections of flower buds.

[0074] Figure 6 Images of mango peduncle cross sections stained with safranin and fast green at 200x magnification were acquired to demonstrate the differences in cell structure, cell type, cell size, and cell morphology in longitudinal sections of flower buds. The cross section of the same mango peduncle 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 callus induction medium: A is the initial stage of induction, when the radicle and callus appear simultaneously; B and C are the callus in the middle stage of induction; D is the callus in the final stage of induction, when some callus begins to differentiate into seedlings. Detailed Implementation

[0076] Various exemplary embodiments 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, features, and embodiments of the present invention.

[0077] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as any smaller range between any other stated value or intermediate value within said range, are also included in this invention, and the upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0078] Unless otherwise stated, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described in this invention, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0079] Various improvements and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention. This will be obvious to those skilled in the art. The specification and embodiments of this invention are merely exemplary.

[0080] The terms “comprising,” “including,” “having,” “containing,” etc., used in this invention are all open-ended terms, meaning that they include but are not limited to.

[0081] Unless otherwise specified, all raw materials used in this invention are conventional commercially available products. Unless otherwise specified, all methods used in this invention are conventional methods in the field. All substances used in this invention are of conventional quality and, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0082] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. Example 1

[0083] Take 3 to 4 sterile tobacco seedlings that are about 8 weeks old, remove the main veins and leaf tip tissue, and cut them into uniform fine shreds of 0.5mm-1.5mm using scissors in a clean bench.

[0084] Place the tobacco filaments into 10 ml of enzymatic hydrolysate containing 0.1 g / L of cellulase, mix gently to ensure full contact between the tobacco filaments and the hydrolysate, place on a horizontal constant temperature shaker, 45 rpm / min, at 26℃, and enzymatically hydrolyze in the dark for 150 min-180 min.

[0085] Take a 50ml centrifuge tube, place a 200-mesh filter at the opening, slowly pour the enzymatically hydrolyzed mesophyll solution into the central area of ​​the filter for filtration, remove the filter, and centrifuge the cell solution collected through the filter at 100rpm / min for 7min. Discard the supernatant after centrifugation.

[0086] The precipitate was resuspended in 1X PBS buffer, and the purity of single cells was determined according to the national standard GB / T 39729-2020 "General requirements for cell purity determination - flow cytometry". The cell purity obtained was 65.9% and the cell clumping rate was 32.5%.

[0087] The viability of the prepared single cells was determined to be 78.9% using a flow cytometry assay kit and Molecular Probe LIVE / DEAD Fixable activity dye. Example 2

[0088] Take 3 to 4 sterile tobacco seedlings that are about 8 weeks old, remove the main veins and leaf tip tissue, and cut them into uniform fine shreds of 0.5mm-1.5mm using scissors in a clean bench.

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

[0090] Cleaned tobacco leaf fibers were inoculated onto MS medium containing 6-benzylaminopurine (0.0025 g / L), naphthaleneacetic acid (0.005 g / L), agar powder (5.0 g / L), and sucrose (CY3 fluorescent label) (30.0 g / L) at pH 6.5 in the dark for 48 hours.

[0091] Callus tissue induced and cultured in the dark for 48 hours was placed in 20.0 ml of enzymatic hydrolysate with a cellulase content of 0.1 g / L, the volume of which was 3 times that of the callus tissue. The culture was carried out at 26.0℃, 50.0 rpm / min, in the dark, with shaking for 30 min to obtain a callus cell suspension.

[0092] The obtained callus cell suspension was used to separate, sort, and count fluorescently labeled cells using a FACS flow cytometer.

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

[0094] The purity of single cells was determined according to the national standard GB / T 39729-2020 "General requirements for cell purity determination - flow cytometry". The number of effective cells obtained was 10,515, the cell purity was 98.5%, and the cell clumping rate was 1.5%.

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

[0096] Beneficial effects: Example 1 used a traditional method for preparing single-cell plants from leaves, resulting in single cells with a cell purity of 65.9%, a cell aggregation rate of 32.5%, and a viability of 78.9%. Example 2, using the technical solution of this invention, produced single cells with a cell purity as high as 98.5%, a cell aggregation rate of only 1.5%, and a single-cell viability as high as 99.95%. Cell purity and single-cell viability were increased by 32.6% and 21.05% respectively compared to the traditional method, while the cell aggregation rate was reduced by 31.0%. Example 3

[0097] Brown rice obtained by removing the husks from mature rice seeds was placed in 50ml centrifuge tubes, sterilized with 75% ethanol for 10 minutes, and then soaked in 30.0%-50.0% sodium hypochlorite solution (active chlorine content of 8.25%) for 20 minutes, with the centrifuge tubes gently shaken during the process. After rinsing with sterile water 4 times in a clean bench, sterilized explants were obtained.

[0098] Explants were inoculated onto a solid culture medium containing N6+ 6-benzylaminopurine (0.005 g / L) + naphthaleneacetic acid (0.005 g / L) + agar powder (5.0 g / L) + glucose (FAM fluorescent labeling) (50.0 g / L), pH=6.0, and induced in the dark at 30°C for 72 hours. Figure 7 ).

[0099] Darkness-induced callus tissue for 72 hours ( Figure 7The callus cells were placed in 15.0 ml of enzyme hydrolysate with a concentration of 0.01 g / L. The volume of the enzyme hydrolysate was 1.5 times that of the callus tissue. The cells were cultured at 28.0 ℃, 60.0 rpm / min, in the dark, with shaking for 60 min to obtain a callus cell suspension.

[0100] The obtained callus cell suspension was used to separate, sort, and count fluorescently labeled cells using a FACS flow cytometer.

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

[0102] The purity of single cells was determined according to the national standard GB / T 39729-2020 "General requirements for cell purity determination - flow cytometry". The number of effective cells obtained was 11,189, the cell purity was 99.5%, and the cell clumping rate was 0.58%.

[0103] The viability of the prepared single cells was measured to be up to 100.0 using a flow cytometry assay kit and Molecular Probe LIVE / DEAD Fixable activity dye.

[0104] Beneficial effects: Enzymatic hydrolysis mainly utilizes a mixed enzymatic hydrolysate of cellulase, pectinase, and dissociation enzyme to treat young plant tissues. However, it has significant limitations for older parts of plants, such as flowers, stems, fruits, and seeds, where it is difficult to obtain intact and viable protoplasts. Example 3, using mature rice seeds as explants, successfully obtained single cells from mature rice seeds with cell purity and viability as high as 99.5% and 100%, respectively, through callus induction. This overcomes the limitation of traditional enzymatic hydrolysis methods in achieving single-cell separation of plant seeds, especially single cells of the embryo and endosperm within mature plant seeds. Example 4

[0105] Remove the outer scales of the peony buds, place the buds in 75% ethanol for 5 minutes on a clean bench, then soak them in 30.0%-50.0% sodium hypochlorite solution (active chlorine content of 8.25%) for 15 minutes, and rinse them three times with sterile water in a clean bench to obtain sterilized peony bud explants.

[0106] Disinfected peony bud explants were inoculated onto a solid medium containing 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 fluorescent labeling), and induced at pH 6.2 in the dark for 48 hours.

[0107] Callus tissue induced in the dark for 48 hours was placed in 10.0 ml of enzymatic hydrolysate with a pectinase content of 0.02 g / L. The volume of the enzymatic hydrolysate was 2.0 times that of the callus tissue. The cells were cultured at 28.0℃, 50.0 rpm / min, in the dark, with shaking for 40 min to obtain a callus cell suspension.

[0108] The obtained callus cell suspension was used to separate, sort, and count fluorescently labeled cells using a FACS flow cytometer.

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

[0110] The purity of single cells was determined according to the national standard GB / T 39729-2020 "General requirements for determination of cell purity - flow cytometry". The number of effective cells obtained was 9892, the cell purity was 99.9%, and the cell clumping rate was 0.10%.

[0111] Using a flow cytometry assay kit and eBioscience FVD eFluor activity dye, the viability of the prepared single cells was found to be as high as 99.98%. Example 5

[0112] Take leaves of *Lysimachia christinae*, remove the main vein and leaf tip tissue, and cut them into uniform leaf segments of 1.5mm-5.0mm. In a clean bench, make two horizontal cuts perpendicular to the leaf veins, ensuring the leaf is not cut open. Place the leaves in 75% ethanol for 10 minutes on the clean bench, then soak them in a 30.0%-50.0% sodium hypochlorite solution (active chlorine content 8.25%) for 15 minutes. Rinse three times with sterile water in the clean bench before use. Take brown rice obtained by hulling mature rice seeds, place it in 50ml centrifuge tubes, disinfect with 75% ethanol for 10 minutes, then soak in a 30.0%-50.0% sodium hypochlorite solution (active chlorine content 8.25%) for 20 minutes, gently shaking the centrifuge tubes during this time. Rinse four times with sterile water in the clean bench to obtain sterilized explants. Sterilized rice seed explants and *Leymus chinensis* leaves were inoculated together on callus culture medium with the abaxial surfaces facing upwards. The induction medium consisted of 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 fluorescent labeling), pH=6.0, and cultured at 28℃ in the dark for 60 hours.

[0113] Callus tissue induced in the dark for 60 hours was placed in 12.0 ml of enzymatic hydrolysate with a pectinase content of 0.02 g / L. The volume of the enzymatic hydrolysate was 1.8 times that of the callus tissue. The cells were cultured at 30.0℃, 60.0 rpm / min, in the dark, with shaking for 60 min to obtain a callus cell suspension.

[0114] The obtained callus cell suspension was used to separate, sort, and count fluorescently labeled cells using a FACS flow cytometer.

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

[0116] The purity of single cells was determined according to the national standard GB / T 39729-2020 "General requirements for cell purity determination - flow cytometry". The number of effective cells obtained was 11,269, the cell purity was 99.98%, and the cell clumping rate was 0.5%.

[0117] Using a flow cytometry assay kit with eBioscience FVD eFluor active dye, the viability of the prepared single cells was found to be as high as 100.0.

[0118] This invention employs plant explant callus culture, particularly effective for hard seeds, fruits, and highly fibrous plant roots, stems, and leaves where enzymatic hydrolysis is less efficient. First, explant callus is cultured. By adding a carbon source containing a fluorescent group to the culture medium, callus cell clusters with fluorescent groups, thin cell walls, and uniform cell morphology and size are generated, easily dissociated by a single enzymatic hydrolysate. The cell suspension formed after dissociation by the single enzymatic hydrolysate is then separated and sorted using flow cytometry. The resulting plant leaf cell purity reaches 98.5%, with a cell clustering rate of only 1.5% and a single-cell viability of 99.95%. Cell purity and single-cell viability are increased by 32.6% and 21.05% respectively compared to traditional methods, while the cell clustering rate is reduced by 31.0%. Using mature rice seeds as explants, and through callus induction, seed single cells with cell purity and viability as high as 99.5% and 100%, respectively, were successfully obtained, overcoming the limitations of traditional enzymatic hydrolysis methods in preparing seed single cells, especially seed embryo and endosperm single cells. Furthermore, by co-inducing with *Lysimachia christinae* leaves and mature rice seeds, the prepared cells achieved a purity of 99.98%, a cell aggregation rate of only 0.5%, and a single-cell viability as high as 100.0%, overcoming the limitations of single-cell preparation caused by inter-species differences.

Claims

1. A method for preparing plant single cells, characterized in that, The procedure consists of the following steps: S1. Take leaves of *Ariocarpus santalinus*, remove the main vein and leaf tip tissue, and cut them into uniform leaf segments of 1.5mm-5.0mm. Make two horizontal cuts perpendicular to the leaf veins in a clean bench, ensuring the leaf is not severed. Place the leaves in 75% ethanol for 10 minutes, then soak them in 30.0%-50.0% sodium hypochlorite solution for 15 minutes. Rinse three times with sterile water in a clean bench before use. S2. Place mature rice seeds (hulled) into 50ml centrifuge tubes, disinfect with 75% ethanol for 10 minutes, then soak in 30.0%-50.0% sodium hypochlorite solution for 20 minutes, gently shaking the centrifuge tubes during this time. Rinse four times with sterile water in a clean bench to obtain sterilized explants. S3. Inoculate the sterilized rice seed explants and *Ariocarpus santalinus* leaves together, with the back facing upwards, onto callus culture medium. The induction medium is 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 fluorescent labeling, pH=6.0, co-inducing culture at 28℃ in the dark for 60 hours; S4, the callus tissue co-inducing in the dark for 60 hours was placed in 12.0 ml of enzymatic hydrolysate with a pectinase content of 0.02 g / L, the enzymatic hydrolysate being 1.8 times the volume of the callus tissue, and cultured at 30℃, 60.0 rpm / min in the dark with shaking for 60 min to obtain a callus cell suspension; S5, the obtained callus cell suspension was used to separate, sort and count fluorescently labeled cells using a FACS flow cytometer. The parameters of the FACS flow cytometer were set as follows: nozzle diameter 60 µm, sheath fluid pressure 20 psi, excitation wavelength 589 nm, emission wavelength 610 nm; S6. The purity of single cells was determined according to the national standard GB / T 39729-2020 "General requirements for cell purity determination - flow cytometry". The number of effective cells obtained was 11269, the cell purity was 99.98%, and the cell clumping rate was 0.5%. S7. The cell viability was detected by flow cytometry using a cell viability assay kit and eBioscience FVD eFluor active dye. The viability of the prepared single cells was as high as 100.0%.

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