Method for separating plant stem cells from plant leaves and related cell line obtained by using method

Abstract

The present invention relates to a method for isolating a stem cell line derived from a tissue of a primary meristem among leaves of a plant, comprising the steps of: (a) sterilizing at least a portion of a leaf containing a central vein or other veins; (b) incising a central vein or other veins of the leaf to expose primary meristem cells from internal vascular tissue of the central vein or other veins; and (c) culturing cells from said exposed cells, which avoids the difficulty of surface sterilization when using other types of explants, and to stem cell lines obtained using said method.

Description

SUMMARY OF THE INVENTION

[0002] The present invention relates to methods for isolating stem cells from plant leaves and establishing stem cell lines, as well as uses of the isolated stem cells and stem cell lines thereof. Background Art

[0003] Plants are nature's chemists, capable of producing a large number of secondary metabolites (natural products), which are the cornerstones of many medicines, cosmetics, foods and agricultural products. The source plant species used to produce extracts containing natural products or to isolate specific purified natural products (NPs) are usually slow-growing, limited in population, and the concentration of the target molecule varies greatly between plants, and its concentration in vivo is usually extremely low. This situation leads to an unmet demand for NPs and extracts, increasing the use of intensive farmland for growing plants producing NPs for industrial applications. Therefore, an alternative plant NP production system is needed.

[0004] One potential approach is to use in vitro cultured plant cells isolated from target NP-producing plant species. The plant cell culture process involves inducing callus formation from plant tissues or organs (explants) grown on synthetic solid media in a culture dish. Callus consists of a large number of dedifferentiated plant cells (DDCs). Subsequently, callus fragments are inoculated into liquid media in flasks to generate DDC suspension cultures.

[0005] Plant cell culture offers an attractive alternative bioNP manufacturing pathway to whole plants. It is sustainable, efficient, agnostic to environmental conditions, standardized, provides a robust product supply, is free of zoonotic viruses, can be optimized to produce commercially relevant levels of target metabolites, and complies with Good Manufacturing Practice (GMP) regulatory guidelines.

[0006] However, this traditional method of culturing and using DDC for NP production results in genetic and epigenetic damage to plant cells. As a result, DDC exhibits many limitations, including slow growth, significant cell aggregation, increased stress sensitivity, unstable viability, and limited NP yield, limiting the widespread commercial use of this approach outside of high-value applications.

[0007] To circumvent the problems associated with typical DDC culture, Lee et al. (Cultured cambial meristematic cells as a source of plant NPs. Nature Biotechnology (2010) 28(11), ) isolated cultured cambium meristem cells (CMC). The cell suspension culture is derived from cambium tissue, which is derived from secondary meristems in plant stems and branches, and the cell suspension culture shows potential as a platform for producing plant NPs. These cells derived from the cambium showed superior growth, reduced cell aggregation, increased stress resistance and increased NP production in long-term culture. It is worth noting that the cambium is a secondary meristem formed in the stems and roots after the differentiation of primary plant tissues. The cambium is responsible for increasing the diameter of the stems and roots and forming woody tissue (Fischer et al., 2019Annu.Rev.Plant Biol.70:293–319).

[0008] WO2007052876A1 discloses a method for isolating a cell line from a plant, wherein the isolated plant cell line is derived from a cambium obtained from a plant branch or stem. The method comprises collecting tissue containing a plant cambium, culturing the tissue, thereby inducing a layer of meristem proliferation from the cambium without dedifferentiation into callus.

[0009] However, surface sterilization of explants from plant stems, branches, roots or storage roots is difficult to carry out successfully, especially for plant samples collected from natural environments, because these ecosystems have rich microbial diversity. For example, due to the irregular surface structure of plant organs, explant samples obtained from these plant organs involve high levels of endophytes and endosymbionts (usually bacteria or fungi), which exist in plants for at least part of the plant life cycle without causing obvious diseases (Hardoim et al. 2015. The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes. Microbiol & Mol Biol Revs. 79 (3): 293-320). Therefore, the sterilization process itself needs to be systematically adjusted to generate sterile cell cultures from different plant species, which is both cumbersome and time-consuming.

[0010] Other explants that can be used for methods of isolating cells are, for example, leaves of plants. EP3942017A1 relates to isolating cell lines from plants of the genus Melissa by inducing callus formation from plant tissues including leaves or whole leaves on a solid plant growth medium and subsequently inoculating in a liquid plant growth medium to generate a relevant plant cell suspension culture. However, the Melissa DDC obtained exhibits many limitations, including: slow growth rate, significant cell aggregation, increased stress sensitivity, unstable vigor, and low NP production. Summary of the invention

[0011] The object of the present invention is to provide a method for isolating and generating plant cell lines that have not dedifferentiated into callus tissue, wherein the method is simple, rapid, more easily standardized and circumvents the difficulties of surface sterilization when using plant secondary meristems derived from stems, branches or storage roots as explants.

[0012] In one aspect, the present invention relates to a method for isolating a stem cell line derived from primary meristem tissue in a plant leaf, comprising the following steps:

[0013] (a) sterilizing at least a portion of a leaf containing a central vein or other veins;

[0014] (b) cutting the central vein or other veins of the leaf to expose primary meristem cells from the inner vascular tissue of the central vein or other veins; and

[0015] (c) culturing cells derived from the exposed cells in a cell induction medium.

[0016] That is, in one aspect of the present invention, a method for isolating or obtaining at least one plant stem cell is provided, the method comprising the following steps:

[0017] (a) sterilizing at least a portion of a leaf containing a central vein or other leaf veins;

[0018] (b) dissecting the central vein or other veins of the leaf to expose primary meristem cells from the inner vascular tissue of the central vein or other veins; and

[0019] (c) obtaining at least one stem cell from the inner vascular tissue of the central vein or other veins.

[0020] In one embodiment, the stem cells are primary phloem meristem cells.

[0021] In one embodiment, the leaves are obtained from plants established or grown in a vertical farm.

[0022] The method for isolating a stem cell line derived from a tissue of a primary meristem in a leaf of a plant according to the present invention allows culturing and isolating cells that do not dedifferentiate into callus tissue or cells that do not dedifferentiate from callus tissue.

[0023] In the method according to the invention, cells are cultured from leaf explants. Preferably, the leaves (such as young leaves) are collected from plant species that naturally produce high-value natural products.

[0024] Thus, the method may further comprise culturing the at least one stem cell to obtain a stem cell line.

[0025] In one embodiment, the cell culture does not include dedifferentiated cells and / or protoplasts, or does not substantially include dedifferentiated cells and / or protoplasts. In one embodiment, the method does not include forming a callus obtained from dedifferentiated cells or forming a callus substantially comprising dedifferentiated cells - and subsequently obtaining stem cells from such callus.

[0026] The culturing step may also include:

[0027] (a) culturing primary meristem cells from the vascular tissue within the central vein or other veins in suspension; and

[0028] (b) optionally selecting a high performance cell line that preferentially responds to a plant immunity elicitor.

[0029] Initiation of suspension culture may include incubating the plate containing the isolated primary meristem cells at 16°C to 30°C in a cycle of 24h / 0h light / dark to 0h / 24h light / dark.

[0030] In another aspect of the present invention, there is provided a plant stem cell or a plant stem cell line obtained or obtainable by the method of the present invention.

[0031] In another aspect of the present invention, there is provided a plant stem cell product or a plant extract obtained or obtainable from the plant stem cells of the present invention.

[0032] In another aspect of the present invention, a plant or part thereof obtained or obtainable from the plant stem cells of the present invention is provided. Plant products and / or plant extracts obtained or obtainable from the plant are also included in the scope of the present invention.

[0033] The stem cell line derived from the primary meristem in the leaves of a plant preferably has the following characteristics:

[0034] (a) the stem cell line is formed from undifferentiated cells, preferably substantially genetically homogeneous cells; and

[0035] (b) the cells of said line are morphologically characterized by the presence of multiple amyloplasts; and

[0036] (c) the stem cell line is a fast-growing cell line; and / or

[0037] (d) optionally, having high phytochemical accumulation in response to induction.

[0038] In another aspect of the present invention, there is provided a method for producing a plant natural product, the method comprising isolating or obtaining at least one plant stem cell or cell as described herein, and isolating / collecting the product.

[0039] In another aspect of the present invention, there is provided a method for producing a stem cell extract, the method comprising isolating or obtaining at least one plant stem cell or cell as described herein, and isolating / obtaining an extract.

[0040] In one embodiment, the method may include:

[0041] (a) isolating at least one stem cell derived from a tissue of a primary meristem in a leaf of a plant; and

[0042] (b) establishing a stem cell line comprising said at least one isolated stem cell, preferably, wherein said stem cell line is substantially genetically homogeneous;

[0043] (c) optionally selecting a high performance cell line; and / or

[0044] (d) optionally inducing the production or secretion of a plant natural product or extract; and

[0045] (e) collecting the plant natural product or extract.

[0046] The high-performance cell line is characterized by a high-performance indicator or a high-performance trait, which is selected from at least one of the following: cell size, proliferation, product yield and / or developmental capacity, wherein preferably, the improvement is relative to another cell / other cells in the cell line.

[0047] Inducing the production or secretion may comprise treating the suspension culture with a plant inducer.

[0048] Plant natural compounds have a wide range of activities against insects, fungi, bacteria and viruses. Therefore, in one embodiment, the plant product can be an insecticide, a fungicide, an antibacterial agent or an antiviral agent. Alternatively, the product can be a therapeutic agent or a nutraceutical. In one embodiment, the plant product can be selected from flavonoids, alkaloids, polyphenols, polysaccharides, quinones and saponins.

[0049] In another aspect of the present invention, a composition is provided, comprising at least one plant stem cell obtained by the method of the present invention, or at least one plant stem cell extract and / or plant product obtained or obtainable from the stem cell or stem cell line of the present invention. The composition may be selected from a cosmetic composition, an agricultural composition, a pharmaceutical composition, a food composition, a beverage composition or a human / animal health care composition.

[0050] In another aspect, there is also provided a method for producing a plant or a plant part thereof and / or a crop breeding method, the method comprising:

[0051] (a) the method according to claim 1, isolating at least one stem cell derived from a tissue of a primary meristem in a leaf of a plant;

[0052] (b) establishing a stem cell line comprising said at least one isolated stem cell, preferably, wherein said stem cell line is substantially genetically homogeneous;

[0053] (c) optionally selecting a high-performance cell line; and

[0054] (d) growing a new plant or plant part thereof from at least one stem cell in the stem cell line and / or at least one stem cell in the high performance cell line.

[0055] The method may further comprise growing the plant or plant part using vertical farming practices.

[0056] The plant according to all aspects of the invention as described herein may be a dicot, a monocot or a gymnosperm. In one embodiment, the plant is a vascular plant. DETAILED DESCRIPTION

[0057] The present invention will now be further described. In the following paragraphs, different aspects of the present invention will be defined in more detail. Unless there is a clear indication to the contrary, each aspect defined can be combined with any other one or more aspects. Specifically, any feature that is expressed as preferred or advantageous can be combined with any other one or more features that are expressed as preferred or advantageous.

[0058] Unless otherwise indicated, the practice of the present invention will employ conventional techniques within the skill of the art such as botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and DNA recombination technology, bioinformatics, etc. These techniques are fully explained in the literature.

[0059] The plant according to all aspects of the invention described herein may be a dicot, a monocot or a gymnosperm. In one embodiment, the plant is a vascular plant.

[0060] In one embodiment, the plant is a crop. By crop is meant any plant grown on a commercial scale for human or animal consumption or use. In another embodiment, the plant is Arabidopsis thaliana.

[0061] In one embodiment, the plant can be selected from asparagus, grasses, palm, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, cilantro, sunflower, peanut, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe, yew (Taxus), eggplant, pineapple, sumac, chickpea, rosemary, lychee, licorice (Glycyrrhiza glabra), spinach, soybean, brassicas, carrot, corn, rice, Thuja, juniper, pine, garlic, cucumber, chili, pepper, lettuce, peach, watermelon, grape, apple, onion, orange, banana, pea, mango, citrus, tea, sugarcane, cotton, barley, sorghum, wheat, rice, quassia and magnolia, and members of the genus Quillija and Cupressaceae.

[0062] In one embodiment, the plant may be selected from the group consisting of Brassicas, legumes, cereals, citrus, root vegetables, tuber and root crops, fruits (including berries and soft fruits), and fruit, nut and seed bearing trees.

[0063] For the purposes of the present invention, the term "plant stem cell" refers to a plant cell that is capable of self-renewal and differentiation into specialized cells. Thus, through mitosis, plant stem cells are able to maintain a stem cell population and divide to generate precursor cells that subsequently differentiate into tissues and organs. This asymmetric division can occur at the cellular or population level.

[0064] The terms "plant stem cells" and "stem cells" are used interchangeably hereinafter.

[0065] "Plant stem cells" may also refer to plant stem cells that have not been substantially differentiated, are taken from meristematic tissue, and are maintained as a substantially homogenous population under culture conditions. Therefore, the stem cells described in the present invention are different from plant cells obtained from differentiated somatic tissues and are stimulated or dedifferentiated to a more potent state.

[0066] In one embodiment, the plant stem cells are totipotent or pluripotent. Totipotency refers to the ability of a cell to generate all cell types and establish an entire organism, while pluripotency refers to the ability of a cell to generate most but not all cell types.

[0067] In one embodiment, the plant stem cells are derived from primary meristem cells and / or secondary meristem cells of a plant.

[0068] "Meristematic tissue" refers to a tissue that includes a population of plant stem cells. Meristematic cells are organized into meristems, which are regions of meristem that contribute to plant growth. "Primary meristems" are established during embryo formation and contribute to the initial growth, i.e., length or height, of a plant. Examples of primary meristems include the shoot apical meristem (SAM) and the root apical meristem (RAM).

[0069] Meristems that originate from primary meristems and form after germination are called "secondary meristems," such as axillary meristems. Secondary meristems help increase the width of plant stems and roots, thereby forming thicker, stronger tissues that can support plant growth. Meristem cells together constitute the plant stem cell niche.

[0070] In one embodiment, plant stem cells are derived from primary meristem cells, preferably from primary meristem cells in and / or around plant vasculature, more preferably, the vasculature is a leaf vasculature. These stem cells are referred to herein as vascular stem cells or VSCs.

[0071] In a preferred embodiment, plant stem cells are derived from or obtained from primary meristem cells around or in the central vein of a leaf. The central vein of a leaf is equivalent to the main vein or midrib. Therefore, stem cells are obtained by exposing the midrib and / or central vein and obtaining stem cells.

[0072] Veins can be defined as the supporting structure of the entire leaf, providing support and structure to the leaf. Veins include xylem, a conductive tissue responsible for transporting water and minerals throughout the leaf and the wider plant. Veins also include phloem, a tissue that transports glucose produced by photosynthesis. Therefore, veins can include xylem and / or phloem in addition to meristem cells.

[0073] Other veins are veins extending from the central vein (also called the midrib). From the veins come the veinlets. Therefore, "other veins" refers to veins or veinlets other than the central vein.

[0074] Phloem is formed by protophloem, which matures into metaphloem and finally becomes phloem. Cells comprising protophloem are called protophloem cells. Early phloem is formed by stem cells in meristem, so cells of early protophloem may include meristem stem cells or cells having characteristics of both protophloem and meristem cells. Such cells may be called protophloem meristem cells. Protophloem meristem cells may be defined by the presence of one or more marker cells. Suitable marker cells include APL (altered phloem development) (Bonke et al 2003), SWEET11 (Gebauer et al 2017) and BZIP9 (basic leucine zipper 9).

[0075] In one embodiment, the leaves are selected from plants grown or grown in a vertical farm. Vertical farms or vertical farming refer to cultivation practices that allow plant varieties to be cultivated on multiple overlapping levels with the goal of maximizing the number of plants per cubic meter. Vertical farms are structures that produce the highest quality plant varieties because the cultivation process takes place in a closed environment where all environmental parameters (temperature, humidity, CO2, light and nutrients) are controlled. Therefore, selecting leaves from plants grown or grown in vertical farms allows highly controlled and selected cell material to be obtained for the isolation of stem cells and / or stem cell lines. Similarly, the use of vertical farming technology to extract plants or plant parts from stem cells can obtain samples of the highest quality.

[0076] Cutting open plant tissue to expose plant stem cells

[0077] Before cutting open plant tissue, it needs to be sterilized. The purpose of the sterilization step is to remove microorganisms on the surface of the plant so that it can be used to isolate target cells.

[0078] Suitable sterilizing agents are well known in the art, such as sodium hypochlorite (bleach), calcium hypochlorite, hydrogen peroxide, ethanol, etc. Preferably, sodium hypochlorite is used at a concentration of 0.5% to 1.0% for 30 to 40 minutes. Calcium hypochlorite powder should be dissolved in water before use and then filtered. Usually, the concentration used is 3.25%. The concentration of hydrogen peroxide used is 3%.

[0079] Ethanol can be used for sterilization, for example at a concentration of 70% to 95%.

[0080] It is necessary to remove microorganisms from the explants used because these microbial cells grow much faster than plant cells and will quickly contaminate the cell isolation method. At the same time, useful sterilizing agents such as ethanol or bleach treatments can be toxic to plant cells at high concentrations or long contact times. Therefore, it is desirable to minimize the effects of these sterilizing agents by maintaining the shortest incubation time for these compounds and, preferably, the lowest effective concentration. It is worth noting that the surface of the leaves is smoother than that of the stems, which facilitates a milder and more gentle sterilization procedure and increases the possibility of isolating a viable, healthy cell population. Therefore, when using leaves as explants, they can be kept in the solvent for as short as 1 to 15 minutes while still removing most of the harmful microorganisms and ensuring that the explants are not damaged.

[0081] Thus, in one embodiment, there is a method of isolating plant stem cells from a leaf, the method comprising sterilizing at least a portion of the leaf containing the central vein or other veins.

[0082] In another embodiment, there is a method of isolating or establishing a plant stem cell line, the method comprising sterilizing at least a portion of a leaf containing a central vein or other veins.

[0083] Similarly, in another embodiment, there is a stem cell line obtained by a method comprising sterilizing at least a portion of a leaf containing a central vein or other veins.

[0084] In a preferred embodiment, the leaves are sterilized with a sterilizing agent, preferably, the sterilizing agent is selected from sodium hypochlorite (bleach), calcium hypochlorite, hydrogen peroxide and / or ethanol.

[0085] After sterilization, carefully cut (cut open) the central vein or other veins of the leaf to expose the primary meristem cells that form the inner vascular tissue. In a preferred embodiment, the plant stem cells are derived from the primary meristem cells around the central vein of the leaf. The central vein of the leaf is equivalent to the main vein or midrib.

[0086] In another embodiment, the plant stem cells may be derived from the lateral veins or secondary veins of a leaf.

[0087] There are practical advantages to isolating cells from the central vein: the central vein is usually thicker than the lateral and secondary veins, making it easier to manipulate, allowing more cells to be exposed. Preferably, the cut is made along the length of the central vein or other veins, so in the case of the central vein, the cut direction is from the tip to the base of the leaf or from the base to the tip of the leaf. In a preferred embodiment, the leaf is cut in half along and through the central vein.

[0088] By quantifying the presence or absence of morphological, metabolic and / or genetic markers in the cell population, the nature of the cells or cell populations isolated from the meristem cells can be confirmed as plant stem cells. Stem cell markers and characteristics are well known to those skilled in the art and described in the literature (see Miyashima et al. 2013), but non-limiting examples are included herein. Morphological markers include small size, round shape, large nuclei, sparse cytoplasm and prominent nucleoli. These characteristics can be determined by mass cytometry as described herein. Genetic markers associated with plant stem cells include, but are not limited to, WUSCHEL-like homeobox 4 (WOX4), CLAVATA3 / ESR-related 41 / 44 (CLE41 / 44), PHLOEM INTERCALATED WITH XYLEM (PXY) / TDIF RECEPTOR (TDR), and cytokinin response factor 1 (CYTOKININ RESPONSE 1) (CRE1) / WOODEN LEG (WOL) / Arabidopsis histidine kinase (AHK). The expression of stem cell markers and other markers of interest can be quantified by conventional methods known to those skilled in the art. For example, reverse transcriptase-polymerase chain reaction (RT-PCR) can reverse transcribe mRNA isolated from a single cell into cDNA, which is then amplified by PCR to quantify gene expression. Then, cells can be identified by adjusting mRNA abundance.

[0089] This careful leaf dissection process will expose meristem cells in the vascular tissue derived from the primary meristem, such as protophloem meristem cells. Subsequently, these cells or explant regions containing meristem cells are collected and subjected to a culture step to induce proliferation in the presence of a suitable culture medium (e.g., a cell induction medium as described herein).

[0090] Cultivate isolated cells and establish cell lines

[0091] For the purposes of the present invention, the term "plant stem cell culture" refers to the in vitro cultivation, maintenance and proliferation of plant stem cells isolated by the methods described herein. Plant stem cell cultures can refer to stem cells in liquid and / or solid phase cultures. Thus, plant stem cell cultures refer to stem cells in a suitable culture medium, such as cell induction medium on a solid platform and / or stem cells in liquid suspension culture. In a preferred embodiment, the plant stem cell culture includes stem cells in liquid suspension culture. In a preferred embodiment, plant stem cells are cultured on solid and / or liquid culture medium.

[0092] The culturing step according to the present invention involves steps performed after the target cells are exposed from the leaves. More specifically, the leaves of the target plant species are sterilized and then the central vein or other veins of the leaves are cut to expose a thin layer of the desired stem cells. For example, the veins are cut to expose the primary protophloem cells. These and other primary meristem cells have self-renewal, undifferentiated properties and show faster growth rates compared to traditional differentiated cells. In the culturing step, the cut leaves are placed on a cell induction medium to promote the proliferation of the meristem cells.

[0093] Cell induction medium refers to a medium that stimulates cell proliferation and induces callus formation. Callus refers to a visible mass of undifferentiated cells. As used herein, callus does not include dedifferentiated cells and is not derived from dedifferentiated cells.

[0094] Cultivation of exposed leaf veins induces the formation of callus, a visible mass of undifferentiated cells. The length of time from explanting cells to the formation of callus varies depending on the source of the cells. DDC forms visible callus about 7 days after explantation. Vascular plant stem cells form callus in less than 7 days, usually 6 days. Therefore, the stem cell population associated with the present invention produces visible callus earlier than DDC. Cells obtained from the central vein or other veins of the leaf will form callus recognizable to the naked eye within 3 to 5 days, while cells obtained from the native phloem will form callus within 6 days of culture. Therefore, the isolated stem cells of the present invention are different from cells obtained from differentiated cells that form callus through dedifferentiation.

[0095] The composition of the cell induction medium is well known to those skilled in the art and can be based on, for example, the papers of Murashige and Skoog (1962) and Gamborg et al. (1968). The specific composition will vary depending on the plant species used. The medium disclosed by Murashige and Skoog is hereinafter referred to as MS medium, and the medium disclosed by Gamborg et al. is hereinafter referred to as B5 medium.

[0096] For example, the cell induction medium includes macronutrients, micronutrients and / or plant hormones. In a preferred embodiment, the cell induction medium contains the components that make up the B5 medium or MS medium and one or more of 2,4-D, kinetin, NAA, 6-BA and other plant growth regulators. In an even more preferred embodiment, the cell induction medium contains at least 0 to 10 mg / L 2,4-D, at least 0.01 to 1 mg / L kinetin, at least 0.01 to 1 mg / L NAA and at least 0 to 10 mg / L 6-BA.

[0097] In another embodiment of the present invention, a specific medium composition for isolating meristem cells from the central vein or other veins of a leaf is optimized to promote the proliferation of a target cell type of a specific plant species. This is achieved by testing different concentrations and ratios of essential components, including a suitable base, carbon and nitrogen sources, vitamins, medium optimization additives, and plant hormones (different ratios of auxin: cytokinin).

[0098] In more detail, in order to promote proliferation, the explants are placed on a cell induction medium and cultured under specific temperature and light / dark conditions. It should be noted that the proliferation of cells from the central vein or other veins is visible to the naked eye within 3 to 5 days of culture, while in contrast, the growth of dedifferentiated cells from other regions of the explant forms callus visible to the naked eye only after 7 days. In one embodiment of the present invention, a faster growing primary phloem cell group can be identified within 6 days of culture. Subsequently, the cell group is excised from the explant and transferred to a new petri dish containing a cell induction medium.

[0099] Next, cells are isolated from the leaves, i.e., the desired stem cells are identified and isolated from the explants. That is, after callus formation, some or all of the stem cells can be excised from the explants and transferred to a petri dish containing solid and / or liquid cell induction medium.

[0100] Therefore, in one embodiment, a method for isolating one or more plant stem cells or establishing a stem cell line is provided, the method comprising culturing at least one stem cell from the central vein or other veins. In one embodiment, culturing comprises culturing on a solid and / or liquid cell induction medium.

[0101] Stem cells can be cultured on a cell induction medium under specific temperature and light / dark conditions. For example, a 24h / 0h light / dark to 0h / 24h light / dark cycle can be used, more preferably, an 18h / 6h light / dark cycle. At the same time, a temperature of 16°C to 30°C can be used. Preferably, the cells are cultured at a temperature of 25±1°C.

[0102] The above embodiments are also applicable to other methods described herein, such as stem cell lines obtainable by a method, methods for producing plant natural products or extracts, etc.

[0103] Further, the isolated stem cells are cultured on different petri dishes with cell line induction medium to initiate suspension culture. Optionally, a high-performance cell line that responds to a plant immunity inducer is selected.

[0104] Subsequently, the cells can be inoculated into a glass flask containing a suspension start medium (SIM) and cultured in a liquid to establish a plant stem cell suspension culture. Preferably, the glass flask and the suspension start medium are sterile. The suspension start medium is also referred to as a plant suspension medium. The flask can be incubated at a stirring rate of 110-1200RPM under appropriate light / dark conditions at 21°C to 25°C. The growth of the cultured cells can be continuously monitored using an inverted stereoscope and measured after 14 days of incubation.

[0105] Further, suspension culture is initiated. The isolated stem cell mass is cultured on a solid cell induction medium under specific temperature and light / dark conditions. For example, a 24h / 0h light / dark to 0h / 24h light / dark cycle can be used, more preferably, an 18h / 6h light / dark cycle. At the same time, a temperature of 16°C to 30°C can be used. Preferably, the cells are cultured at a temperature of 25±1°C.

[0106] Individual cell populations can be used to inoculate sterile glass flasks containing suspension start medium (SIM). Flasks can be incubated at 21°C to 25°C under appropriate light / dark conditions with a stirring rate of 110-130 RPM. The growth of the cultured cells can be continuously monitored using an inverted stereoscope and measured after 14 days of incubation.

[0107] The measurement of cell growth can be calculated, for example, by transferring 25 ml of cell suspension to a 50 ml sterile conical flask and allowing the cells to settle vertically for 15 minutes. Record the cell set volume (Cell Set Volume, CSV15), and it is expected that the cell volume will increase at least 10 times after two weeks of incubation. After the growth assessment is completed, the cells are subcultured in a fresh SIM culture medium with a CSV15:SIM ratio of 1:10 (for example, 5 ml CSV15 is subcultured in a 250 ml sterile glass flask to a final volume of 50 ml culture medium). The subculture process is repeated every 14 days until the subculture volume reaches 100 ml, and a constant growth rate in the subculture every 14 days is observed. At this stage, the suspension culture established comprises a homogeneous meristem cell population.

[0108] In one embodiment, cell growth is measured every 14 days of incubation.

[0109] In one embodiment, measuring cell growth comprises inoculating a known volume of suspension priming medium (SIM) with a stem cell suspension of known density / volume at one-fifth of the volume, and measuring the density occupied by stem cells after a 14-day incubation period. Thus, cell growth can be defined as an increase in cell density, such as an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% or more over a 14-day culture period.

[0110] In another embodiment, measuring cell growth comprises measuring cell sedimentation volume at day 1 and day 14, wherein measuring cell sedimentation volume comprises transferring a known volume of suspension initiation medium (SIM) with at least one fifth of the volume of a stem cell suspension of known density / volume, allowing the cells to sediment for 15 minutes, and measuring the volume of the mixture (cell sedimentation volume, CSV). Thus, cell growth can be defined as an increase in cell CSV, such as an increase of 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000% or more over a 14 day culture period.

[0111] In another embodiment, measuring cell growth comprises measuring cell number on day 1 and day 14. In one embodiment, cell growth is defined as an increase in the total number of cells, such as at least a 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, or 800% increase in the total number of cells over a 14 day culture period.

[0112] The cells contained in the plant stem cell suspension culture flask established by the method described herein include stem cell lines.

[0113] Thus, in one aspect of the present invention, there is provided a culture medium optimized for maintaining and / or proliferating isolated stem cells and / or stem cell line populations.

[0114] In one embodiment, the culture medium is optimized for maintaining a substantially homogeneous population of plant stem cells under culture conditions.

[0115] In one embodiment, the culture medium is a stem cell suspension medium comprising the composition of Table 2 or Table 4.

[0116] In another or additional embodiment, the culture medium is a co-cultivation medium comprising the above plant stem cell suspension medium supplemented with 50 μM acetosyringone.

[0117] In other aspects of the present invention, a stem cell line established by the above stem cell culture method is provided.

[0118] The isolated stem cell populations of the present invention have a high degree of genetic stability and homogeneity, and can provide high-quality stem cell lines that are free or substantially free of deleterious genetic aberrations induced by the dedifferentiation process required by other methods.

[0119] In addition, optionally, a selection strategy can be applied to enable identification of cell lines that produce high yields of the target natural product. Such a selection strategy enables identification of high-performance cell lines at an early stage in the cell line development process.

[0120] Therefore, in another aspect of the present invention, there is a method for selecting high-performance stem cells or stem cell lines, the method comprising: screening the high-performance trait in at least one or more single plant stem cells; and selecting the one or more single plant stem cells with high-performance traits.

[0121] In one embodiment, cells are screened and / or selected at the single cell level.

[0122] "High performance indicator or trait", "high performance cell or cell line" refers to a cell or cell line with improved plant performance in terms of cell size, proliferation, product yield or developmental capacity, wherein preferably the improvement is relative to another cell / other cells in the cell line. Therefore, the two phrases "high performance trait" and "improved plant performance" can be used interchangeably.

[0123] In a preferred embodiment, the high performance trait is high yield of a target natural product in response to a plant immunity inducer. Thus, in one embodiment, the method further comprises treating the suspension culture with a plant immunity inducer and screening for production of at least one, for example, phytochemical.

[0124] In one embodiment, the plant inducer is a plant immune activator.

[0125] Plant immune response inducers or activators (or simply "plant inducers") are molecules that can be recognized by plant cells and trigger plant defense responses. Inducers can be derived from pathogens and include, for example, oligosaccharides, peptides, glycopeptides, glycolipids, lipophilic inducers, toxins (including coronatine) and polysaccharides (such as chitin and its derivatives). In a preferred embodiment, bacterial flagellin homologues are used as inducers of immune responses. Immune inducers known to those skilled in the art include allergic pathogenic protein (Harpin) (HrpZ), flagellin, cold shock protein, elongation factor (EF-Tu), lipopolysaccharide (LPS), peptidoglycan, oligogalacturonic acid anhydride, lipopeptide, dimethyl sulfide, pseudomonad, tri-N-alkylated benzylamine derivatives (NABD), 2,4-diacetylphloroglucinol (DAPG), oligogalacturonic acid anhydride, extracellular ATP and / or DL-β-aminobutyric acid (BABA), etc. The immune inducer can be synthetic, such as salicylic acid and its analogs, propylenethiazole (PBZ), 1,6-dichloroisonicotinic acid (INA), benzothiadiazole (BTH), thiazolidine (TDL), isotianil, N-cyanomethyl-2-chloroisonicotinamide (NCI), 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA), etc. Abiotic stress inducers, such as osmotic stress inducers, can also be used to stimulate the immune response of stem cells.

[0126] In a preferred embodiment, the plant inducer is selected from salicylic acid, methyl jasmonate, coronatine, chitosan and / or an osmotic stress inducer.

[0127] In a preferred embodiment, the phytochemical belongs to the phytochemical family of phenols, flavonoids and / or terpenes.

[0128] In one embodiment, the yield of the natural product in response to the plant immune response inducer is measured. The assay for measuring natural products is known to the technician and is described in the literature (Altemimi et al., 2017). A colorimetric assay can be used, which uses a reagent that can change color when an analyte is present. Well-known instruments such as high pressure liquid chromatography can be used to purify natural products. Different types of spectroscopic techniques, such as UV-visible spectroscopy, infrared spectroscopy, nuclear magnetic resonance and mass spectrometry, can then be used to identify the purified compound.

[0129] In one embodiment, the abundance of the target gene transcript is measured after exposure to the inducer. In a preferred embodiment, the transcript is selected from plant hormone signal-related genes, such as pathogen-related protein 1 (PATHOGEN-RELATED1) or plant defensin 1.2 (PLANT DEFENSIN1.2). In a preferred embodiment, transcript abundance is measured by RT-qPCR. In another embodiment, the transcript abundance in response to the immune inducer is measured by RNA sequencing. In a preferred embodiment, transcript abundance is measured at the single cell level.

[0130] In another preferred embodiment, the high performance trait is high proliferation potential. In one embodiment, screening for the high performance trait comprises measuring iododeoxyuridine incorporation (IdU, similar to fluorescent BrdU). In another embodiment, screening for the high performance trait comprises measuring cyclin B1, cyclin A, and phosphorylated histone H3 using complementary fluorophore-antibody conjugates. Detection of these markers can be achieved by flow cytometry, or combined with mass spectrometry flow cytometry, as reported in Behbehani et al., 2012.

[0131] In another preferred embodiment, the high performance trait corresponds to cell lifespan. Cell lifespan can be screened using marker-assisted methods. In one embodiment, screening for the high performance trait comprises measuring the expression of telomerase and / or WOX4 and / or CLE41 / 44 and / or CRE1 and / or WOL and / or AHK, wherein high expression is associated with high performance.

[0132] Selection refers to the selection of one or more stem cells or stem cell lines for further subculturing, screening and / or genetic modification of the selected cells or cell lines.

[0133] More specifically, the present invention describes a method for isolating multiple suspension culture lines and selecting them based on their growth rate parameters. Suitable growth parameters and measurement methods have been discussed above. In addition, cell lines with faster growth rates can be selected based on their natural product production capabilities. This multiple selection process can isolate cell populations that grow quickly and produce a high yield of the desired natural product.

[0134] In one embodiment, the selection process consists of treating the suspension culture with plant inducers (such as but not limited to salicylic acid, methyl jasmonate, coronatine and chitosan, osmotic stress inducers), and evaluating the production of specific phytochemical families (including but not limited to phenols, flavonoids or terpenes). In addition, the anti-radical activity (ARA) of the isolated and cultured cells is also a measure to further confirm the biological activity of the synthesized natural product.

[0135] Assays for measuring these natural product families are known to the skilled artisan and described in the literature. However, the selection strategy of the present invention is based on the isolation and cultivation of target meristem cells, including the application of colorimetric tests to evaluate the response to plant elicitors, followed by the selection of cell lines with the highest production of the target natural product.

[0136] In another embodiment of the present invention, the concentration and mixture composition of the plant inducer can be further optimized to increase the yield of the target natural product in the selected high-performance plant stem cell suspension strain. Therefore, after identifying a cell population with a higher yield of the natural product, the cell line can be further screened for a secondary inducer by testing 0.001 μM to 1,000 μM of the inducer. In addition, as part of this selection process, the growth phase of the culture during induction and the duration of the induction treatment are also optimized.

[0137] According to the method described in the present invention, cells can be cultured into callus without dedifferentiation. That is, the method may include cultivation, wherein the cell culture does not include dedifferentiated cells and / or protoplasts, or substantially does not include dedifferentiated cells and / or protoplasts. Methods for producing cell suspension cultures from different tissues and different plant species are known. However, in the above method, different tissues are used as explants as a whole, rather than being selectively dissected to separate veins from leaves. Cell suspension cultures starting from the entire tissue are usually the result of epidermal cells, palisade cells, guard cells, etc. undergoing a dedifferentiation process, thereby recovering to pluripotent cells that can undergo mitotic cell division. One problem with this is that, due to the dedifferentiation process, the dedifferentiated cells produced can accumulate a large amount of harmful genetic and epigenetic modifications. According to the present invention, cells around the veins are separated, which have the ability to continue to divide and maintain pluripotent activity without undergoing a dedifferentiation process.

[0138] In plants, only the meristem cells / stem cells found in the primary meristems of plants responsible for the growth of leaves, buds, flowers and roots, or in the secondary meristems required to increase the diameter of stems and roots, can actively divide, i.e., have mitotic activity. Once the meristem cells differentiate into specific cell types, such as palisade cells, epidermal cells and mesophyll cells in leaves, these differentiated cells can no longer divide. Therefore, starting cell suspension culture from whole organs or tissues is the result of the reactivation of mitosis by the differentiated cells in these plant organs or tissues after undergoing a dedifferentiation process. These dedifferentiated plant cells regain the ability to divide. However, this dedifferentiation process can cause a large number of harmful genetic and epigenetic modifications, resulting in poor performance of dedifferentiated cells in suspension culture in terms of growth, cell aggregation and production of natural products. According to the present invention, plant stem cells that are naturally endowed with the ability to continue to divide and maintain pluripotent activity are separated.

[0139] In particular, the present invention is not based on the isolation of callus tissue originating from the whole leaf. The method of the present invention relies on exposing a thin layer of cells located in the central vein of the leaf or the periphery of other veins, and then isolating leaf stem cells, which are immortalized, undifferentiated, grow faster and show a special morphology (large number of starch granules and small size). Due to the fast growth rate of these leaf stem cells, they can be separated from the dedifferentiated cells, thereby obtaining a homogeneous cell population. In addition, the method according to the present invention is fast, easy to implement and can be used for a variety of plant genera.

[0140] Furthermore, the isolation of homogenous stem cell populations allows the sorting of cell lines based on their ability to produce secondary metabolites under selection with immune activators. This selection allows the identification of high-performing cell lines at an early stage during development.

[0141] When isolating meristem cells from parts such as stems (especially woody plants), a major problem is the increased risk of contamination due to the presence of high levels of endophytes (microorganisms) on the stem surface. In order to overcome this problem, the method according to the invention allows the isolation of stem-like meristem cells from the central veins of leaves rather than from the stems. Compared to the stems, the surface of the leaves is usually smoother, which facilitates the surface sterilization process and reduces the rate of microbial contamination on solid culture media.

[0142] In one aspect of the invention, plant stem cells or stem cell lines obtained or obtainable by any or all of the methods described herein are provided. In addition, plants, plant parts or cell lines obtained or obtainable by the methods described herein are provided. In one embodiment, products or extracts of plant stem cells or stem cell lines are obtained or obtainable by the methods described herein. Compositions comprising stem cells, stem cell lines, plant products and / or plant extracts obtained or obtainable by the methods described herein are also provided. In one embodiment, the stem cell line has not yet been dedifferentiated into callus.

[0143] In another aspect, provided is a stem cell line obtained from the primary meristem in a leaf of a plant, having the following characteristics:

[0144] a. the stem cell line is formed by undifferentiated cells; and

[0145] b. the morphology of the stem cells of the line is characterized by the presence of multiple amyloid bodies; and

[0146] c. the stem cell line is a fast-growing cell line; and

[0147] d. Optionally, the stem cell line has high phytochemical accumulation in response to induction.

[0148] A fast growing cell line refers to a cell line that grows faster than a control cell (eg, a differentiated leaf cell or a dedifferentiated cell).

[0149] In additional or alternative embodiments, the plant stem cell line comprises a substantially genetically homogeneous population of undifferentiated cells.

[0150] In another aspect, a method for producing a plant or plant part and / or a crop breeding method is provided, the method comprising

[0151] a. isolating at least one stem cell from the tissue of the primary meristem in the leaf of a plant;

[0152] b. establishing a stem cell line comprising said at least one isolated stem cell, preferably, wherein said stem cell line is substantially genetically homogeneous;

[0153] c. Optionally, selecting a high-performance cell line; and

[0154] d. growing a new plant or plant part from at least one stem cell in said stem cell line.

[0155] In one embodiment, the method further comprises growing the plant or plant part using vertical farming practices. In a preferred embodiment, the high performance cell line is a high yielding and / or high proliferative cell line.

[0156] Also provided is a composition comprising stem cells obtained or obtainable from the above stem cell line, or an extract or product thereof.

[0157] As described above, the plant stem cells and cell lines presented herein have greater proliferative properties and genetic homogeneity compared to dedifferentiated cells and cell lines.

[0158] In a preferred embodiment, the stem cell line is obtained by a method according to the invention.

[0159] In another aspect of the present invention, a method for producing a plant natural product or extract is provided, the method comprising

[0160] a. isolating at least one stem cell from the tissue of the primary meristem in the leaf of a plant;

[0161] b. establishing a stem cell line comprising said at least one isolated stem cell, preferably, wherein said stem cell line is substantially genetically homogeneous;

[0162] c. Optionally, selecting a high performance cell line; and / or

[0163] d. Optionally, inducing the production or secretion of a plant natural product or extract; and

[0164] e. Collecting the plant natural products or extracts.

[0165] In another aspect of the invention there is a composition comprising at least one plant stem cell, preferably comprising a plurality of stem cells, preferably from a stem cell line obtainable or obtained by the methods described herein.

[0166] In additional or alternative embodiments, the composition may comprise at least one plant stem cell extract and / or at least one plant stem cell product, preferably from a stem cell line obtainable or obtained by the methods described herein.

[0167] In one embodiment, the composition comprises at least one plant stem cell obtained or obtainable by the method of the present invention, or at least one plant natural product and / or extract obtained or obtainable from the stem cell or stem cell line of the present invention, preferably by selecting a high-performance cell line. That is, the composition comprises at least one plant stem cell or cell line showing a high-performance indicator or trait, or a plant natural product and / or extract obtained from the cell or cell line.

[0168] In a preferred embodiment, the composition is used in cosmetics or as a cosmetic. The cosmetics can be used for humans or animals. Therefore, in one embodiment, a composition for cosmetics is disclosed, the composition comprising at least one stem cell, preferably a plurality of stem cells and / or a plant stem cell product and / or a plant stem cell extract, preferably from a stem cell line obtainable or obtained by the methods described herein. The cosmetic composition can be formulated in any known manner, such as a cream, lotion, water, cleanser, shampoo, oil, etc. The concentration of the extracts typically ranges from 0.01%, 0.02%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% to 5% (w / w).

[0169] It is well known that many plant stem cells, extracts and products have powerful antioxidant and / or anti-inflammatory effects. For example, grapes (Vitis vinifera), lilacs (Syringa vulgaris), Swiss apples (Uttwiler spatlauber), etc. Therefore, the composition may also include other components, such as phytochemicals, antioxidants and / or anti-inflammatory components. Examples of antioxidants include tocopherols, lecithin, ascorbic acid, vitamin E, vitamin C, and coenzyme Q10, anthocyanins and curcumin, etc. Examples of anti-inflammatory compounds include aloe vera, turmeric, calendula extract, witch hazel, niacinamide, sea buckthorn oil, vitamin C, vitamin D and vitamin E, oats or ginger. Other components may be emulsifiers and / or gel formers, such as glycyrrhizin. In another embodiment, the composition is used for agricultural compositions. In one embodiment, the composition may be a monofunctional or multifunctional plant stem cell growth composition. The specific use of this composition includes increasing water absorption, reducing excessive use of pesticides and improving crop quality and / or performance. The agricultural composition may also include, but is not limited to: plant stem cells (preferably in a liquid matrix), antibacterial agents, starch, trace metal elements that can be used as fertilizer synergists, plant growth regulators, photosynthesis agents and / or water retaining agents, etc. In addition, the plant stem cell matrix liquid may be selected from one or more of agar, polyphenols, flavonoids, nucleic acids and protein hydrolysates. In addition, the antibacterial agent may be selected from one or more of amino acids, metal salts, chitosan powder and chitin. In addition, trace element fertilizer synergists may include one or more of iron, manganese, boron, zinc, copper, molybdenum, chlorine, cobalt, etc. In addition, plant growth regulators include one or more of auxin, gibberellin, chlormequat, cytokinin and abscisic acid. In addition, photosynthesis agents may be sodium bisulfite, etc. In addition, water retaining agents are humic acid, etc.

[0170] In another embodiment, the composition is used in a pharmaceutical composition. In one embodiment, the pharmaceutical composition is used to treat or prevent proliferative diseases (such as cancer), angiogenic diseases and cardiovascular diseases, inflammatory diseases, bone formation diseases, metabolic diseases, and nervous system diseases. Plant cell extracts for pharmaceutical compositions are well known in the art and have been widely reviewed, for example in Li, et al. Stem Cell Research & Therapy (2022) 13: 472.

[0171] In another embodiment, the composition is used in a food composition and / or a beverage composition.

[0172] For any of the above compositions, the composition may further include at least one human stem cell, extract or product thereof.

[0173] The present invention can be further described by the following non-limiting illustrations:

[0174] A method for isolating a stem cell line derived from primary meristem tissue in a plant leaf, comprising the following steps:

[0175] (a) sterilizing at least a portion of the leaf containing the central vein or other veins

[0176] (b) cutting the central vein or other veins of the leaf to expose primary meristem cells from the vascular tissue inside the central vein or other veins; and

[0177] (c) culturing cells derived from the exposed cells in a cell induction medium.

[0178] A stem cell line derived from the tissue of the primary meristem in the leaves of a plant refers to a cell line comprising cells that are self-renewing, undifferentiated and can give rise to other cells, e.g., have a faster growth rate and smaller size than other plant cells. In the context of the present invention, plant stem cells are equivalent to mammalian stem cells.

[0179] The sterilization step is a step to remove any microorganisms that may contaminate the surface of the plant explants used to isolate the target cells.

[0180] The sterilizing agent that can be used is common in the technical field, and can be, for example, sodium hypochlorite (bleach), calcium hypochlorite, hydrogen peroxide, ethanol, etc. Preferably, sodium hypochlorite with a concentration of 0.5% to 1.0% is used for sterilization for 30 to 40 minutes. Calcium hypochlorite powder should be dissolved in water before use and then filtered. Usually, its use concentration is 3.25%. The use concentration of hydrogen peroxide is 3%.

[0181] Ethanol can be used for sterilization, for example at a concentration of 70% to 95%.

[0182] It is imperative to remove as much microorganisms as possible from the explants used, as these microbial cells grow much faster than plant cells and can quickly contaminate the cell isolation method. At the same time, useful sterilizing agents such as ethanol or bleach treatments can be toxic to plant cells at high concentrations or with prolonged contact. Therefore, it is desirable to minimize the effects of these sterilizing agents by maintaining the shortest incubation time with these compounds, and preferably, the lowest effective concentration. It is worth noting that the surface of leaves is smoother than that of stems, which facilitates a milder and more gentle sterilization procedure and increases the likelihood of isolating a viable, healthy cell population. Therefore, when using leaves as explants, they can be kept in the solvent for as little as 1 to 15 minutes while still removing most of the harmful microorganisms and ensuring that the explant is not damaged.

[0183] After sterilization, carefully cut (cut open) the central vein or other veins of the leaf to expose the primary meristem cells that form the internal vascular tissue. The central vein of the leaf corresponds to the main vein or midrib, has a central linear structure, and extends from the base of the leaf to the tip of the leaf along the length of the leaf. Other veins refer to other veins on the leaf except the central vein, such as lateral veins or secondary veins. Preferably, the vein selected for exposing cells is the central vein. This is because the central vein is usually thicker than the lateral veins and secondary veins, which is easier to operate and allows more cells to be exposed. Preferably, the cutting is carried out along the length direction of the central vein or other veins, so with respect to the central vein, the cutting direction is from the tip of the leaf to the base of the leaf or from the base of the leaf to the tip of the leaf. In a preferred embodiment, the leaf is cut into two halves along the central vein and through the central vein.

[0184] This careful leaf dissection process will expose meristem cells derived from the primary meristem, such as protophloem meristem cells. These cells are then subjected to a culture step to induce proliferation in the presence of a suitable cell induction medium, as further described.

[0185] After exposure of the target cells, the explant region containing the meristem cells is placed on a cell induction medium for a culture step as described in the previous step.

[0186] In one embodiment of the method, the cells have not undergone dedifferentiation into callus tissue.

[0187] In another embodiment, the culturing step comprises:

[0188] (a) isolating primary meristem cells from the central vein or other inner vascular tissue of the leaf vein;

[0189] (b) initiating suspension culture; and

[0190] (c) Optionally, selecting a high-performance cell line that responds to a plant immunity inducer

[0191] In one embodiment of the method, the initiating suspension culture step comprises incubating the plate comprising the isolated primary meristem cells at 16°C to 30°C in a 24h / 0h light / dark to 0h / 24h light / dark cycle.

[0192] In one embodiment of the method, the cell induction medium comprises macronutrients, micronutrients and / or phytohormones.

[0193] In one embodiment of the method, the cell induction medium contains the components constituting B5 medium or MS medium and one or more of 2,4-D, kinetin, NAA, 6-BA and other plant growth regulators.

[0194] In one embodiment of the method, the cell induction medium contains 0 to 10 mg / L 2,4-D, 0.01 to 1 mg / L kinetin, 0.01 to 1 mg / L NAA, and 0 to 10 mg / L 6-BA.

[0195] In another embodiment of the present invention, a specific medium composition for isolating meristem cells from the central vein or other veins of a leaf is optimized to promote the proliferation of a target cell type of a specific plant species. This is achieved by testing different concentrations and ratios of essential components, including a suitable base, carbon and nitrogen sources, vitamins, medium optimization additives, and auxins (different ratios of auxin: cytokinin).

[0196] The culturing step according to the present invention involves a step performed after exposing the target cells. More specifically, before culturing, the leaves of the target plant species are sterilized, and then the central veins or other veins of the leaves are cut open to expose a thin layer of the desired stem cells. For example, the veins are cut to expose the primary protophloem cells. Compared with traditional differentiated cells, these and other primary meristem cells have the characteristics of self-renewal, undifferentiation, and show a faster growth rate. In the culturing step, the cut leaves are placed on a cell induction medium to promote the proliferation of meristem cells. Next, cells are separated from the leaves, that is, the desired stem cells are identified and separated from the explant. Further, a suspension culture is initiated, in which the separated stem cells are cultured on different petri dishes with a cell line induction medium. Optionally, a high-performance cell line that responds to a plant immune inducer is selected.

[0197] In another aspect, the stem cell line is derived from tissue of the primary meristem in the leaf of a plant having the following characteristics:

[0198] (a) The stem cell line is formed by undifferentiated cells

[0199] (b) The morphological feature of the stem cell line is the presence of multiple amyloid bodies

[0200] (c) The stem cell line is a fast-growing cell line

[0201] (d) optionally, having high phytochemical accumulation in response to induction.

[0202] In one embodiment, the above-mentioned stem cell line is obtained by any of the above-mentioned methods.

[0203] A cosmetic composition, agricultural composition, pharmaceutical composition, food composition, beverage composition or human / animal health care composition comprising cells derived from the cell line according to any one of the above embodiments.

[0204] Example

[0205] The following examples of the present invention are described. The isolation and induction of plant stem cells from primary meristems, and the subsequent production of suspension cultures and cell line selection are directed to Glycyrrhiza glabra, Taxus baccata, Q. saponaria, and Q. indica, but by extension, can also be used for any leafy plant. The following examples are intended to be illustrative only and not limiting.

[0206] Example 1. Preparation of plant materials and isolation of plant stem cells from Glycyrrhiza glabra.

[0207] Surface sterilization of plant tissues. Collect leaves of Glycyrrhiza glabra. After collecting the leaves, sterilize the leaves surface with 70% ethanol (10 minutes), 10% bleach (containing 5% sodium hypochlorite) (10 minutes), and 1% bleach (containing 5% sodium hypochlorite) (10 minutes). After surface sterilization, wash with distilled water 3 to 4 times.

[0208] Preparation of internal vascular tissue in Glycyrrhiza glabra leaves. Place Glycyrrhiza glabra leaves horizontally on a hard surface. Cut the leaves vertically with a scalpel or razor, and the incision should pass through the midrib or other veins of the leaves to expose the internal vascular tissue containing the target plant stem cells. Place the cut leaves on a culture medium.

[0209] After culturing the leaf explants for 3 to 7 days, visible callus is observed on the exposed leaf main vein or other leaf veins. At this time point, the rest of the leaf can be removed. The meristem stem cells from the proliferation of the original phloem are cultured separately in different petri dishes with a culture medium (such as a cell line induction medium). In order to induce cells and callus, MS (Murashige & Skoog medium) is used. Other available culture media, such as B5 (Gamberg's B5 medium), WPM (Lloyed & McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre) are also known in the art. All of these culture media can be used. Table 1 lists the content of MS. The culture is grown at a temperature of 25 ± 1 ° C under a 16h / 8h light / dark cycle.

[0210] Table 1: MS medium for culturing dissected leaf explants

[0211]

[0212] The white, friable callus with the best growth rate in the callus tissue is subcultured to a new medium every 14 days. The meristem cells derived from the exposed main vein or other leaf veins grow fast and have no "browning". These cells are then easily selected as stable cell lines. Due to the influence of light conditions, the callus produced by Glycyrrhiza uralensis is white-green. After 6 months of culture in liquid culture medium, the callus tissue remains as a single cell or small cell mass.

[0213] Establishment of suspension culture of Glycyrrhiza glabra

[0214] Cells derived from exposed Glycyrrhiza glabra leaf veins were cultured in flasks containing the liquid culture medium listed in Table 2:

[0215] Table 2: Suspension initiation medium for initiating and maintaining stem cell lines

[0216]

[0217] The cells were cultured at 25±1°C in a 16h / 8h light / dark cycle on a rotary shaker at 120 rpm and subcultured every two weeks.

[0218] Example 2. Preparation of plant materials and isolation of plant stem cells from Indian quassia

[0219] Surface sterilization of plant tissues. Collect leaves of the Indian quassia plant. After collecting the leaves, sterilize the leaves surface with 70% ethanol (10 minutes), 10% bleach (containing 5% sodium hypochlorite) (10 minutes), and 1% bleach (containing 5% sodium hypochlorite) (10 minutes). After surface sterilization, wash with distilled water 3 to 4 times.

[0220] Preparation of internal vascular tissue in Indian quassia leaves. Place the Indian quassia leaves horizontally on a hard surface. Use a scalpel or razor to cut the leaves vertically, through the midrib or other veins of the leaves, to expose the internal vascular tissue containing the target plant stem cells. Place the cut leaves on a culture medium.

[0221] After 3 to 7 days in culture, callus begins to form and expand on the exposed main vein or other leaf vein cuts, while the rest of the leaf can be removed.

[0222] The callus tissue was cultured separately in different petri dishes using cell line induction medium. To induce cells and callus tissue, MS (Murashige & Skoog medium) was used. Other available culture media, such as B5 (Gamberg's B5 medium), WPM (Lloyed & McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre) are also known in the art. All of these culture media can be used.

[0223] The content of MS is shown in Table 1. The culture was grown in the dark at 25±1℃. The white, friable callus with better growth rate was subcultured to a new medium every 14 days. The target meristem cells derived from the exposed main veins grew fast and had no browning, and these cells were easily selected as stable cell lines. The callus obtained from Indian quassia was white-yellow and remained as single cells or small cell clusters after 6 months of culture.

[0224] Establishment of suspension culture of Quassia indica

[0225] Cultures derived from exposed Glycyrrhiza glabra leaf veins were grown in flasks containing the liquid medium listed in Table 2.

[0226] The cells were cultured in the dark at 25±1°C on a rotary shaker at 120 rpm and subcultured every two weeks.

[0227] Example 3. Preparing plant materials from Taxus chinensis and / or Quillaja sapodilla and isolating plant stem cells.

[0228] Surface sterilization of plant tissues. Collect leaves of European yew and / or Chilean soapberry plants. Surface sterilize with 70% ethanol (10 minutes), 10% bleach (containing 5% sodium hypochlorite) (10 minutes), and 1% bleach (containing 5% sodium hypochlorite) (10 minutes) in sequence. After surface sterilization, wash with distilled water 3 to 4 times.

[0229] Preparation of vascular tissue in leaves of Taxus chinensis and / or Quillaja chinensis. Place leaves of Taxus chinensis and / or Quillaja chinensis horizontally on a hard surface. Cut the leaves vertically with a scalpel or razor, and the cut should pass through the midrib or other veins of the leaves to expose the internal vascular tissue containing the target plant stem cells. Place the cut leaves on a culture medium.

[0230] After 3 to 7 days of culture, callus tissue begins to form and expand on the exposed leaf main vein section, while the rest of the leaf can be removed. The callus tissue is cultured separately in different petri dishes using cell line induction medium.

[0231] To induce cells and callus, B5 (Gamberg's B5 medium) was used. Other available culture media, such as MS (Murashige & Skoog medium), WPM (Lloyed & McCown medium), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre medium) are also known in the art. All of these culture media can be used.

[0232] The content of B5 is shown in Table 3. The cultures were grown in the dark at 25 ± 1 °C.

[0233] The white, friable callus with the best growth rate was subcultured onto new medium every 14 days. The target meristem cells derived from the exposed main veins grew fast and did not brown, and these cells were easily selected as stable cell lines. Callus cultured from Taxus chinensis and / or Quillaja chinensis was white-yellow and remained as single cells or small cell clusters after 6 months of culture.

[0234] Table 3: Cell induction medium (B5 medium)

[0235]

[0236] Establishment of suspension cultures of Taxus chinensis and / or Quillaja chinensis

[0237] Cultures derived from exposed Glycyrrhiza glabra leaf veins were grown in flasks containing the liquid medium listed in Table 4.

[0238] Table 4: Suspension starter medium

[0239]

[0240] The cells were cultured at 25±1°C in a 16h / 8h light / dark cycle on a rotary shaker at 120 rpm and subcultured every two weeks.

[0241] Example 4. Comparison between dedifferentiated cells isolated from plant leaves and primary meristem cells isolated using the method of the present invention.

[0242] Lugol's iodine staining and microscopic analysis were performed to evaluate the phenotypic differences between dedifferentiated cells isolated from plant cells generated from whole leaf organs or random fragments of leaves and plant cells isolated and cultured from the central vein or other veins according to the dissection method of the present invention. This staining process allows visualization of amyloplasts, which are organelles involved in nutrient storage and are usually present in cells that do not perform photosynthesis.

[0243] Taxus baccata and Glycyrrhiza glabra samples were obtained according to the steps described in Example 1 and Example 2. Lugol's staining could not detect the presence of amyloplasts in dedifferentiated cells of Taxus baccata or Glycyrrhiza glabra. However, a large number of amyloplasts were shown in cells obtained by the central vein or other vein dissection methods of the present invention, which clearly distinguished them from conventional dedifferentiated cells.

[0244] Example 5. Comparative analysis of the contamination rates of stem-derived samples and leaf-derived samples used in the present invention.

[0245] Explant samples from the stem and leaves were collected from the tropical tree Quassia indica, which had a high microbial load and high contamination rate after surface sterilization according to standard protocols. After collection, the explants were surface sterilized in sequence with 70% v / v ethanol in water for 10 minutes and then with bleach at the concentrations shown in the table below for 10 minutes. After surface sterilization, the samples were washed 3 to 4 times with distilled water. The number of contaminated explants was obtained by counting the explants with visible microbial growth. The ratio of contaminated explants to the total number of explants was calculated according to standard methods. The results are shown in the following table:

[0246] As can be seen from the table, under similar conditions of sterilizing agent and sterilization time, sterilization of leaves resulted in very low contamination rates compared to treatment of stems, making them particularly useful in methods for isolating stem cells.

[0247] More severe sterilization conditions (higher concentrations of sterilizing agents such as concentrated bleach solutions and / or longer sterilization times) may reduce the risk of contamination of stem or leaf samples, but may damage the samples and negatively affect the viability of the cells to be isolated.

[0248] Table 5: Comparison of contamination rates of stems and leaves after sterilization

[0249]

Claims

1. A method for obtaining at least one plant stem cell, the method comprising the following steps: a. sterilizing at least a portion of a leaf containing a central vein or other veins; b. cutting open the central vein or other veins of the leaf to expose the primary meristem cells of the vascular tissue from the central vein or other veins; as well as c. obtaining at least one stem cell from the inner vascular tissue of the central vein or other veins.

2. The method of claim 1, wherein the method further comprises culturing the at least one stem cell to obtain a stem cell line.

3. The method of claim 1 or 2, wherein the method does not comprise forming a callus obtained from dedifferentiated cells or forming a callus consisting essentially of dedifferentiated cells.

4. The method of any one of claims 1 to 3, wherein the cell culture comprises no dedifferentiated cells and / or protoplasts, or comprises essentially no dedifferentiated cells and / or protoplasts.

5. The method of any one of claims 2 to 4, wherein the culturing step further comprises: a. starting a suspension culture comprising cultured primary meristem cells from the internal vascular tissue of the central vein or other veins; as well as b. Optionally, selecting a high performance cell line that preferentially responds to a plant immunity inducer.

6. The method of any one of claims 2 to 5, wherein the cell culture comprises no dedifferentiated cells and / or protoplasts, or comprises essentially no dedifferentiated cells and / or protoplasts.

7. The method of any one of claims 2 to 6, wherein the culturing step further comprises: (a) isolating the primary meristem cells from the inner vascular tissue of the central vein or other veins; (b) initiating a suspension culture comprising cultured primary meristem cells from the inner vascular tissue of the central vein or other veins; as well as (c) Optionally, selecting a high performance cell line that preferentially responds to a plant immunity inducer.

8. The method of claim 7, wherein the initiation of the suspension culture step comprises incubating the plate containing the isolated primary meristem cells at 16°C to 30°C in a cycle of 24h / 0h light / dark to 0h / 24h light / dark.

9. Plant stem cells or plant stem cell lines obtained or obtainable by the method of any one of claims 1 to 8.

10. A plant product or a plant stem cell extract obtained or obtainable from the plant stem cells according to claim 9.

11. A plant or a part thereof obtained or obtainable from the plant stem cell according to claim 9.

12. A stem cell line derived from the primary meristem in the leaves of a plant, wherein the stem cell line has the following characteristics: a. The stem cell line is formed by undifferentiated cells, which are preferably substantially genetically homogeneous cells; and b. The morphology of the cells of the line is characterized by the presence of multiple amyloplasts; and c. the stem cell line is a fast-growing cell line; and d. Optionally, having high phytochemical accumulation in response to induction.

13. A method for producing a plant natural product or extract, the method comprising: a. isolating at least one stem cell derived from the primary meristem tissue in the leaves of a plant, wherein preferably, the at least one stem cell is derived from the primary meristem cells of the vascular tissue of the central vein or other veins; and b. culturing the at least one stem cell to establish at least one stem cell line, preferably, wherein the stem cell line is substantially genetically homogeneous; c. optionally selecting a high performance cell line; and / or d. optionally inducing the production or secretion of a plant natural product or extract; and e. Collecting the plant natural products or extracts.

14. The method of claim 13, wherein the high-performance cell line is characterized by a high-performance indicator or a high-performance trait, wherein the high-performance indicator or the high-performance trait is selected from at least one of the following: cell size, proliferation, product yield and / or developmental capacity, wherein preferably, the improvement is relative to another cell / other cells in the cell line.

15. The method of claim 13, wherein inducing production or secretion of a plant natural product comprises administering at least one plant inducer to the stem cell culture.

16. The method of claim 13, wherein the plant product or extract is selected from the group consisting of flavonoids, alkaloids, polyphenols, polysaccharides, quinones and saponins.

17. The method of any one of claims 1 to 8 and 13 to 16, or the plant stem cell of claim 9, wherein the plant is selected from a dicot, a monocot or a gymnosperm.

18. The method of claim 17, wherein the plant is selected from the group consisting of: In one embodiment, the plant can be selected from asparagus, grass, palm, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, cilantro, sunflower, peanut, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe, yew (Taxus), eggplant, pineapple, sumac, chickpea, rosemary, litchi, liquorice (Glycyrrhiza glabra), spinach, soybean, Brassica, carrot, corn, rice, thuja, juniper, pine, garlic, cucumber, chili, pepper, lettuce, peach, watermelon, grape, apple, onion, orange, banana, pea, mango, citrus, tea, sugarcane, cotton, barley, sorghum, wheat, rice, Simaroubaceae and magnolia, and members of the genera Quillaja and Cupressaceae.

19. A composition comprising at least one plant stem cell according to claim 9 or at least one plant product or plant stem cell extract according to claim 10.

20. The composition of claim 18, wherein the composition is selected from the group consisting of a cosmetic composition, an agricultural composition, a pharmaceutical composition, a food composition, a beverage composition, or a human / animal health care composition.

21. A method for producing plants or plant parts thereof and / or a crop breeding method, the method comprising: a. The method according to claim 1, isolating at least one stem cell derived from the tissue of the primary meristem in the leaf of the plant; b. establishing a stem cell line comprising said at least one isolated stem cell, preferably, wherein said stem cell line is substantially genetically homogeneous; c. optionally selecting a high performance cell line; and d. growing a new plant or plant part thereof from at least one stem cell in the stem cell line and / or at least one stem cell in the high performance cell line.

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