Method for isolating plant stem cells from plant leaves and related cell lines obtained using this method

Abstract

The present invention relates to a method for isolating a stem cell line derived from tissues from the primary meristem in plant leaves, the method comprising: (a) sterilizing at least a part of a leaf containing a central vein or other leaf veins; (b) cutting the central vein or other leaf veins of the leaf to expose primary meristem cells from the internal vein tissue of the central vein or other leaf veins; and (c) culturing cells from the exposed cells, which avoids the difficulties associated with surface sterilization when other types of explants are used, and also relates to the stem cell line obtained using this method.

Description

Technical Field

[0001] The present invention relates to a method for isolating stem cells from plant leaves and establishing a stem cell line, and the use of the isolated stem cells and their stem cell lines.

Background Art

[0002] Plants are natural chemists and can produce a vast number of secondary metabolites (natural products) that form the basis of many pharmaceuticals, cosmetics, foods, and agricultural products. Source plant species used to generate natural product-containing extracts or for the isolation of specific purified natural products (NPs) are often slow-growing, their populations are limited, the concentration of target molecules varies greatly between plants, and their in vivo concentration is usually extremely low. This situation has led to unmet demand for NPs and extracts, and an increasing use of intensive farmland for the cultivation of NP-producing plants for industrial applications. Therefore, alternative production systems for plant NPs have been needed.

[0003] A potential solution is the use of in vitro cultured plant cells isolated from target NP-producing plant species. The plant cell culture process consists of inducing callus formation from plant tissues or organs (explants) grown on synthetic solid media in Petri dishes. Callus consists of a mass of dedifferentiated plant cells (DDCs). Fragments of the callus are then inoculated into liquid media in flasks to generate DDC suspension cultures.

[0004] Plant cell culture offers an attractive alternative biological NP production route for the whole plant. It is sustainable, efficient, independent of environmental conditions, standardized, provides a stable supply of products, does not contain animal-origin infectious viruses, and can be optimized to produce target metabolites at commercially appropriate levels and comply with the regulatory guidelines of pharmaceutical manufacturing standards (GMP).

[0005] However, this classical approach of culturing and using DDC for NP production causes genetic and epigenetic damage to plant cells. Therefore, DDC exhibits significant limitations, including slow growth rate, pronounced cell aggregation, increased stress sensitivity, unstable viability and limited NP production, restricting the wide commercial use of this approach to all but the highest value applications.

[0006] To avoid the problems associated with typical DDC culturing, Non-Patent Document 1 isolated cultured cambial meristem cells (CMC). The cell suspension cultures were derived from cambial tissues from secondary meristems within plant stems and leaves and showed potential as a platform for producing plant NPs. These cambium-derived cells exhibited excellent growth in long-term culture, reduced cell aggregation, improved stress tolerance and increased NP yields. Importantly, the cambium is a secondary meristem formed in stems and roots after the differentiation of primary plant tissues. The cambium is involved in the increase in diameter of stems and roots and the formation of woody tissues (Non-Patent Document 2).

[0007] Patent Document 1 discloses a method for isolating cell lines from plants, and the isolated plant cell lines are derived from cambium obtained from plant leaves or stems. This method includes the steps of collecting a tissue containing the cambium of a plant, culturing the tissue, thereby inducing a layer grown from the cambial secondary meristem without undergoing dedifferentiation into callus.

[0008] However, surface sterilization of explants from plant stems, roots, roots or storage roots is difficult to achieve successfully, especially for plant samples collected from natural environments, because these ecosystems have rich microbial diversity. For example, due to their irregular surface structures, explant samples taken from these plant organs are associated with high levels of endophytes, endosymbionts, often bacteria or fungi, which are present within the plant in at least part of their life cycle without causing obvious diseases (Non-Patent Document 3). Therefore, the sterilization process itself needs to be systematically adapted to produce sterilized cell cultures from different plant species, which is labor-intensive and time-consuming.

[0009] Other explants that can be used in methods for isolating cells are, for example, the leaves of plants. Patent Document 2 relates to the isolation of cell lines from plants of the genus Melissa and the generation of related plant cell suspension cultures by inducing callus formation from plant tissues including leaf pieces or whole leaves on solid plant growth media and subsequent inoculation of liquid plant growth media. However, the obtained Melissa DDC shows significant limitations including slow growth rate, significant cell aggregation, increased stress sensitivity, unstable viability, and low NP production yield.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0011]

Non-Patent Document 1

[0012] An object of the present invention is to provide a method for isolating and generating a plant cell line that has not undergone dedifferentiation into callus, which method is easy, rapid, more easily standardized, and avoids the difficulty of surface sterilization when using plant secondary meristems derived from stems, twigs or storage roots as explants. [Means for Solving the Problems]

[0013] In one aspect, the present invention relates to a method for isolating a stem cell line derived from tissue from the primary meristem in a leaf of a plant, the method comprising the following steps: (a) sterilizing at least a part of the leaf including the central vein or other leaf veins; (b) cutting the central vein or other leaf veins of the leaf to expose primary meristem cells from the internal vein tissue of the central vein or other leaf veins; and (c) culturing cells from the exposed cells in a cell induction medium.

[0014] 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: (a) sterilizing at least a part of a leaf including the central vein or other leaf veins; (b) incising the central vein or other leaf veins of the leaf to expose primary meristem cells from the internal vein tissue of the central vein or other leaf veins; and (c) obtaining at least one stem cell from the internal vein tissue of the central vein or other leaf veins.

[0015] In one embodiment, the stem cells are promeristem cells.

[0016] In one embodiment, the leaf is obtained from a plant cultivated or grown in a vertical farm.

[0017] The method for isolating a stem cell line derived from a tissue from the primary meristem of a leaf of a plant according to the present invention enables the culture and isolation of cells that do not dedifferentiate into callus or have not dedifferentiated from callus.

[0018] In the method according to the present invention, cells are cultured from leaf explants. Preferably, the leaf (e.g., a young leaf) is collected from a plant species that naturally produces valuable natural products.

[0019] Therefore, the method can further include the step of culturing at least one stem cell to obtain a stem cell line.

[0020] In one embodiment, the cell culture does not contain or substantially does not contain dedifferentiated cells and / or protoplasts. In one embodiment, the method does not include the formation of callus obtained from or substantially containing dedifferentiated cells, and subsequent acquisition of stem cells from such callus.

[0021] The culturing step may further include: (a) culturing primary meristem cells from the internal vein tissue of the central vein or other leaf veins in suspension; and (b) Optionally, preferably in response to a plant immune inducer, a step of selecting a high-performance cell line.

[0022] The start of suspension culture may include the step of incubating a plate containing isolated primary meristem cells at 16 - 30 °C in a 24 hours / 0 hours light / dark to 0 hours / 24 hours light / dark cycle.

[0023] In another aspect of the present invention, there are provided plant stem cells or plant stem cell lines obtainable or obtained by the method of the present invention.

[0024] In another aspect of the present invention, there are provided plant stem cell products or extracts obtainable or obtained from the plant stem cells of the present invention.

[0025] In another aspect of the present invention, there are provided plants or parts thereof obtainable or obtained from the plant stem cells of the present invention. Plant products and / or extracts obtainable or obtained from the plants are also included within the scope of the present invention.

[0026] A stem cell line derived from the primary meristem of a plant leaf preferably has the following characteristics: (a) Formed from undifferentiated cells, preferably substantially genetically homogeneous cells; (b) The cells of the cell line are morphologically characterized by the presence of multiple amyloplasts; and (c) It is a high-growth cell line; and / or (d) Optionally, it has high phytochemical accumulation in response to induction.

[0027] In another aspect of the present invention, there is provided a method for producing a plant natural product, the method including the steps of isolating or obtaining at least one plant stem cell described herein, and isolating / collecting the product. In another aspect of the present invention, there is provided a method for producing a stem cell extract, the method including the steps of isolating or obtaining at least one plant stem cell described herein, and isolating / obtaining the extract.

[0028] In one embodiment, the method may include the following: (a) Isolating at least one stem cell derived from tissue from the primary meristem in a plant leaf; (b) Establishing a stem cell line comprising at least one isolated stem cell, preferably, 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 product or extract.

[0029] The high-performance cell line may be characterized by a high-performance indicator or trait selected from at least one of cell size, proliferation, product yield and / or developmental ability, preferably, the improvement is with respect to another / other cell / cell population in the cell line.

[0030] The step of inducing production or secretion may include treating a suspension culture with a plant inducer.

[0031] Plant natural compounds exhibit a wide range of activities against insects, fungi, bacteria and viruses. Thus, in one embodiment, the plant product may be an insecticide, fungicide, antibacterial or antiviral agent. Alternatively, the product may be a therapeutic agent or dietary supplement. In one embodiment, the plant product may be selected from flavonoids, alkaloids, polyphenols, polysaccharides, quinonoids and saponins.

[0032] In another aspect of the present invention, there is provided a composition 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 from or obtainable from the stem cells or stem cell lines of the present invention. The composition may be selected from cosmetics, agricultural products, pharmaceuticals, foods, beverages or human / animal healthcare compositions.

[0033] In a further aspect, there is provided a method of producing a plant or a part of the plant and / or a method of crop breeding, the method comprising: (a) isolating at least one stem cell derived from tissue from the primary meristem in the leaves of a plant according to the method of claim 1; (b) establishing a stem cell line comprising at least one isolated stem cell, preferably, the stem cell line is substantially genetically homogeneous; (c) optionally, selecting a high-performance cell line; and (d) growing a new plant or a part of the plant from at least one stem cell of the stem cell line and / or at least one stem cell of the high-performance cell line.

[0034] The method may further comprise growing a plant or a part of the plant using vertical farming practices.

[0035] Plants according to all aspects of the invention described herein can be dicotyledonous plants, monocotyledonous plants or gymnosperms. In one embodiment, the plant is a vascular plant.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further described below. In the following sections, different aspects of the invention are defined in more detail. Each aspect so defined can be combined with any one or more other aspects unless the contrary is clearly indicated. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0037] The practice of the present invention, unless otherwise indicated, uses conventional techniques within the scope of those skilled in the art, such as botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, etc. Such techniques are well described in the literature.

[0038] Plants according to all aspects of the invention described herein can be dicotyledonous plants, monocotyledonous plants or gymnosperms. In one embodiment, the plant is a vascular plant.

[0039] In one embodiment, the plant is a crop plant. A crop plant means any plant cultivated on a commercial scale for human or animal consumption or use. In another embodiment, the plant is Arabidopsis thaliana.

[0040] In one embodiment, the plant can be selected from asparagus, grass, palm, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, coriander, sunflower, peanut, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe vera, yews (taxus), eggplant, pineapple, sumac, chickpea, rosemary, lychee, licorice (Glycyrrhiza glabra), spinach, soybean, cruciferous plants, carrot, corn, rice, ginkgo, elm, pine, garlic, cucumber, pepper, lettuce, peach, watermelon, grape, apple, onion, mandarin, banana, pea, mango, orange, tea, sugarcane, cotton, barley, sorghum, wheat, rice, quassia, magnolia, and species of Quillija and family Cupressaceae.

[0041] In one embodiment, the plant can be selected from cruciferous, leguminous, gramineous, citrus, root vegetables, tuber and rhizome crops, berries and soft fruits and fruits including fruits, nuts and trees having seeds.

[0042] For the purposes of the present invention, the term "plant stem cell" refers to a plant cell capable of self-renewal and differentiation into specialized cells. Thus, through mitosis, plant stem cells can maintain a stem cell population, divide to generate progenitor cells, and then differentiate into tissues and organs. This asymmetric division can occur at the cell or population level.

[0043] The terms "plant stem cell" and "stem cell" are used interchangeably hereinafter.

[0044] "Plant stem cell" may also mean a plant stem cell that has not previously undergone substantial differentiation, has been removed from meristematic tissue, and is maintained as a substantially homogeneous population under culture conditions. Thus, the stem cells described in the present invention are obtained from differentiated somatic tissues and are different from plant cells that have been stimulated or dedifferentiated into a more potent state.

[0045] In one embodiment, the plant stem cell exhibits totipotency or pluripotency. Totipotency refers to a cell that has the ability to give rise to all cell types and establish a complete organism, and pluripotency refers to a cell that has the ability to give rise to most, but not all, cell types.

[0046] In one embodiment, the plant stem cell is derived from the primary and / or secondary meristematic tissues of a plant.

[0047] "Meristematic tissue" means a tissue containing a population of plant stem cells. Meristematic tissue is organized into meristems, which are regions of meristematic tissue that contribute to plant growth. "Primary meristem" is established during embryogenesis and contributes to the primary growth of the plant, i.e., length or height. Examples of primary meristems include the shoot apical meristem (SAM) and the root apical meristem (RAM).

[0048] Meristematic tissues that are derived from primary meristems and established after embryogenesis can be called "secondary meristems" such as axillary meristems. Secondary meristems contribute to the growth of the width of stems and roots in plants, resulting in a thicker and stronger tissue that can support the growing plant. Also, meristematic tissue contains a plant stem cell niche.

[0049] In one embodiment, the plant stem cell is derived from a primary meristem, preferably from a primary meristem found within and / or surrounding the vascular bundles of a plant, more preferably of a leaf. These stem cells are referred to herein as vascular stem cells or VSCs.

[0050] In a preferred embodiment, the plant stem cells are derived from or obtained from the primary meristem around or from the central vein of the leaf. The central vein of the leaf corresponds to the main vein or midrib. Thus, the stem cells are obtained by exposing the midrib and / or central vein and obtaining the stem cells.

[0051] The leaf veins can be defined as a support structure across the leaf blade, supporting the leaf and providing structure. The leaf veins include xylem, which is a conductive tissue that transports water and minerals to the leaf and the broader plant. The main vein also includes phloem, which is a tissue that transports glucose produced during photosynthesis. Thus, the leaf veins can include xylem and / or phloem in addition to the meristem.

[0052] Other leaf veins mean the leaf veins that originate from the central vein (also known as the midrib). Smaller leaf veins arise from the leaf veins. Thus, "other leaf veins" refers to leaf veins or smaller leaf veins that are not the central vein.

[0053] Phloem is formed from the protophloem, matures into the metaphloem, and finally becomes the phloem. Cells containing the protophloem are called protophloem cells. The initial phloem is formed by stem cells in the meristem, and thus, the cells of the initial protophloem can include stem cells in the meristem, or cells that share the characteristics of both protophloem and meristem cells. Such cells can be referred to as protophloem meristem cells. Protophloem meristem cells can be defined by the presence of one or more marker cells. Examples of suitable marker cells include APL (ALTERED PHLOEM DEVELOPMENT) (Bonke et al 2003), SWEET11 (Gebauer et al 2017), and BZIP9 (basic leucine zipper 9).

[0054] In one embodiment, the leaves are selected from plants grown or cultivated in a vertical farm. A vertical farm or vertical farming refers to a cultivation practice that enables the cultivation of plant species at multiple overlapping levels with the aim of maximizing the number of plants per cubic meter. Since the cultivation process takes place within a closed environment where all environmental parameters (temperature, humidity, CO2, light, and nutrients) are controlled, a vertical farm is a structure that produces the highest quality plant species. Therefore, by selecting leaves from plants grown or cultivated in a vertical farm, highly controlled and selected cell material for the isolation of stem cells and / or stem cell lines can be obtained. Similarly, by using vertical farming techniques to produce plants or parts of plants from stem cells, the highest quality specimens can be obtained.

[0055] Incision of plant tissue to expose plant stem cells Before incising the plant tissue, it is subjected to a sterilization process. The sterilization process aims to remove microorganisms from the surface of the plant used for the isolation of target cells.

[0056] Suitable sterilizing agents are generally known in the art and can be, for example, sodium hypochlorite (bleach), calcium hypochlorite, hydrogen peroxide, ethanol, etc. Preferably, sodium hypochlorite is used at a concentration of 0.5 - 1.0% for 30 - 40 minutes for sterilization. Calcium hypochlorite powder should be dissolved in water before use and subsequently filtered. Typically, it is used at a concentration of 3.25%. Hydrogen peroxide can be used at a concentration of 3%.

[0057] Ethanol can be used for sterilization, for example, at a concentration of 70 - 95%.

[0058] These microbial cells grow much more rapidly than plant cells and quickly contaminate methods for isolating cells, so it is essential to remove the microorganisms from the used explants. At the same time, useful sterilizing agents such as ethanol or bleaching treatments can be toxic to plant cells at high concentrations or long exposure times. Therefore, it is desirable to minimize the effects of these sterilizing agents by minimizing the incubation time and, preferably, maintaining the effective concentration of these chemicals at a minimum throughout the sterilization procedure. Importantly, leaves have a smoother surface compared to the surface of the stem, which allows for a less stimulating and gentler sterilization procedure, increasing the probability of isolating a viable and healthy cell population. Thus, when leaves are used as explants, they can be held in a solvent for a short period of about 1 - 15 minutes while still removing most of the harmful microorganisms and ensuring that the explants are not damaged.

[0059] Accordingly, in one embodiment, a method for isolating plant stem cells from a leaf is provided, the method comprising the step of sterilizing at least a portion of the leaf including the midrib or other leaf veins.

[0060] In another embodiment, a method for isolating or establishing a plant stem cell line is provided, the method comprising the step of sterilizing at least a portion of the leaf including the midrib or other leaf veins.

[0061] Similarly, in another embodiment, a stem cell line obtainable by a method comprising the step of sterilizing at least a portion of the leaf including the midrib or other leaf veins is provided.

[0062] In a preferred embodiment, the leaf is sterilized with a sterilizing agent preferably selected from sodium hypochlorite (bleach), calcium hypochlorite, hydrogen peroxide and / or ethanol.

[0063] After sterilization, the central vein or other leaf veins of the leaf are carefully cut (incised) to expose the meristematic cells that form the internal vascular tissue. In a preferred embodiment, the plant stem cells are derived from the meristem around the central vein of the leaf. The central vein of the leaf corresponds to the main vein or midrib.

[0064] In a further embodiment, the plant stem cells can be derived from the lateral or secondary leaf veins of the leaf.

[0065] Isolating cells from the central vein has practical advantages; typically, it is thicker than the lateral secondary veins, easier to manipulate, and allows more cells to be exposed. Preferably, the central vein or other leaf veins are cut along their length, and thus in the case of the central vein, in the direction from the tip to the base or from the base towards the tip of the leaf. In a preferred embodiment, the leaf is cut into two along and through the central vein.

[0066] The properties of cells or cell populations isolated from a meristem can be confirmed as plant stem cells by quantifying the presence or absence of morphological markers, metabolic markers, and / or gene markers within the cell population. Stem cell markers and characteristics are known to those skilled in the art and are described in the literature (see Miyashima et al. 2013), and non-limiting examples are included herein. Morphological markers include small size, roundness, large nuclei, insufficient cytoplasm, and prominent nucleoli. Such characteristics can be determined by the mass spectrometry methods described herein. Gene markers associated with plant stem cells include, but are not limited to, WUSCHEL-like HOMEOBOX4 (WOX4), CLAVATA3 / ESR-related 41 / 44 (CLE41 / 44), PHLOEM INTERCALATED WITH XYLEM (PXY) / TDIF RECEPTOR (TDR), and 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 routine methods known to those skilled in the art. For example, reverse transcriptase-polymerase chain reaction (RT-PCR) can quantify gene expression by reverse transcribing mRNA isolated from a single cell into cDNA and subsequently amplifying it by PCR. The identity of the cells can then be informed by adjustment of the mRNA abundance.

[0067] This careful leaf dissection process exposes meristematic cells, such as prefoliar meristematic cells, in the vascular tissue derived from the primary meristem. Subsequently, the regions of the explants containing these cells or meristematic cells are collected and subjected to a culturing process to induce growth in the presence of a suitable culture medium, such as the cell induction medium described herein.

[0068] Culture of isolated cells and establishment of cell lines For the purposes of the present invention, the term "plant stem cell culture" refers to the in vitro culture, maintenance, and proliferation of plant stem cells isolated by the methods described herein. A plant stem cell culture can refer to the stem cells in the liquid and / or solid phase of the culture. Thus, a plant stem cell culture refers to stem cells in a suitable medium such as a cell induction medium on a solid platform and / or stem cells in a liquid suspension culture. In a preferred embodiment, the plant stem cell culture comprises stem cells in a liquid suspension culture. In a preferred embodiment, the plant stem cells are cultured on a solid and / or liquid medium.

[0069] The culturing process according to the present invention relates to a process performed after the exposure of target cells from leaves. More specifically, the leaves of the target plant species are sterilized, and the central vein or other leaf veins of the leaves are incised to expose a thin layer of the desired stem cells. For example, the leaf veins are cut to expose the primary prefoliar cells. These and other meristematic cells are self-renewing, undifferentiated, and exhibit a faster growth rate than classical differentiated cells. In the culturing process, the cut leaves are placed on a cell induction medium to promote the growth of the meristematic cells.

[0070] A cell induction medium means a medium that stimulates the growth of cells and induces the formation of callus. Callus means a visible mass of undifferentiated cells. As used herein, callus does not contain or is not derived from dedifferentiated cells.

[0071] Culturing the exposed leaf veins induces the formation of callus, which is a visible mass of undifferentiated cells. The length of time between the explantation of the cells and callus formation varies depending on the source of the cells. DDC forms visible callus on the 7th day and thereafter after explantation. Vascular plant stem cells form callus earlier than 7 days, typically in 6 days. Thus, the stem cell population associated with the present invention produces visible callus earlier than DDC. Cells obtained from the central vein or other leaf veins of the leaf form visually distinguishable callus within 3 - 5 days, and cells obtained from the prefoliar leaf form callus within 6 days of culturing. Thus, the isolated stem cells of the present invention are different from cells obtained from differentiated cells that have dedifferentiated to form callus.

[0072] The composition of the cell induction medium is known to those skilled in the art and can be based on, for example, the papers by Murashige and Skoog (1962) and Gamborg et al. (1968). The specific composition varies depending on the plant species used. The medium disclosed by Murashige and Skoog is hereinafter referred to as the MS medium, and the medium disclosed by Gamborg et al. is hereinafter referred to as the B5 medium.

[0073] For example, the cell induction medium contains macronutrients, micronutrients, and / or plant hormones. In a preferred embodiment, the cell induction medium contains the components constituting the B5 medium or the MS medium, as well as 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 of 2,4-D, at least 0.01 to 1 mg / L of kinetin, at least 0.01 to 1 mg / L of NAA, and at least 0 to 10 mg / L of 6-BA.

[0074] In a further embodiment of the present invention, the specific medium composition used to isolate meristematic cells from the midrib or other leaf veins of a leaf is optimized to promote the growth of the target cell type of a specific plant species. This is achieved by testing different concentrations and ratios of basic components including appropriate bases, carbon and nitrogen sources, vitamins, medium optimization additives, and plant hormones at different auxin:cytokinin ratios.

[0075] More specifically, to promote growth, the dissected explants are placed on a cell induction medium and incubated under specific temperature and light / dark conditions. In particular, the growth of cells from the central vein or other leaf veins is visually distinguishable within 3 to 5 days of incubation, as opposed to the growth of dedifferentiated cells from other regions of the explants that form visually distinguishable callus from the 7th day. In one embodiment of the present invention, the faster-growing front leaf cell population is identified within 6 days of incubation. Subsequently, this cell population is excised from the explant and transferred to a new Petri dish containing the cell induction medium.

[0076] Next, the cells are isolated from the leaves, which means identifying and separating the desired stem cells 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 a solid and / or liquid cell induction medium.

[0077] Thus, in one embodiment, a method for isolating plant stem cells or establishing a stem cell line is provided, the method comprising the step of culturing at least one stem cell from the central vein or other leaf veins. In one embodiment, the culturing comprises culturing on a solid and / or liquid cell induction medium.

[0078] The stem cells can be cultured on a cell induction medium under specific temperature and light / dark conditions. For example, a light / dark cycle of 24 hours / 0 hours to 0 hours / 24 hours, more preferably a light / dark cycle of 18 hours / 6 hours, can be employed. At the same time, a temperature of 16 - 30 °C can be used. Preferably, the cells are cultured at a temperature of 25 ± 1 °C.

[0079] The above embodiments are applicable to other methods described herein, such as methods for obtaining stem cell lines, methods for producing plant natural products or extracts, etc.

[0080] Furthermore, suspension culture is initiated by culturing the isolated stem cells on different Petri dishes in a cell line induction medium. Optionally, high-performance cell lines are selected in response to plant immune elicitors.

[0081] Subsequently, the cells can be inoculated into a glass flask containing suspension initiation medium (SIM) and cultured in liquid to establish a plant stem cell suspension culture. Preferably, the glass flask and the suspension initiation medium are sterile. The suspension initiation medium is also called a plant suspension medium. The flask can be incubated at 21 - 25°C with a stirring speed of 110 - 1200 RPM under appropriate light and dark conditions. The growth of the cultured cells can be constantly observed using an inverted stereomicroscope and measured after 14 days of incubation.

[0082] Furthermore, suspension culture is initiated. The isolated stem cell clusters are cultured on a solid cell induction medium under specific temperature and light / dark conditions. For example, a light / dark cycle of 24 hours / 0 hours can be adopted to a light / dark cycle of 0 hours / 24 hours, more preferably a light / dark cycle of 18 hours / 6 hours. At the same time, a temperature of 16 - 30°C can be used. Preferably, the cells are maintained at a temperature of about 25 ± 1°C.

[0083] An independent cell population can be inoculated into a sterile glass flask containing suspension initiation medium (SIM). The flask can be incubated at 21 - 25°C with a stirring speed of 110 - 130 RPM under appropriate light and dark conditions. The growth of the cultured cells can be constantly observed using an inverted stereomicroscope. Cell growth can be measured after 14 days of incubation.

[0084] The measurement of cell growth can be calculated, for example, by transferring 25 ml of cell suspension into a sterile 50 ml conical flask and allowing the cells to settle vertically for 15 minutes. Record the cell set volume (CSV15), which is expected to reach at least a 10-fold increase during a 2-week incubation. After growth evaluation, the cells are subcultured in fresh SIM medium at a 1:10 CSV15:SIM ratio (e.g., subculture 5 ml of CSV15 into a medium with a final volume of 50 ml in a 250 ml sterile glass flask). The subculture process may be repeated every 14 days until a subculture volume of 100 ml is reached, and a constant growth rate is observed during each 14-day subculture. At this stage, the established suspension culture consists of a homogeneous population of meristematic cells.

[0085] In one embodiment, cell growth is measured every 14 days of incubation.

[0086] In one embodiment, the measurement of cell growth includes inoculating a known volume of suspension initiation medium (SIM) with 1 / 5 volume of a stem cell suspension of known density / volume, and measuring the density occupied by the stem cells after a 14-day incubation period. Thus, cell growth can be defined as an increase in cell density within a 14-day culture period, for example, an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, or 200% or more.

[0087] In another embodiment, the measurement of cell growth includes measuring the cell set volume on day 1 and then on day 14. The step of measuring the cell set volume includes transferring at least 1 / 5 of the volume of a stem cell suspension of known density / volume into a known volume of suspension initiation medium (SIM), setting the cells for 15 minutes, and measuring the volume of the mixture (cell set volume, CSV). Thus, cell growth can be defined as an increase in the CSV of the cells, for example, an increase of 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1000% or more within a 14-day culture period.

[0088] In another embodiment, the measurement of cell proliferation includes the step of measuring the cell number on day 1 and then on day 14. In one embodiment, cell proliferation is defined as an increase in the total cell number during a 14-day culture period, for example, an increase in the total cell number of at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, or 800%.

[0089] The cells contained within the established plant stem cell suspension culture flask include stem cell lines via the methods described herein.

[0090] Accordingly, in one aspect of the present invention, a culture medium optimized for the maintenance and / or proliferation of an isolated stem cell and / or a population of stem cell lines is provided.

[0091] In one embodiment, the culture medium is optimized to maintain a substantially homogeneous population of plant stem cells under culture conditions.

[0092] In one embodiment, the culture medium is a stem cell suspension medium comprising the composition of Table 2 or Table 4.

[0093] In an alternative or additional embodiment, the medium is a co-culture medium comprising the above-described plant stem cell suspension medium supplemented with 50 μM acetosyringone.

[0094] In a further aspect of the present invention, a stem cell line established from the above-described stem cell culture method is provided.

[0095] The high genetic stability and homogeneity of the isolated stem cell population of the present invention provide high-quality stem cell lines that do not have or substantially do not have undesirable genetic abnormalities induced by the dedifferentiation processes required by alternative methods.

[0096] Furthermore, optionally, a selection strategy can be applied to enable the identification of cell lines that produce target natural products in high yields. This selection strategy enables the identification of high-performance cell lines from the early stages of the cell line development process.

[0097] Accordingly, in a further aspect of the present invention, a method for selecting a high-performance stem cell or stem cell line is provided, the method comprising the steps of screening for said high-performance trait in at least one single plant stem cell; and selecting said single plant stem cell or a cell having the high-performance trait.

[0098] In one embodiment, the cells are screened and / or selected at the single cell level.

[0099] The high-performance indicator or trait, high-performance cell or cell line means a cell or cell line with improved plant performance with respect to cell size, proliferation, product yield or generative ability, preferably, said improvement is relative to another / other cells in the cell line. Accordingly, the terms "high-performance trait" and "improvement in plant performance" are used interchangeably.

[0100] In a preferred embodiment, the high-performance trait is a high yield of target natural product in response to a plant immune elicitor. Accordingly, in one embodiment, the method further comprises the steps of treating a suspension culture with a plant elicitor and screening for at least one, for example, the production of phytochemicals.

[0101] In one embodiment, the plant elicitor is a plant immunopotentiator.

[0102] A plant immune response inducer or activator (or simply "plant inducer") is a molecule that can be recognized by plant cells and induces a plant defense response. 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, a bacterial flagellin homolog is used as an inducer of the immune response. Immune elicitors are known to those skilled in the art and include harpin (HrpZ), flagellin, cold shock protein, elongation factor (EF-Tu), lipopolysaccharide (LPS), peptidoglycan, oligogalacturonide, lipopeptide, dimethyl sulfide, pseudobacterin, tri-N-alkylated benzylamine derivative (NABD), 2,4-diacetylphloroglucinol (DAPG), oligogalacturonide, extracellular ATP and / or DL-β-aminobutyric acid (BABA), etc. Immune inducers may be synthetic, such as salicylic acid and its analogs, probenazole (PBZ), 1,6-dichloro-isonicotinic acid (INA), benzothiadiazole (BTH), thiazinyl (TDL), isothiazinyl, N-cyanomethyl-2-chloroisonicotinamide (NCI), 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA), etc. For example, abiotic stress inducers for osmotic stress can also be used to stimulate the immune response of stem cells.

[0103] In a preferred embodiment, the plant inducer is selected from salicylic acid, methyl jasmonate, coronatine, chitosan and / or an osmotic stress inducer.

[0104] In a preferred embodiment, the phytochemicals belong to phytochemicals of phenols, flavonoids and / or terpenoids.

[0105] In one embodiment, the yield of natural products in response to plant immune response elicitors is measured. Assays for measuring natural products are known to those skilled in the art and are described in the literature (Altemimi et al., 2017). A colorimetric assay can be used that employs a reagent that undergoes a measurable color change in the presence of the analyte. Well-known instruments such as high-performance liquid chromatography can be used for the purification of natural products. The purified compound can then be identified by various spectroscopic techniques such as UV-visible, infrared, nuclear magnetic resonance, and mass spectrometry.

[0106] In one embodiment, the abundance of the target gene transcript is measured after exposure to the elicitor. In a preferred embodiment, the transcript is selected from genes related to plant hormone signaling, such as PATHOGEN-RELATED1 or PLANT DEFENSIN1.2. In a preferred embodiment, the abundance of the transcript is measured using RT-qPCR. In a further embodiment, the abundance of the transcript in response to the immune elicitor is measured by RNA sequencing. In a preferred embodiment, the abundance of the transcript is measured at the single cell level.

[0107] In another preferred embodiment, the high-performance trait is high proliferative capacity. In one embodiment, screening for said high-performance trait involves measuring iododeoxyuridine incorporation (IdU, similar to fluorescent BrdU). In another embodiment, screening for said high-performance trait involves measuring cyclin B1, cyclin A, and phosphorylated histone H3 using a complementary fluorophore-antibody conjugate. Detection of such markers can be achieved by flow cytometry or can be combined in mass cytometry as reported by Behbehani et al., 2012.

[0108] In another preferred embodiment, the high-performance trait corresponds to cell lifespan. The lifespan can be screened using a marker-assisted approach. In one embodiment, screening for the high-performance trait includes measuring the expression of telomerase and / or WOX4 and / or CLE41 / 44 and / or CRE1 and / or WOL and / or AHK, where high expression correlates with high performance.

[0109] Selection means selecting stem cells or cells or stem cell lines for further subculturing, screening, and / or genetic modification of the selected cells or cell lines.

[0110] More specifically, the present invention describes a method of isolating a plurality of suspension culture strains and selecting them according to their growth rate parameters. Appropriate growth parameters and measurement methods have been discussed above. Further, faster-growing cell lines can be selected according to their natural product production ability. By this multiple selection process, a cell population with a high growth rate and a high yield of the desired natural product is isolated.

[0111] In one embodiment, the selection process consists of the steps of treating the suspension culture with plant elicitors (e.g., but not limited to, salicylic acid, methyl jasmonate, coronatine, and chitosan, osmotic stress inducer), and evaluating the production of specific phytochemical families including but not limited to phenolics, flavonoids, or terpenoids. Further, the anti-radical activity (ARA) of the isolated and cultured cells is an additional measure for confirming the biological activity of the synthesized natural products.

[0112] Assays for measuring these natural product families are known to those skilled in the art and are described in the literature. However, the selection strategy in the present invention is based on the isolation and culture of target meristematic cells, applies colorimetric tests in parallel to evaluate the response to plant elicitors, and subsequently selects the cell line showing the highest yield of the target natural product.

[0113] In another embodiment of the present invention, in order to increase the production of target natural products in a selected high-performance plant stem cell suspension cell line, the concentration and composition mixture of plant elicitors can be further optimized. Thus, after the identification of a cell population with higher production of natural products, this cell line is further subjected to a secondary elicitor screening by testing elicitors in the range of 0.001 μM to 1,000 μM. Furthermore, the growth stage of the culture at the time of elicitation and the duration of the elicitation treatment are optimized as part of this selection process.

[0114] According to the method described in the present invention, cells can be cultured without undergoing dedifferentiation into callus. That is, the method can include a culture in which the cell culture does not contain or substantially does not contain dedifferentiated cells and / or protoplasts. Methods for generating cell suspension cultures from different tissues and different plant species are known. However, in such methods, different tissues are used as explants as a whole and do not undergo selective dissection to isolate leaf veins from leaves. The initiation of cell suspension culture from the whole tissue is typically the result of, for example, epidermal cells, palisade cells, guard cells, etc. undergoing a dedifferentiation process, thereby returning to pluripotent cells that can undergo mitotic cell division. The problem associated with this is that the resulting dedifferentiated cells accumulate significant harmful genetic and epigenetic modifications due to the dedifferentiation process. According to the present invention, the cells surrounding the leaf veins are isolated so that they can continuously divide without undergoing a dedifferentiation process and have the ability to retain pluripotent activity.

[0115] In plants, only meristematic cells / stem cells found in either the primary meristems of plants involved in the growth of leaves, buds, flowers and roots or the secondary meristems required for the increase in the diameter of stems and roots are able to divide actively, i.e., are mitotically active. When meristematic cells differentiate into specific cell types, such as palisade cells, epidermal cells and mesophyll cells within leaves, these differentiated cells can no longer divide. Therefore, the initiation of cell suspension cultures from either whole organs or tissues results from the differentiated cells present in these plant organs or tissues undergoing a dedifferentiation process, which leads to the reactivation of their mitosis. These dedifferentiated plant cells then regain the ability to divide. However, this dedifferentiation process results in significant detrimental genetic and epigenetic modifications and does not lead to optimal performance of the dedifferentiated cells in suspension cultures with respect to proliferation, cell aggregation and the production of natural products. According to the present invention, plant stem cells that divide continuously and naturally have the ability to retain pluripotent activity are isolated.

[0116] In particular, the present invention is not based on the isolation of callus derived from whole leaves. The method of the present invention relies on exposing a thin layer of cells located around the central vein or other leaf veins within the leaf and then isolating leaf stem cells that are immortal, undifferentiated, grow at a faster rate and exhibit a specific morphology (numerous amyloplasts, small size). The faster growth rate of these leaf stem cells enables their isolation from dedifferentiated cells, resulting in a homogeneous cell population. Furthermore, the method according to the present invention is rapid, easy to implement and applicable to a wide variety of plant genera.

[0117] Furthermore, the isolation of a homogeneous population of stem cells enables the classification of cell lines according to their ability to produce secondary metabolites in response to immunostimulatory factor screening. This selection enables the identification of high-performance cell lines from the early stages of the growth process.

[0118] For example, a major concern when isolating meristematic cells from a stem (especially a woody plant) is the increased risk of contamination by high levels of endophytes (microorganisms) present on the stem surface. To overcome this problem, the method according to the present invention enables the isolation of stem cell-like meristematic cells from the central vein of a leaf rather than from the stem. Leaves typically have a smoother surface compared to the stem, which facilitates the surface sterilization process and reduces the microbial contamination rate on solid media.

[0119] In one aspect of the invention, there is provided a plant stem cell or stem cell line obtainable or obtained by any or all of the methods described herein. Further provided is a plant, a part of a plant or a cell line obtainable or obtained by the methods described herein. In one embodiment, a product or extract of a plant stem cell or stem cell line is obtainable or obtained by the methods described herein. Also provided is a composition comprising stem cells, stem cell lines, plant products and / or plant extracts obtainable or obtained from the methods described herein. In one embodiment, the stem cell line has not undergone dedifferentiation into callus.

[0120] In a further aspect, there is provided a stem cell line obtained from a tissue derived from the primary meristem in a leaf of a plant having the following characteristics: a. formed from undifferentiated cells; and b. the stem cells of the line are morphologically characterized by the presence of a plurality of amyloplasts; and c. is a high-proliferation cell line; and d. optionally, has high phytochemical accumulation in response to elicitation.

[0121] A high-proliferation cell line means a cell line with enhanced proliferation compared to control cells, such as differentiated leaf cells or dedifferentiated cells.

[0122] In additional or alternative embodiments, the plant stem cell line comprises a substantially genetically homogeneous population of undifferentiated cells.

[0123] In a further aspect, there is provided a method of producing a plant or a part of a plant and / or a method of crop breeding, the method comprising: a. isolating at least one stem cell derived from tissue from the primary meristem in a leaf of a plant; b. establishing a stem cell line comprising at least one isolated stem cell, preferably wherein the stem cell line is substantially genetically homogeneous; c. optionally, selecting a high-performance cell line; and d. growing a new plant or a part of a plant from at least one stem cell of the stem cell line.

[0124] In one embodiment, the method further comprises growing a plant or a part of a plant using vertical growing practices. In a preferred embodiment, the high-performance cell line is a high-yield and / or highly proliferative cell line.

[0125] Also provided is a composition comprising a stem cell, an extract or a product thereof obtainable or obtained from the above-mentioned stem cell line.

[0126] As described above, the plant stem cells and cell lines presented herein exhibit greater proliferative ability and genetic homogeneity compared to dedifferentiated cells and cell lines.

[0127] In a preferred embodiment, the stem cell line is obtained by the method according to the invention.

[0128] In a further aspect of the invention, there is provided a method for producing a plant natural product or extract, the method comprising: a. isolating at least one stem cell derived from tissue from the primary meristem in a leaf of a plant; b. establishing a stem cell line comprising at least one isolated stem cell, preferably wherein the stem cell line is substantially genetically homogeneous; c. optionally, selecting a high-performance cell line; and / or d. Optionally, a step of inducing the production or secretion of a plant natural product or extract; and e. A step of collecting the plant natural product or extract.

[0129] In a further aspect of the present invention, there is provided a composition comprising at least one plant stem cell, preferably a plurality of stem cells, preferably a stem cell line obtained or obtainable by the method described herein.

[0130] 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 a plant stem cell product derived from a stem cell line obtained or obtainable by the method described herein.

[0131] 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 cells or stem cell lines 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 exhibiting high-performance indicators or traits, or a plant natural product and / or extract obtained from said cell or cell line.

[0132] In a preferred embodiment, the composition is for use in or as a cosmetic. The cosmetic may be for use on humans or animals. Thus, in one embodiment, a composition for use in a cosmetic is disclosed, which comprises at least one stem cell, preferably a plurality of stem cells and / or plant stem cell products and / or plant stem cell extracts, preferably derived from a stem cell line that can be obtained or is obtained by the method described herein. The cosmetic composition can be formulated in any known manner, for example, as a cream, lotion, water, cleansing liquid, shampoo, oil, etc. The concentration of the extract is usually between 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.

[0133] In the art, it is well known that many plant stem cells, extracts and products have strong antioxidant and / or anti-inflammatory effects. For example, Vitis vinifera, Syringa vulgaris, Uttwiler spatlauber, etc. Thus, the composition may also contain additional components, such as phytochemicals, antioxidants and / or anti-inflammatory components. Examples of antioxidants include, for example, tocopherol, lecithin, ascorbic acid, vitamin E, vitamin C, coenzyme Q10, anthocyanin, curcumin, etc. Examples of anti-inflammatory compounds include aloe, turmeric, safflower extract, witch hazel, niacinamide, sea buckthorn oil, vitamins C, D and E, oatmeal or ginger. Further components may be emulsifiers and / or gelling agents, such as glycyrrhizin. In another embodiment, the composition is for use in agricultural compositions. In one embodiment, the composition can be a single-functional or multi-functional plant stem cell growth composition. Specific uses of such compositions include an increase in water absorption rate, a reduction in the use of excessive pesticides, and use in increasing the quality and / or performance of crops. The agricultural composition may further include, but is not limited to: preferably plant stem cells in a liquid matrix, antibacterial agents, starch, trace metal elements that can act as fertilizer synergists, plant growth regulators, photosynthetic agents and / or water retention agents, etc. Further, the plant stem cell matrix solution can be selected from one or more of agar, polyphenols, flavonoids, nucleic acids, and protein hydrolysates. Further, the antibacterial agent can be selected from one or more of amino acids, metal salts, chitosan powder, and chitin. Further, the trace element fertilizer synergist may include one or more of iron, manganese, boron, zinc, copper, molybdenum, chlorine, cobalt, etc. Further, the plant growth regulator includes one or more of auxin, gibberellin, chlormequat, cytokinin, and abscisic acid. Further, the photosynthetic agent may be sodium bisulfite, etc. Further, the water retention agent is humic acid, etc.

[0134] In another embodiment, the composition is for use in a pharmaceutical composition. In one embodiment, the pharmaceutical composition is for use in the treatment or prevention of proliferative diseases (e.g., cancer), angiogenesis and cardiovascular diseases, inflammatory diseases, bone-forming diseases, metabolic diseases, and neurological diseases. Plant cell extracts for use in pharmaceutical compositions are well known in the art and are widely reviewed, for example, in Li, et al. Stem Cell Research & Therapy (2022) 13:472.

[0135] In another embodiment, the composition is for use in a food and / or beverage composition.

[0136] For any of the above compositions, the composition may further comprise at least one human stem cell, its extract or product.

[0137] The present invention may be further illustrated by the following non-limiting description: A method for isolating a stem cell line derived from tissue from the primary meristem in a plant leaf, the method comprising the following steps: (a) sterilizing at least a part of the leaf containing the central vein or other leaf veins; (b) cutting the central vein or other leaf veins of the leaf to expose primary meristem cells from the internal vein tissue of the central vein or other leaf veins; and (c) culturing the cells from the exposed cells in a cell induction medium.

[0138] A stem cell line derived from tissue from the primary meristem in a plant leaf is understood to be a cell line that is self-renewing, undifferentiated, and capable of producing other cells that, for example, have a faster growth rate and a smaller size compared to other plant cells. In the context of the present invention, plant stem cells are comparable to mammalian stem cells.

[0139] The sterilization step is a step of removing any type of microorganism that contaminates the surface of the plant explant used for the isolation of the target cells.

[0140] Sterilizing agents that can be used are common in the art and can be, for example, sodium hypochlorite (bleaching agent), calcium hypochlorite, hydrogen peroxide, ethanol, etc. Preferably, sodium hypochlorite is used at a concentration of 0.5 - 1.0% for 30 - 40 minutes for sterilization. Calcium hypochlorite powder should be dissolved in water before use and then filtered. Typically, it is used at a concentration of 3.25%. Hydrogen peroxide can be used at a concentration of 3%.

[0141] Ethanol can be used, for example, at a concentration of 70 - 95% for sterilization.

[0142] These microbial cells grow much more rapidly than plant cells and quickly contaminate the method of isolating the cells, so it is essential to remove as many microorganisms as possible from the explants used. At the same time, useful sterilizing agents or bleaching treatments such as ethanol can be toxic to plant cells at high concentrations or long exposure times. Therefore, it is desirable to minimize the effects of these sterilizing agents by maintaining the shortest incubation time, preferably the minimum effective concentration of these chemicals throughout the sterilization procedure. Importantly, leaves have a smoother surface compared to the surface of the stem, which allows for a less irritating and gentler sterilization procedure, increasing the probability of isolating a viable and healthy cell population. Therefore, when leaves are used as explants, they can be held in the solvent for a short time of about 1 - 15 minutes while still removing most of the harmful microorganisms and ensuring that the explants are not damaged.

[0143] After sterilization, carefully cut (incise) the central vein or other leaf veins of the leaf to expose the meristematic 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 along the length of the leaf from the base of the leaf towards the tip of the leaf. Other leaf veins are understood to be other leaf veins present in the leaf near the central vein, such as lateral leaf veins or secondary leaf veins. Preferably, the leaf vein selected to expose the cells is the central vein. This is because typically, the central vein is thicker than the lateral secondary leaf veins, making the operation easier and enabling more cells to be exposed. Preferably, the central vein or other leaf veins are cut along their length, and thus in the case of the central vein, in the direction from the tip to the base or from the base towards the tip of the leaf. In a preferred embodiment, the leaf is cut into two along the central vein and through it.

[0144] This careful leaf incision process exposes meristematic cells, such as prefoliar meristematic cells, in the vascular tissue derived from the meristem. Subsequently, these cells are subjected to a culturing process where, as further described, proliferation is induced in the presence of an appropriate cell induction medium.

[0145] After exposing the target cells as described in the previous step, the area of the explant containing the meristematic cells is subjected to a culturing process on a cell induction medium.

[0146] In one embodiment of the method, the cells do not undergo dedifferentiation into callus.

[0147] In a further embodiment, the culturing process includes the following: (a) isolating the meristematic cells from the internal vein tissue of the central vein or other leaf veins; (b) initiating suspension culture; and (c) optionally, selecting a high-performance cell line in response to a plant immune elicitor.

[0148] In one embodiment of the method, the start of suspension culture involves incubating a plate containing isolated primary meristem cells at 16 - 30 °C in a 24 hours / 0 hours light / dark - 0 hours / 24 hours light / dark cycle.

[0149] In one embodiment of the method, the cell induction medium contains macro, micronutrients and / or plant hormones.

[0150] In one embodiment of the method, the cell induction medium contains components that make up B5 medium or MS medium, as well as one or more of 2,4 - D, kinetin, NAA, 6 - BA and other plant growth regulators.

[0151] In one embodiment of the method, the cell induction medium contains 0 - 10 mg / L of 2,4 - D, 0.01 - 1 mg / L of kinetin, 0.01 - 1 mg / L of NAA and 0 - 10 mg / L of 6 - BA.

[0152] In a further embodiment of the invention, the specific medium composition used to isolate meristem cells from the central vein or other leaf veins of the leaf is optimized to promote the growth of the target cell type of a specific plant species. This is achieved by testing different concentrations and ratios of basic components, including appropriate bases, carbon and nitrogen sources, vitamins, medium optimization additives and plant hormones, at different auxin:cytokinin ratios.

[0153] The culturing process according to the present invention relates to a process carried out after the exposure of target cells. More specifically, before culturing, the leaves of the target plant species are sterilized, and the central vein or other leaf veins of the leaves are incised to expose a thin layer of the desired stem cells. For example, the leaf veins are cut to expose the primary prefoliar cells. These and other primary meristematic cells are self-renewing, undifferentiated, and exhibit a faster growth rate than classical differentiated cells. In the culturing process, the cut leaves are placed on a cell induction medium to promote the growth of meristematic cells. Next, the cells are isolated from the leaves, which means identifying the desired stem cells and separating them from the explants. Further, a suspension culture is initiated in which the isolated stem cells are cultured on different Petri dishes in a cell line induction medium. Optionally, high-performance cell lines are selected in response to plant immune elicitors.

[0154] In a further aspect, there is provided a stem cell line obtained from a tissue derived from the primary meristem in the leaves of a plant having the following characteristics: (a) Formed from undifferentiated cells; (b) Morphologically characterized by the presence of a plurality of amyloplasts; (c) A high-speed growth cell line; (d) Optionally, having high phytochemical accumulation in response to induction.

[0155] In one embodiment, the above stem cell line is obtained by any of the above methods.

[0156] Cosmetic, agricultural, pharmaceutical, food, beverage or human / animal healthcare compositions comprising cells derived from a cell line according to any of the above embodiments.

Examples

[0157] The examples of the present invention will be described below. The isolation and induction of plant stem cells derived from the primary meristem, and the subsequent generation of suspension cultures, along with cell line selection, are described for G. glabra, T. baccata, Q. saponaria, Q. indica, but by extension, can be utilized for any foliar plant. The following examples are provided by way of illustration and not limitation.

[0158] Example 1. Preparation of plant materials from G. glabra and isolation of plant stem cells. Surface sterilization of plant tissues. Leaves of G. glabra plants were collected. After the leaves were collected, they were surface sterilized sequentially with 70% ethanol for 10 minutes, 10% bleach (containing 5% sodium hypochlorite) for 10 minutes, and 1% bleach (containing 5% sodium hypochlorite) for 10 minutes. After surface sterilization, they were washed 3 - 4 times with distilled water.

[0159] Preparation of internal vascular tissue from leaves of G. glabra. The leaves of G. glabra were placed horizontally on a hard surface. The leaves were cut vertically with a sharp object such as a scalpel or razor, and it was necessary to cut across the leaf veins or other leaf veins of the leaves to expose the internal vascular tissue containing the target plant stem cells. The incised leaves were placed on the medium.

[0160] On the 3rd to 7th day after culturing the leaf explants, visible callus was observed on the exposed main veins or other leaf veins of the leaves. At this point, the remaining leaves could be removed. The proliferative meristem stem cells derived from the previous leaves were individually cultured on different Petri dishes using a culture medium such as a cell line induction medium. For the purpose of cell and callus induction, MS (Murashige & Skoog medium) was used. Other available media are known in the art, for example: B5 (Gamberg's B5 medium), WPM (Lloyed & McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre), etc. The application of all these media is possible. The components of MS are shown in Table 1. The cultures were grown at 25 ± 1 °C under a 16 - hour / 8 - hour light / dark cycle.

Table 1

[0161] Among the calli, white and fragile calli with good growth rates were subcultured onto fresh medium every 14 days. The proliferation rate of meristematic cells derived from the exposed main vein or other leaf veins was fast and there was no "browning", and then these cells could be easily selected as stable cell lines. The calli obtained from G. glabra were white - green due to light conditions and were maintained as single cells or small cell clusters after 6 months of culture in liquid medium.

[0162] Establishment of G. glabra suspension culture Cells derived from the exposed leaf veins of G. glabra were cultured in flasks containing the liquid media listed in Table 2:

Table 2

[0163] The cells were cultured on a rotary shaker at 120 rpm at 25 ± 1 °C under a 16 - hour / 8 - hour light / dark cycle with a subculture interval of 2 weeks.

[0164] Example 2. Preparation of plant materials and isolation of plant stem cells from Q. indica. Surface sterilization of plant tissues. Leaves of Q. indica plants were collected. After the leaves were collected, they were surface - sterilized sequentially with 70% ethanol for 10 minutes, 10% bleach (containing 5% sodium hypochlorite) for 10 minutes, and 1% bleach (containing 5% sodium hypochlorite) for 10 minutes. After surface sterilization, they were washed 3 - 4 times with distilled water.

[0165] Preparation of internal vascular tissue from leaves of G. indica. Leaves of G. indica were placed horizontally on a hard surface. The leaves were cut vertically with a sharp object such as a scalpel or knife, and it was necessary to cut across the leaf veins or other leaf veins of the leaves to expose the internal vascular tissue containing the target plant stem cells. The dissected leaves were placed on the medium.

[0166] On the 3rd to 7th day of culture, while the calli began to form and expand on the exposed main vein or other leaf vein cuts of the leaf, the remaining part of the leaf could be removed.

[0167] The calli were cultured individually on different Petri dishes using a cell line induction medium. For the purpose of cell and callus induction, MS (Murashige & Skoog medium) was used. Other available media are known in the art, for example: B5 (Gamberg's B5 medium), WPM (Lloyed & McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre), etc. The application of all these media is possible.

[0168] The components of MS are shown in Table 1. The cultures were grown in the dark at 25 ± 1 °C. Among the calli, white and fragile calli with a good growth rate were subcultured into fresh medium every 14 days. The growth rate of the target meristematic cells derived from the exposed main vein was fast without browning, and these cells could be easily selected as stable cell lines. The calli obtained from Q.Indica had a white to yellow color and were maintained as single cells or small cell clusters after 6 months of culture.

[0169] Establishment of Q.indica suspension culture The cultures derived from the exposed leaf veins of G.glabra were cultured in flasks containing the liquid media listed in Table 2.

[0170] The cells were cultured on a rotary shaker at 120 rpm at 25 ± 1 °C in the dark at a subculture interval of 2 weeks.

[0171] Example 3. Preparation of plant materials and isolation of plant stem cells in T.baccata and / or Q.saponaria. Surface sterilization of plant tissues. Leaves of T.baccata and / or Q.saponaria plants were collected. After collecting the leaves, they were surface sterilized successively with 70% ethanol for 10 minutes, 10% bleach (containing 5% sodium hypochlorite) for 10 minutes, and 1% bleach (containing 5% sodium hypochlorite) for 10 minutes. After surface sterilization, they were washed 3 - 4 times with distilled water.

[0172] Preparation of vascular tissue from the leaves of T. baccata and / or Q. saponaria. The leaves of T. baccata and / or Q. saponaria were placed horizontally on a hard surface. The leaves were cut vertically with a sharp object such as a scalpel or razor blade, and it was necessary to cut across the leaf veins or other leaf veins of the leaves in order to expose the internal vascular tissue containing plant stem cells. The dissected leaves were placed on the medium.

[0173] On the 3rd to 7th day of culture, while the callus begins to form and expand on the exposed main vein or other leaf vein cuts of the leaf, the remaining part of the leaf can be removed. The callus was cultured individually on different Petri dishes using a cell line induction medium.

[0174] For the purpose of cell and callus induction, B5 (Gamberg's B5 medium) was used. Other available media are known in the art, for example: MS (Murashige & Skoog medium), WPM (Lloyed & McCown), SM (schenk & Hildebrand medium), LP (Quoirin & Lepiovre), etc. The application of all these media is possible.

[0175] The components of B5 are shown in Table 3. The cultures were grown in the dark at 25 ± 1 °C.

[0176] Among the callus, white and fragile callus with a good growth rate were subcultured onto fresh medium every 14 days. The growth rate of the target meristematic cells derived from the exposed main vein was fast without browning, and these cells could be easily selected as stable cell lines. The callus obtained from T. baccata and / or Q. saponaria was white to yellow and was maintained as either single cells or small cell clusters of cells after 6 months of culture.

Table 3

[0177] Establishment of suspension cultures of T. baccata and / or Q. saponaria Cultures derived from the exposed leaf veins of G. glabra were cultured in flasks containing the liquid media listed in Table 4.

Table 4

[0178] The cells were cultured on a rotary shaker at 120 rpm at 25 ± 1 °C with a 16 h / 8 h light / dark cycle at a subculture interval of two weeks.

[0179] Example 4. Comparison between dedifferentiated cells isolated from plant leaves and primary meristematic cells isolated by the method according to the invention. Iodine Lugol staining and microscopic analysis were performed to evaluate phenotypic differences between dedifferentiated cells isolated by the production of plant cells from whole leaf organs or random leaf pieces and plant cells isolated and cultured from the central vein or other leaf vein incision methods according to the invention. This staining procedure enables visualization of amyloplasts, organelles involved in nutrient storage that are typically present in cells not undergoing photosynthesis.

[0180] Samples of T. baccata and G. glabra were obtained according to the procedures shown in Examples 1 and 2. Iodine Lugol staining was unable to detect the presence of amyloplasts in either Taxus baccata or Glyzhyrryza glabra dedifferentiated cells. However, the cells obtained by the central vein or other leaf vein incision methods of the present invention showed numerous amyloplasts and were clearly distinguishable from classical dedifferentiated cells.

[0181] Example 5. Comparative analysis of the contamination rates of samples derived from stems and samples derived from leaves used in the present invention. Samples of stem and leaves were collected from Quassia indica, a tropical tree, which showed a high microbial load and high contamination rate after surface sterilization according to the standard protocol. After collecting the explants, they were surface sterilized with 70% v / v aqueous ethanol solution for 10 minutes and bleached for 10 minutes at the concentrations shown in the following table. After surface sterilization, the samples were washed 3 - 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 total explants was calculated by the standard method. The results are shown in the following table.

[0182] As can be seen from the table, the sterilization of leaves results in a very low contamination rate under the same sterilizing agent and sterilization time conditions compared to the treatment of stems, which is particularly useful in the method of isolating stem cells.

[0183] More stringent sterilization conditions (higher concentration of sterilizing agent, such as concentrated bleach solution and / or longer sterilization time) can improve the risk of contamination on stem or leaf samples, but can damage the samples and have an adverse effect on the viability of the isolated cells.

Table 5

Claims

1. A method for obtaining at least one plant stem cell, comprising: a. sterilizing at least a part of a leaf including a midrib or other leaf veins; b. incising the midrib or other leaf veins of the leaf to expose meristematic tissue cells from the midrib or other leaf vein tissues; and c. obtaining at least one stem cell from the inner vein tissue of the midrib or other leaf veins A method comprising the above steps.

2. The method according to claim 1, further comprising culturing the at least one stem cell to obtain a stem cell line.

3. The method according to claim 1 or 2, characterized in that the method does not include or substantially does not include the formation of callus obtained from dedifferentiated cells or substantially containing dedifferentiated cells.

4. The method according to any one of claims 1 to 3, characterized in that the cell culture does not contain or substantially does not contain dedifferentiated cells and / or protoplasts.

5. The culturing step comprises: a. starting a suspension culture containing the cultured meristematic tissue cells from the inner vein tissue of the midrib or other leaf veins; and b. optionally, preferably in response to a plant immune inducer, selecting a high-performance cell line The method according to any one of claims 2 to 4, further comprising the above steps.

6. The method according to any one of claims 2 to 5, characterized in that the cell culture does not contain or substantially does not contain dedifferentiated cells and / or protoplasts.

7. The culturing step comprises: a. isolating the meristematic tissue cells from the inner vein tissue of the midrib or other leaf veins; b. starting a suspension culture containing the cultured meristematic tissue cells from the inner vein tissue of the midrib or other leaf veins; and c. optionally, preferably in response to a plant immune inducer, selecting a high-performance cell line The method according to any one of claims 2 to 6, further comprising the above steps.

8. The step of starting the suspension culture comprises incubating a plate containing the isolated meristematic tissue cells at 16 to 30 °C in a 24 h / 0 h light / dark to 0 h / 24 h light / dark cycle. The method according to claim 7, characterized by including the above steps.

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

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

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

12. A stem cell line derived from the primary meristem of a plant leaf, the stem cell line having the following characteristics: a. Formed from undifferentiated cells, preferably substantially genetically homogeneous cells; and b. The cells of the cell line are morphologically characterized by the presence of a plurality of amyloplasts; and c. A high-proliferation cell line; and d. Optionally, having high phytochemical accumulation in response to induction Characterized in that it has, a stem cell line.

13. A method for producing a plant natural product or extract, a. A step of isolating at least one stem cell derived from a tissue from the primary meristem in a plant leaf, preferably, the at least one stem cell is derived from primary meristem cells from the central vein or other leaf vein tissues, the step; b. A step of culturing at least one stem cell to establish at least one stem cell line, preferably, the stem cell line is substantially genetically homogeneous, the step; c. Optionally, a step of selecting a high-performance cell line; and / or d. Optionally, a step of inducing the production or secretion of a plant natural product or extract; and e. A step of collecting the plant natural product or extract Including, a method.

14. The high-performance cell line is characterized by a high-performance index or trait selected from at least one of cell size, proliferation, product yield and / or developmental ability, preferably, the improvement is with respect to another / other cell / cell group in the cell line, characterized in that, the method according to claim 13.

15. The step of inducing the production or secretion of the plant natural product includes a step of applying at least one plant inducer to the stem cell culture, characterized in that, the method according to claim 13.

16. The plant product or extract is selected from flavonoids, alkaloids, polyphenols, polysaccharides, quinonoids and saponins, characterized in that, the method according to claim 13.

17. The plant is selected from dicotyledonous plants, monocotyledonous plants or gymnosperms, characterized in that, the method according to any one of claims 1 to 8 and 13 to 16, or the plant stem cells according to claim 9.

18. The plant is selected from one embodiment, and the plant is asparagus, grass, palm, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, coriander, sunflower, peanut, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe vera, yews (taxus), eggplant, pineapple, smac, chickpea, rosemary, lychee, licorice (Glycyrrhiza glabra), spinach, soybean, cruciferous plant, carrot, corn, rice, ginseng, fern, pine, garlic, cucumber, pepper, lettuce, peach, watermelon, grape, apple, onion, mandarin, banana, pea, mango, orange, tea, sugarcane, cotton, barley, sorghum, wheat, rice, quassia, magnolia, and a method according to claim 17, characterized in that it is selected from the Quillija species and the Cupressaceae family.

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 according to claim 18, characterized in that the composition is selected from cosmetics, agricultural, pharmaceutical, food, beverage or human / animal healthcare compositions.

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

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