GB2628406A - Methods of using vascular plant stem cells - Google Patents

Abstract

A method of introducing genetic modifications to at least one cultured plant stem cell, wherein the plant stem cell is a vascular stem cell, comprising a. isolating at least one stem cell or cells from a tissue of a meristem of a plant, preferably the primary meristem in a leaf; b. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; and c. introducing said genetic modification into the cell or cells is claimed. Alternatively claimed are methods of producing a genetically altered plant or screening for crop protection agents comprising \parobtaining a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population, wherein preferably the stem cell line is obtained from primary meristem tissue in a leaf of a plant; preferably introducing at least one genetic modification into said stem cells and screening for said target alterations or agents.

Description

Methods of using vascular plant stem cells

FIELD OF THE INVENTION

The present invention relates to plant stem cells, particularly to methods to transform and screen transformed, as well as untransformed, plant stem cells in a high-throughput manner for further culturing or regeneration of whole plants or plant parts.

BACKGROUND

The progression of the climate crisis and the increasing global population has created an unmet demand for new plant varieties with desirable traits. Current processes used to generate new plant varieties are too time-consuming or are associated with negative genetic variations. Desirable traits include higher yield, less consumption of resources including water and fertilizer, improved nutrient content, heightened tolerance to environmental stress, and greater resistance to disease and pests.

Farmers and plant breeders have long relied on traditional breeding techniques to develop plant varieties with desirable traits. In traditional plant breeding, genetic variation is introduced into a plant line through cross pollination between genetically distinct individual. Genetic crosses result in progeny with different combinations of gene alleles. Offspring from two inbred parental lines often have increased vigour (aka "hybrid vigour") and other desirable characteristics. Over several generations, the offspring of genetic crosses can be selected for those with favourable traits, leading to the development of new plant varieties. However, traditional breeding poses several limitations including time consumption, access to narrow genetic variation, lack of precision, restrained by genetic linkage and difficulties in scaling.

Today, Genetic Modification (GM) is used to (for example) introduce exogenous genes of known function into plant varieties and Gene Editing (GE) is used to (for example) directly mutate endogenous genes of interest with limited off-target effects. Whilst these approaches improve breeding efficiency, they still require lengthy periods of plant growth and screening to confirm the presence of an active transgene or the efficacy of a gene edit, and to establish if a genetic change results in a phenotypic difference that could underpin a novel or enhanced plant trait. Therefore, new technologies that support the rapid transformation and identification of plants with desirable traits in a high-throughput manner will be required to meet the high demand for improved elite crops.

The transformation of plant cell types with high differentiation potential presents an opportunity to accelerate the screening and generation times for new plant varieties. The transformation of plant callus tissue formed from dedifferentiated plant cells (DDC) has been used to this end (Ochoa-Villarreal et al. 2015). DDCs originate from differentiated plant somatic tissue and are chemically stimulated into a developmentally potent state. However, the utility of DDCs is limited because their production in tissue culture induces somaclonal variation from an accumulation of random genetic and epigenetic changes in culture (Roberts and Kolewe, 2010). The alterations seen in DDCs can be genetically degradative and result in long term cell senescence. The undesirable genetic variation varies by the somatic tissue, further potentially reducing the safety and confidence in DDCs for producing new plant varieties.

Therefore, current processes used to generate new plants varieties remain inadequate, and the need for novel technologies remains.

SUMMARY OF INVENTION

We have developed methods for the genetic modification, high-throughput screening and selection of desirable plant stem cells at the single cell level. The methods presented here allow the identification and evaluation of trait expression at the single plant cell level. Also presented are methods to select genetically modified high-fidelity cells, and regenerate a genetically faithful plant thereof. Our invention reduces the time required to generate new plant varieties, because only the cells containing desirable traits are selected and optionally regenerated into plant parts or whole plants. Moreover, the plant stem cells and cell lines presented herein display greater proliferation relative to dedifferentiated cells and cell lines, further decreasing the time of plant variety generation. The genetic homogeneity of the plant stem cell lines presented improves the fidelity of the regenerated plants, also reducing the time required to test and evaluate them. Transformed plant stem cells, plant stem cell lines and regenerated plant parts and plants thereof are included within this invention. Also included are the products of the latter. Our work reveals a new strategy for plant breeding that is applicable to all plants of interest.

In one aspect of the invention, there is provided a method of introducing at least one genetic modification to at least one plant stem cell of a stem cell culture, wherein the plant stem cell is a vascular stem cell(s), the method comprising a. isolating at least one stem cell or cells from a tissue from a primary meristem of a plant, preferably the primary meristem in a leaf; b. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; and c. introducing at least one genetic modification into the cell or cells.

In one embodiment, the method does not comprise dedifferentiation of the isolated cell or cells into a callus.

In a further embodiment, the method further comprises selecting for at least one high-performance stem cell in the stem cell culture, the method comprising: a. optionally applying at least one selection pressure to at least one stem cell, wherein said selection pressure corresponds to a high-performance indicator or trait; b. screening for a high-performance trait in the at least one single plant stem cell; and c. selecting said single plant stem cell or cells with the high-performance trait.

In one embodiment, the plant may be a vascular plant. In another embodiment, the plant may be selected from a dicotyledon, a monocotyledon or a gymnosperm.

In one embodiment, the at least one plant stem cell is isolated from meristematic tissue.

In a further embodiment, the at least one plant stem cell is isolated from a primary and/or secondary meristematic tissue.

In one embodiment, the at least one plant stem cell is derived from primary meristematic tissue, preferably from primary meristematic tissue found within and/or surrounding the vasculature of a leaf.

In a preferred embodiment, the at least one plant stem cell is derived from primary meristematic tissue around the central vein or lateral or secondary vein of a leaf.

In one embodiment, the method of genetic modification comprises introducing at least one mutation into the genome of at least one plant stem cell.

In one embodiment, the method of genetic modification comprises introducing at least one exogenous nucleic acid into at least one plant stem cell. In one embodiment, introducing exogenous nucleic acid into the plant stem cell(s) uses transformation.

In one embodiment, the method of genetic modification comprises gene editing.

In a preferred embodiment, the method of gene editing comprises use of CRISPR.

In a further preferred embodiment, the method of gene editing comprises use of CRISPR-Cas9, wherein preferably Cas9 is fused to a base editor.

In an alternative embodiment, the method of gene editing comprises use of TALENS or a Zinc Nuclear Finger (ZNF) domain protein.

In one embodiment, the method of genetic modification comprises transformation. In a preferred embodiment, the method of transformation is Agrobacterium tumefaciens mediated transformation. In an additional or alternative embodiment, the method of transformation is biolistic-based transformation.

In another embodiment, the genetic modification is introduced using transformation, wherein preferably the method comprises introducing exogenous nucleic acid into the plant stem cell(s).

In another aspect of the invention, there is provided a plant stem cell or stem cell line obtained or obtainable by the methods described herein. In one embodiment, products or extracts of the plant stem cell or stem cell line are obtained or obtainable by the methods described herein. In one embodiment, the stem cell line has not gone through dedifferentiation into callus.

In one aspect of the invention, there is provided a method of high-throughput screening of plant stem cells for a target trait, the method comprising a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; b. optionally applying at least one selection pressure to at least one stem cell of the plant stem cell culture wherein said selection pressure corresponds to the target trait; c. screening for said target trait in the at least one single plant stem cell or cells; d. selecting said single plant stem cell or cells with the target trait; and e. optionally regenerating a plant, plant part or plant cell line from said single plant stem cell or cells.

In one embodiment, the method further comprises introducing a genetic modification and then optionally, a selection pressure to the at least one stem cell.

In one embodiment, the plant stem cell(s) is a primary and/or secondary meristematic stem cell(s). In one embodiment, the plant stem cell(s) is a vascular stem cell(s).

In one embodiment, culturing said plant stem cell line comprises culturing said stem cells on a solid and/or liquid medium.

In one embodiment, the stem cell culture does not substantially comprise dedifferentiated cells and/or protoplasts.

In one embodiment, genetic modification comprises introduction of at least one exogenous nucleic sequence.

In one embodiment, genetic modification comprises gene editing.

In one embodiment, genetic modification comprises carrying out CRISPR-Cas gene editing to introduce at least one mutation into a least one target gene and/or promoter.

In one embodiment, genetic modification comprises introducing at least one CRISPR enzyme, wherein the CRISPR enzyme is fluorescently tagged. In one embodiment, the CRISPR enzyme is selected from a DNA base editor, prime editor or nuclease. The method may further comprise selecting cell or cells expressing the fluorescent tag/marker. By selecting cells expressing the fluorescent tag/marker cells that are gene edited are selected.

In one embodiment, the method comprises screening said plant stem cell or cells for a phenotype of the target trait. The phenotype of the target trait may be an improvement in plant performance. Alternatively, or additionally, the method comprises screening said plant stem cell or cells for expression of the target trait.

In one embodiment, screening for a target trait occurs in stem cells in a callus, wherein callus is formed or substantially formed of cells that have not been dedifferentiated into a callus. Alternatively, screening for a target trait occurs at the single cell level.

In one embodiment, the genetic modification and/or selection pressure corresponds to the target trait.

In one embodiment, applying a selection pressure comprises applying a selection agent, wherein the selection agent allows selection of gene edited or transformed plant stem cell or cells.

In a preferred embodiment, the target trait is screened for before and/or after the application of a selection pressure to the at least one stem cell of the plant stem cell culture.

In a preferred embodiment, the target trait is detectable at the single cell level.

In one embodiment, the target trait corresponds to a genetic modification introduced into at least one plant stem cell.

In one embodiment, the target trait may be identified in a screening process, through the measurement of a marker indicative of said target trait.

In one embodiment, the marker of a target trait may be at least one lipid, carbohydrate, glycolipid, protein, glycoprotein, lipoprotein or nucleic acid sequence.

In one embodiment, the marker corresponds to a genetic modification and/or selection pressure for a target trait.

In one embodiment, the marker comprises a selectable marker.

In one embodiment, the marker comprises an antibiotic resistance marker. In one embodiment, the antibiotic resistance marker is selected from kanamycin, G418, bleomycin, streptomycin, spectinomycin, hygromycin, rifampicin and/or a 13-lactam.

In one embodiment, the marker comprises an antimetabolite resistance marker. In one embodiment, the antimetabolite marker is selected from methotrexate and/or sulphonamide resistance.

In one embodiment, the marker comprises a herbicide resistance marker. In a preferred embodiment, the herbicide is selected from at least one of a imidazolinone herbicide, glufosinate-ammonium, L-phosphinothricin, glyphosate, and/or a sulfonylurea. Preferably, the imidazolinone herbicide is chlorsulfuron.

In one embodiment, the marker comprises a reporter gene.

In one embodiment, the marker comprises a fluorescent marker.

In one embodiment, the marker comprises a fluorescently tagged CRISPR enzyme. In one embodiment, screening comprises screening for said fluorescently tagged CRISPR enzyme.

In one embodiment, screening comprises using routine methods to identify the presence, absence or level of a target trait, through the measurement of a marker indicative of said target trait.

In one embodiment, screening comprises a positive selection approach, wherein stem cells with the target trait are removed from a population and retained for downstream analysis. In another embodiment, screening comprises a depletion approach, wherein stem cells without the target trait are removed from the overall population. In a further embodiment, screening includes a negative selection approach wherein several stem cell types are removed to leave one stem cell type in the population.

In one embodiment, the target trait is herbicide resistance and a selection agent comprising a herbicide is applied in a screening process.

In one embodiment, screening comprises the use of DNA-barcoded antibodies.

In one embodiment, screening additionally comprises nucleic acid sequencing.

In one embodiment, screening comprises mass cytometry.

In one embodiment, screening comprises measuring gene transcript abundance, preferably by means of reverse transcription polymerase chain reaction (rt-PCR).

In one embodiment, screening comprises multi-omic screening, wherein stem cells are screened using genomic, epigenomic, metabolomic and/or proteomic technologies.

In a preferred embodiment, the screening further comprises cell sorting.

In one embodiment, screening comprises florescence activated cell sorting (FAGS).

In one embodiment, screening additionally comprises nucleic acid sequencing.

In one embodiment, screening additionally comprises metabolomics screening or transcriptome sequencing.

In one embodiment, screening comprises whole genome or amplicon next generation sequencing.

In one embodiment, screening occurs on an opto-fluidic device, preferably an opto-fluidic chip. In a preferred embodiment, the microfluidic device is a Beacon (Berkeley Lights) single-cell optofluidic system. In a most preferred embodiment, the microfluidic device is an OptoSelect chip.

In one embodiment, selection comprises selection for a marker.

In one embodiment, the method comprises introducing into at least one stem cell of the plant stem cell culture at least one exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence further comprises a nucleic acid sequence encoding a marker, and wherein the method further comprises screening and/or selecting for said marker.

In a further, optional aspect of the invention, there is provided a method for selecting high-performance cell lines, the method comprising: a. optionally applying at least one selection pressure to at least one stem cell of the plant stem cell line wherein said selection pressure corresponds to a high-performance indicator or trait; b. screening for said high-performance trait in the at least one single plant stem cell or cells; c. selecting said single plant stem cell or cells with the high-performance trait.

In one embodiment, the high-performance trait is selected from high proliferative potential, cell longevity and/or stem cell identity.

In a preferred embodiment, the high-performance trait is a high yield of target natural product in response to an environmental stimulus. In a preferred embodiment, the environmental stimulus is a plant immune elicitor. In one embodiment, the selection pressure comprises treating the suspension culture with a plant elicitor and screening for the production of at least one phytochemical.

In one aspect of the invention, there is provided a culture medium that is optimised for the maintenance and/or proliferation of a population of isolated stem cells and/or stem cell line.

In a further aspect of the invention there is provided a plant obtained or obtainable by the above-described methods.

Accordingly, in one embodiment, there is provided a transformed plant stem cell, plant part or plant obtained or obtainable by the above-described methods.

In one embodiment, there is provided a genetically edited plant stem cell, plant part or plant obtained or obtainable by the above-described methods. In one embodiment, products of said plant or plant thereof are obtained or obtainable from the above-described methods.

A plant according to all aspects of the invention described herein may be a monocot, a dicot or gymnosperm plant.

In one embodiment, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a most preferred embodiment, the plant is a vascular plant.

In one embodiment, the plant may be selected from brassicas, legumes, cereals, citrus, root vegetables, tuber and rhizome crops, fruits including berries and soft fruits and fruit, nut and seed bearing trees.

In one embodiment, the plant may be selected from asparagus, grasses, palms, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, coriander, sunflower, peanuts, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe vera, yews (taxus) aubergine, pineapple, sumac, chickpea, rosemary, lychee, liquorice (Glycyrrhiza glabra), spinach, soybean, brassicas, carrot, corn, rice, Thuja, Juniper, Pine, Garlic, cucumber, chillies, peppers, lettuce, peaches, watermelons, grapes, apples, onions, mandarins, bananas, peas, mangos, oranges, tea, sugarcane, cotton, barley, sorghum, wheat, rice, quassia and Magnolia, as well as members of the Quillija species and Cupressaceae family.

DESCRIPTION OF THE FIGURES

Figure 1 shows detection of a plasmid in transformed vascular stem cell lines by PCR.

Figure 2 shows the detection of epitope-tagged recombinant MYB3-HA proteins in Taxus cell cultures using western blot. The number under the blot indicates the lines from Fig1, and protein ladder size 46 kD is indicated with an arrow. The top panel shows a longer exposure, the bottom panel a shorter exposure.

Figure 3 shows that non-transformed G. glabra stem cells are sensitive to 5 mg/L of hygromycin. At this concentration cell lysis occurs and the media became cloudy and necrotic.

Figure 4 shows the identification of successful G. glabra stem cell transformants on selection media. A) Identification of successful G. glabra stem cell transformed with pRGB32.U3.F3H. Untransformed G. glabra stem cell callus is indicated by red arrow. B. Identification of successful G. glabra stem cells transformed with pRGB32.U3.FNSII. Untransformed G. glabra stem cell callus is indicated by red arrow.

Figure 5 shows the identification of a 200 by deletion in transformed G. glabra stem cells, blue arrow indicates wild type gene fragment, red arrow indicates fragment with deletion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. Such nucleic acids or polynucleotides can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

The aspects of the invention involve recombinant DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.

For the purposes of the invention, a "genetically altered plant" or "mutant plant" is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, a mutant plant is a plant that has been altered compared to the naturally occurring WT plant using a mutagenesis method, such as any of the mutagenesis methods described herein. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the plant genome has been altered compared to wild type sequences using a mutagenesis method. In another embodiment, a mutant plant is a plant that has been altered compared to the naturally occurring WT plant using exogenous gene insertion.

In one aspect, there is a genetically altered plant or part thereof obtained or obtainable by the methods described herein. In one embodiment, the products of said plant or part thereof are also obtained or obtainable.

A plant according to all aspects of the invention described herein may be a monocot or a dicot plant or a gymnosperm. In one embodiment the plant is a vascular plant.

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

In one embodiment, the plant may be selected from asparagus, grasses, palms, rose, cactus, potato, tomato, coconut, broccoli, fig, sweet potato, coriander, sunflower, peanuts, strawberry, ginger, quinoa, tulip, ginger, pomegranate, aloe vera, yews (taxus) aubergine, pineapple, sumac, chickpea, rosemary, lychee, liquorice (Glycyrrhiza glabra), spinach, soybean, brassicas, carrot, corn, rice, Thuja, Juniper, Pine, Garlic, cucumber, chillies, peppers, lettuce, peaches, watermelons, grapes, apples, onions, mandarins, bananas, peas, mangos, oranges, tea, sugarcane, cotton, barley, sorghum, wheat, rice, quassia and Magnolia, as well as members of the Quillija species and Cupressaceae family.

In one embodiment, the plant may be selected from a brassica, legume, cereal, citrus, root vegetable, tuber and rhizome crop, fruits including berries and soft fruits and fruit, nut and seed bearing trees.

For the purposes of this invention, the term "plant stem cell" refers to a plant cell capable of self-renewal and differentiating into specialised 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 tissue and organs. This asymmetric division may occur at a cell or population level.

The terms "plant stem cells" and "stem cells" are terms used interchangeably hereafter.

For the purposes of this invention, the term "plant stem cell culture" refers to the in vitro explanting, maintenance and proliferation of plant stem cells isolated by the methods described herein. Plant stem cell cultures may refer to stem cells in the liquid and/or solid phase of culturing. Thus, plant stem cell cultures refer to stem cells within a cell induction medium on solid platform and/or stem cells within a liquid suspension culture. In a preferred embodiment, the plant stem cell culture comprises stem cells within a liquid suspension culture. In a preferred embodiment, plant stem cells are cultured on a solid and/or liquid media.

In the first aspect of the invention, there is provided a method for introducing at least one genetic modification to at least one plant stem cell of a stem cell culture.

In one embodiment, the plant stem cells display totipotency or pluripotency. Totipotency refers to cells with the differentiation potential to give rise to all cell types and establish an entire complete organism, while pluripotency refers to cells with the differentiation potential to give rise to the majority but not all cell types.

In one embodiment, the plant stem cells are derived from primary and/or secondary meristematic tissue of plants.

By "meristematic tissue" is meant the tissue comprising populations of plant stem cells.

Meristematic tissues are organized into meristems, zones of meristematic tissue that contribute to the growth of the plant. "Primary meristems" are established during embryogenesis and contribute to primary growth of a plant, namely the length or height. Examples of primary meristems include the shoot apical meristems (SAM) and the root apical meristems (RAM).

Meristems that derive from primary meristems and are established post-embryonically can be referred to as "secondary meristems", such as axillary meristems. Secondary meristems contribute to growth in width of stems and roots in plants, resulting in thicker, sturdier tissues that can support the growing plant. Together, meristematic tissue comprises the plant stem cell niche.

In one embodiment, the plant stem cells are derived from primary meristematic tissue, preferably from primary meristematic tissue found within and/or surrounding the vasculature of a plant, more preferably the vasculature of a leaf. These stem cells are referred to herein as vascular stem cells or VSCs. For the avoidance of doubt, stem cells within or around all vasculature of a plant is within the scope of this invention. Vascular stem cells may be derived from regions including, but not limited to, a leaf, a petiole, a node, an internode, flower, shoot, root, blade or bud.

In a preferred embodiment, the plant stem cells are derived from primary meristematic tissue around the central vein of a leaf. The central vein of a leaf corresponds to the main vein or midrib.

In an additional embodiment, the plant stem cells may be derived from lateral or secondary veins in a leaf.

In one embodiment the plant stem cells are not derived or obtained from cambium tissue.

For the purposes of this invention, a plant stem cell can be isolated from the meristematic tissue of a plant, preferably within the plant leaf. The nature of an isolated cell or cell population from meristematic tissues can be confirmed as plant stem cells by quantifying the presence or absence of morphological, metabolic and/or genetic markers within the cell population. Stem cell markers and features are well known to those in the art and described in literature (see Miyashima et al. 2013), but non-limiting examples are included herein. Morphological markers include small size, roundness, a large nucleus, scant cytoplasm and prominent nucleoli. Such features can be determined by mass cytometry, described herein. Genetic markers associated with plant stem cells include, include but are not limited to VVUSCHEL-like HOMEOBOX 4 (WOX4 CLAVATA3iESR.-related 41/44 (CLE41/44), PHLOEM INTERCALATED WITH XYLEM (PXYVTDIF RECEPTOR (TDR) and CYTOKININ RESPONSE 1 (CRE1)/WOODEN LEG (WOLVARABIDOPSIS HISTIDINE KINASE (AHK) [others/specifically identified by your work?]. The expression of stem-cell markers, and other markers of interest, can be quantified by routine methods known to those in the art. For example, reverse transcriptase -polymerase chain reaction (RT-PCR) can quantify gene expression by reverse transcribing mRNA isolated from single cells to cDNA and subsequently amplifying it by PCR. The identification of cells can then be informed by adjustments in mRNA abundance.

Thus, by "plant stem cells" is also meant plant stem cells that have not previously undergone substantial differentiation, have been removed from meristematic tissue and are maintained as a substantially homogenous population under culture conditions.

"Plant stem cells" as described in this invention are distinct from plant cells that have been obtained from differentiated, somatic tissue and are stimulated into a more potent state. These de-differentiated plant cells (DDC) are known in the art (Lee et al. 2010).

STEM CELL LINE/CULTURING Plant stem cells may be isolated from a meristem in a partially or fully grown plant.

Preferably, plant stem cells are isolated from meristematic tissue found within a leaf, or a shoot or a root, using methods described below. Methods of establishing a plant stem cell culture and cell line have been described elsewhere, including Murashige and Skoog (1962) and Gamborg et al. (1968), and are exemplified in the Examples described herein.

Briefly, the isolation of plant stem cells comprises surface sterilization of plant tissue, cutting the sterilized leaf along the central vein (midrib) to expose the stem cell population and culturing the leaf to induce callus formation. Single stem cells are transferred from the callus into individual petri dishes for culturing. Media suitable for cell and callus induction ("cell induction media") are known in the art, for example Gamborg's B5 medium, Lloyd & McCown medium, Schenk & Hildebrand medium, Quoirin & Lepiovre medium, TB medium. Calli displaying a good growth rate should be selected. Preferably, the cell induction medium comprises macro, micronutrients and/or plant hormones. In a preferred embodiment, the cell induction medium contains ingredients that compose a B5 or MS medium and one or more of 2,4-D, kinetin, NAA, 6-BA and other plant growth regulators. Even more preferred, the cell induction medium contains from 0 to10 mg/L 2,4D, from 0.01 to 1 mg/L kinetin, from 0.01 to 1 mg/L NAA and from 0 to 10 mg/L 6-BA.

Culturing the exposed vein will induce the formation of a callus, a visible mass of undifferentiated cells. The length of time between explanting cells and callus formation differs according to the source of the cell. DDCs form a visible callus at and after 7 days after explanting. Vascular plant stem cells form a callus earlier than 7 days, typically at 6 days. Therefore, the stem cell populations of relevance to this invention produce a visible callus earlier than DDCs. Cells obtained from the central or other vein of a leaf form a visually identifiable callus within 3 to 5 days, and cells obtained from the protophloem within 6 days of culturing.

Following callus formation, the stem cells are transferred to petri dishes and are cultured on solid cell induction media under specific temperature and light/dark conditions. For example, a 24h/Oh light/dark to Oh/24h light/dark cycle may be employed, more preferably a 18h/6h light/dark cycle. At the same time, a temperature between 16-30 °C may be employed. Preferably the cells are cultured at a temperature of 25±1 °C.

Subsequently, cells are inoculated into a glass flask containing a Suspension Initiation Medium (SIM) and are cultured in liquid to establish a plant stem cell suspension culture.

Suspension initiation medium is also referred to as plant suspension medium. The flask may be incubated at 21 -25 °C under the appropriate light/dark conditions at an agitation rate of 110 -1200 RPMs. The growth of the cultured cells may be constantly monitored utilising an inverted stereoscope and measured after 14 days of incubation.

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

In one embodiment, measuring cell growth comprises inoculating a known volume of suspension initiation medium (SIM) with one fifth the volume of a stem cell suspension of a known density/volume, and measuring the density occupied of the stem cells following a 14 incubation period. Thus, cell growth may be defined as an increase in cell density, for example an increase of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 150%, or 200% or more, within the 14-day culturing period.

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

In another embodiment, measuring cell growth comprises measuring the cell number at day 1 and then at day 14. In one embodiment, cell growth is defined as an increase in total cell number, for example an increase in total cell number of at least 50%, 60%, 70%, 80%, 90% 100%, 200%, 300%, 400%, 500%, 600%, 700%, or 800% within the 14-day culturing period.

After the day-14 growth assessment, cells may be subcultured in fresh SIM at a ratio of 1:10 CSV:SIM for example. For example, 5 ml of CSV15 will be subcultured to a final volume of 50 ml of medium in a 250 ml sterile glass flask. The subculture process may be repeated every 14 days until reaching a 100 ml subculture volume and observing a constant growth rate in each 14-day subculture. At this stage, the established suspension culture is composed of a homogenous population of plant stem cells.

The cells contained within the established plant stem cell suspension culture flask, via the method described herein, comprise a stem cell line.

Thus, in one aspect of the invention, there is provided a culture medium that is optimised for the maintenance and/or proliferation of a population of isolated stem cells and/or stem cell line.

In one embodiment, the culture medium is optimised for maintaining a substantially homogenous population of plant stem cells in culture conditions.

In one embodiment, the culture medium is a stem cell suspension medium comprising: Composition Contents (mg/L) Inorganic salts NH4NO3 1650 H3B03 6.2 CaCl2 332.2 CoC12.6H20 0.025 CuSO4.5H20 0.025 Na2-EDTA 37.26 FeSO4-7H20 27.8 MgSO4 180.7 MnSat.H20 16.9 Na2Mo04.2H20 0.25 KI 0.83 KNOB 1900 KH2PO4 170 ZnSO4. 7E120 8.6 Vitamin Myo-inositol 100 Thiamine-HCI 10 Nicotinic acid 1 Pyridoxine-HCI 1 Amino acid Casein hydrosylate 500 Hormone 6-BA 1 Kinetin 0.1 NAA 0.3 2,4-D 3 Sucrose 30000 In an alternative or additional embodiment, the medium is a co-cultivation medium, comprising the plant stem cell suspension medium above supplemented with 50 pM acetosyringone.

In an alternative or additional embodiment, the medium is a selection medium, comprising the plant stem cell suspension above supplemented with 100 mg/L Activated charcoal, 2 g/L gelrite, 150 mg/L Cefalexin, 50 mg/L Timentin and 10 mg/L Hygromycin.

In an additional aspect of the invention, there is provided a stem cell line established from the stem cell culture described above.

The high genetic stability and homogeneity of the isolated stem cell population of this invention offers a high-quality stem cell line absent or substantially absent of unwanted genetic aberrations induced by the dedifferentiation process required by alternative methods.

In a further, optional aspect of the invention, there is provided a method for selecting high-performance cell or cell line, the method comprising: a. optionally applying at least one selection pressure to at least one stem cell of the plant stem cell line wherein said selection pressure corresponds to a high-performance indicator or trait; b. screening for said high-performance trait in the at least one single plant stem cell or cells; c. selecting said single plant stem cell or cells with the high-performance trait.

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

By high-performance indicator or trait, high-performance cell or cell lines is meant a cell or cell line with an improvement in plant performance, with regard to cell size, proliferation, yield of a product or developmental potency, wherein preferably said improvement is relative to another/other cell/cells in the cell line. Therefore, the phrases high-performance trait' and 'improvement in plant performance' are used interchangeably.

In a preferred embodiment, the high performance trait is a high yield of target natural product in response to a plant immune elicitor. In one embodiment, the selection pressure comprises a plant elicitor. In one embodiment, the selection pressure comprises treating the suspension culture with a plant elicitor and screening for the production of at least one phytochemical.

A plant immune response elicitor (or just "plant elicitor") is a molecule that is can be recognised by a plant cell and triggers a plant defence response. Elicitors may be derived from a pathogen and include, as examples, oligosaccharides, peptides, glycopeptides, glycolipids, lipophilic elicitors, toxins including coronatine, and polysaccharides such as chitin and its derivatives, and the like. In a preferred embodiment, a bacterial flagellin homolog is used as an elicitor for the immune response. Immune elicitors are well-known to those in the art, and include Harpin (HrpZ), Flagellin, Cold shock proteins, Elongation factor (EF-Tu), Lipopolysaccharides (LPS), Peptidoglycan, Oligogalacturonides, Lipopeptides, Dimethylsulfide, Pseudobactin, Tri-N-alkylated benzylamine derivative (NABD), 2,4-diacetylphloroglucinol (DAPG), Oligogalacturonides, Extracellular ATP and/or DL-p-aminobutyric acid (BABA) and the like. Immune elicitors may be synthetic, for example salicylic acid and its analogues, probenazole (PBZ), 1,6-dichloro-isonicofinic acid (INA), benzothiadiazole (BTH), Tiadinil (TDL), isotianil, N-Cyanomethy1-2-chloroisonicotinamide (NCI), 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CM PA) and the like. Abiotic stress inducers such as for osmotic stress, may also be used to stimulate the immune response of stem cells.

In a preferred embodiment, the plant elicitor is selected from salicylic acid, methyl-jasmonate, coronatine, chitosan and/or osmotic stress inducers.

In a preferred embodiment, the phytochemical belongs to the phenolics, flavonoids and/or terpenoid family of phytochemicals.

In one embodiment, the yield of natural product in response to a plant immune response elicitor is measured. Assays to measure natural products are known to the skilled person and are described in the literature (Altemimi et al., 2017). Colorimetric assays, using reagents that undergo a measurable colour change in the presence of the analyte, may be used. Well-known instruments such as High Pressure Liquid Chromatography can be used for the purification of natural products. Different varieties of spectroscopic techniques like UV-visible, infrared, Nuclear Magnetic Resonance, and mass spectroscopy can subsequently identify the purified compounds.

In one embodiment, the abundance of a target gene transcript is measured following exposure to an elicitor. In a preferred embodiment, the transcript is selected from a phytohormone signalling associated gene, for example PATHOGEN-RELATEDI or PLANT DEFENSINI.2. In a preferred embodiment, transcript abundance is measured using RT-qPCR. In a further embodiment, transcript abundance in response to immune elicitors is measured by RNA sequencing. In a preferred embodiment, transcript abundance is measured at the single-cell level.

In another preferred embodiment, the high performance trait is high proliferative potential. In one embodiment, screening for said high performance trait comprises measuring iodo-deoxyuridine incorporation (IdU, analogous to the fluorescent BrdU). In another embodiment, screening for said high performance trait comprises measuring cyclin B1, cyclin A, and phosphorylated histone H3 using complimentary fluorophoreantibody conjugates. The detection of such markers can be achieved through flow cytometry, or combined in mass cytometry as reported in Behbehani et al., 2012.

In another preferred embodiment, the high performance trait corresponds to cell longevity. Longevity can be screened for using a marker-assisted approach. In one embodiment, screening for said 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 correlates to high performance.

By selecting is meant electing a stem cell or stem cell line for further sub-culturing, screening and/or genetically modifying selected cells or lines.

GENETIC MODIFICATION

In one aspect of the invention, there is provided a method of introducing at least one genetic modification to at least one plant stem cell, preferably of a stem cell line.

In another aspect of the invention is a plant stem cell(s) obtained or obtainable by the method of introducing at least one genetic modification to at least one plant stem cell, preferably of a stem cell line.

In one embodiment, the method of genetic modification comprises introducing at least one mutation into the genome of all plant stem cells of a stem cell culture.

In one embodiment, the method of genetic modification comprises introducing at least one exogenous nucleic acid sequence into at least one plant stem cell.

In one embodiment, the method of introducing at least one genetic modification to at least one plant stem cell, preferably of a plant stem cell line, comprises transformation.

In one embodiment, the method of introducing at least one genetic modification to at least one plant stem cell, preferably of a plant stem cell line, comprises genetic editing.

TRANSFORMATION

As shown in Figure 1, we have found that you can successfully transform vascular plant stem cells with exogenous nucleic acid to express recombinant proteins (as shown in Figure 2).

Accordingly, in one aspect of the invention, there is provided a method of genetically transforming at least one plant stem cell, the method comprising introducing at least one nucleotide construct.

In a preferred embodiment, the at least one plant stem cell is of a plant stem cell culture.

Transformation methods for generating a genetically altered plant or protoplast are known in the art. The construct is introduced into said plant through a process called transformation. The terms "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Isolated plant stem cells capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom.

The exogenous nucleotide construct may be transiently or stably introduced into a host stem cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

Transformation of plants is a routine technique. Advantageously, any of several transformation methods may be used to introduce an exogenous nucleotide construct into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from isolated plant stem cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the stem cell, particle gun bombardment, transformation using viruses or pollen and microinjection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment (biolistic transformation), infection with (non-integrative) viruses and the like.

Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation. Agrobacterium tumefaciens mediated transformation protocols are well-known and are described within literature. Suitable protocols and methods can be found at Methods in Molecular Biology, 2015, Agrobacterium Protocols, Volume 1, 3rd Edition, Ed: Ken Wang.

In another preferred embodiment, transformation comprises biolistic transformation. In this method, a foreign DNA or RNA construct is coated onto gold or tungsten particles. The particles are released from a gene gun by high-pressure helium gas and directly penetrate the host cell. Suitable protocols and methods are widely available within the art, for example., (Ismagul et al., 2018; Miller et al. 2021).

Examples of plant expression vectors comprise those which are described in details in: Becker et al. (1992); Bevan M.W, (1984); Vectors for Gene Transfer in Higher Plants, 1993.

In another aspect of the invention, there is provided a plant stem cell or stem cell line, obtained by the methods above.

GENE EDITING

In one aspect of the invention, there is provided a method of genetically editing at least one plant stem cell, the method comprising introducing at least one mutation into at least one plant stem cell.

By genetic or gene editing is meant targeted genome editing, a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. By another definition, genetic editing also or alternatively means introducing at least one mutation into at least one plant stem cell, by the methods described herein.

In a preferred embodiment, the method of introducing at least one genetic modification to at least one plant stem cell, preferably of a plant stem cell culture, comprises a type II CRISPR-Cas system.

Suitable methods for producing the CRISPR nucleic acids and vectors system are known, and for example are published in Molecular Plant (Ma et al., 2015, Molecular Plant, D01:10.1016/j.molp.2015.04.007), which is incorporated herein by reference.

In a preferred embodiment, the method of gene editing comprises CRISPR-Cas9.

Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNA, the precrRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted (Wiles et al., 2015).

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Accordingly, using techniques known in the art, such as chopchop.cbu.uib.no/ it is possible to design sgRNA molecules that targets a gene sequence.

By "sgRNA" (single-guide RNA) is meant the combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (that links the tracrRNA and crRNA into a single molecule). "sgRNA" may also be referred to as "gRNA" and in the present context, the terms are interchangeable. The sgRNA or gRNA provide both targeting specificity and scaffolding/binding ability for a Cas nuclease. A gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.

In a further embodiment, the nucleic acid sequence encoding a sgRNA molecule is operable linked to a regulatory sequence, such as a plant promoter. A suitable plant promoter may be a constitutive or strong promoter or may be a tissue-specific promoter. In one embodiment, suitable plant promoters are selected from, but not limited to, cestrum yellow leaf curling virus (CmYLCV) promoter or switchgrass ubiquitin 1 promoter (PvUbi1), wheat U6 RNA polymerase III (TaU6), CaMV35S, wheat U6 or maize ubiquitin (e.g. Ubi1) promoters. A suitable promoter may respond to biotic or abiotic stress conditions, for example the heat-inducible tomato hsp80 promoter (US 5,187,267) and the chilli-inducible potato alpha-amylase promoter (WO 96/12814). Plant gene expression can also be achieved via a chemically inducible promoter. This has been explained in depth in Gatz 1997. Examples of such promoters are the salicylic acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter and an ethanol-inducible promoter.

The nucleic acid construct may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme. By "CRISPR enzyme" is meant an RNA-guided DNA endonuclease that can associate with the CRISPR system. Specifically, such an enzyme binds to the tracrRNA sequence. In one embodiment, the CRIPSR enzyme is a Cas protein ("CRISPR associated protein), preferably Cas 9 or Cpf1 or MAD7, more preferably Cas9. The Cas9 enzyme may be modified as described below. In a specific embodiment Cas9 is codon-optimised Cas9. In another embodiment, the CRISPR enzyme is a protein from the family of Class 2 candidate x proteins, such as C2c1, C2C2 and/or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In an alternative embodiment, the Cas protein may be from any one of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiles or Treponema denticola. In a preferred embodiment, the CRISPR enzyme is operably linked to a regulatory sequence -either the same or a different regulatory sequence as for the sgRNA sequence. Again, suitable regulatory sequences are described above.

In one aspect of the invention, there is provided a plant stem cell or stem cell line, transfected with a CRISPR-Cas9 construct.

In one embodiment, the method of gene editing comprises the use of a non-Cas, programmable endonuclease.

In another aspect of the invention, there is provided a plant stem cell or stem cell line, obtained by the methods above.

In another aspect of the invention, there is provided a plant stem cell or stem cell line, obtained by the methods above, transfected with at least one nucleic acid construct as described herein. Cas9 and sgRNA may be combined or in separate expression vectors (or nucleic acid constructs, such terms are used interchangeably). In other words, in one embodiment, a plant stem cell is transfected with a single nucleic acid construct comprising both sgRNA and a CRISPR enzyme as described in detail above. In an alternative embodiment, a plant stem cell is transfected with two nucleic acid constructs, a first nucleic acid construct comprising at least one sgRNA as defined above and a second nucleic acid construct comprising a CRISPR enzyme or a functional variant or homolog thereof. The second nucleic acid construct may be transfected below, after or concurrently with the first nucleic acid construct. The advantage of a separate, second construct comprising a CRISPR enzyme is that the nucleic acid construct encoding at least one sgRNA can be paired with more than one CRISPR enzyme as described herein, and therefore is not limited to a single CRISPR enzyme function (as would be the case when both the CRISPR enzyme and sgRNA are encoded on the same nucleic acid construct). In one embodiment, the nucleic acid construct comprising a CRISPR enzyme is transfected first and is stably incorporated into the genome, before the second transfection with a nucleic acid construct comprising at least one sgRNA nucleic acid. In an alternative embodiment, a plant stem cell is transfected with mRNA encoding a CRISPR enzyme and co-transfected with at least one nucleic acid construct as defined herein.

In a preferred embodiment of any aspect of the invention described herein, sgRNA can be used with a modified Cas9 protein, such as nickase Cas9 or nCas9 or a "dead" Cas9 (dCas9) fused to a "Base Editor" -such as an enzyme, for example a deaminase such as cytidine deaminase, or TadA (tRNA adenosine deaminase) or ADAR or APOBEC.

These enzymes are able to substitute one base for another. As a result, no DNA is deleted, but a single substitution is made (Kim et al., 2017; Gaudelli et al. 2017). Alternatively, the method may use sgRNA together with a template or donor DNA constructs, to introduce a targeted SNP or mutation. In this embodiment, the introduction of a template DNA strand, following a sgRNA-mediated snip in the double-stranded DNA, can be used to produce a specific targeted mutation (i.e. a SNP) in the gene using homology directed repair.

Accordingly, in one embodiment, the method of gene editing comprises CRISPR-Cas9, wherein preferably Cas9 is fused to a base editor.

In one embodiment, the method of gene editing comprises prime editing. Prime editing is a search-and-replace genome editing technology that is characterised by the absence of double-stranded breaks or donor DNA.

Prime editors (PEs), exemplified by PE1, use a reverse transcriptase fused to an RNA-programmable nickase and a prime editing guide RNA (pegRNA) to copy genetic information directly from an extension on the pegRNA into the target genomic locus. In one embodiment, prime editing comprises a pegRNA, and a fusion protein consisting of Cas9 H840A nickase fused to a modified RT enzyme. Through this system, prime editing can mediate targeted insertions, deletions, all 12 possible base-to-base conversions, and combinations thereof without requiring DSBs or donor DNA templates.

Transformation methods for generating a genetically altered plant of the invention are known in the art. Thus, according to the various aspects of the invention, a CRISPR construct as defined herein is introduced into a plant and expressed as a transgene. The methods available for the introduction or transformation of a CRISPR construct have been described elsewhere.

The CRISPR construct may be transiently or stably introduced into a plant stem cell and may be maintained non-integrated, for example, as a plasmid. In one preferred embodiment, the CRISPR construct is stably integrated into the genome of the plant stem cell target. The resulting transformed plant stem cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

HIGH THROUGHPUT SCREENING

In one aspect of the invention, there is provided a method of high-throughput screening of plant stem cells for a target trait, the method comprising a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; b. optionally applying at least one selection pressure to at least one stem cell of the plant stem cell culture wherein said selection pressure corresponds to the target trait; c. screening for said target trait in the at least one single plant stem cell or cells; d. selecting said single plant stem cell or cells with the target trait; and e. optionally regenerating a plant, plant part or plant cell line from said single plant stem cell or cells.

The method described herein may also be considered a method of high throughput phenotyping.

In one embodiment, the method further comprises introducing a genetic modification and then optionally, a selection pressure to the at least one stem cell. By genetic modification is meant the embodiments described earlier within the application, and includes genetic editing and transformation.

In one embodiment, the selection pressure is selected from abiotic stress and/or biotic stress.

By plant stem cells, is meant all embodiments described earlier within the application.

In one embodiment, culturing said plant stem cell line comprises culturing said stem cells on a solid and/or liquid media.

In one embodiment, the method screening for a target trait occurs in stem cells in a callus. Preferably, the callus is formed or substantially formed of cells that have not been dedifferentiated. In another embodiment, screening is not carried out on a callus.

In one embodiment, the screening for the target trait occurs at the single cell level.

In one embodiment, the screening of the target trait occurs in suspension culture.

In one embodiment, the genetic modification and/or selection pressure corresponds to the target trait.

The definitions of plant stem cell, plant stem cell line, plant stem cell culture and genetic modification have been provided elsewhere in this application. Embodiments pertaining to said terms apply to all aspects of this invention.

By selection pressure is meant the application of at least one stress, also called a selection agent, to at least one plant stem cell or plant stem cell line. The terms selection agent and selective agent are used interchangeably throughout.

The at least one stress or selection agent will create an environment in which only plant stem cells or stem lines with or without a named characteristic are identifiable under set conditions. In one embodiment, the selection pressure is selected from abiotic and/or biotic stresses. Abiotic stresses are physical stresses, and biotic stresses are adverse effects on plants caused by other living organisms. Non-limiting examples of abiotic stresses include low or high temperature, deficient or excess water, high salinity, heavy metals, ultraviolet radiation, high or low oxygen concentrations. Non-limiting examples of biotic stresses include viruses, fungi, bacteria, fungi, parasites, insects and weeds. Products or said living organisms can equally act as a biotic stress.

In one embodiment, the selection agent is selected from a herbicide, antibiotic and/or an metabolite.

In another embodiment, the selection agent is a herbicide. Herbicides are well described in the art. Preferably, the herbicide is selected from glufosinate, glufosinate-ammonium, bromoxynil, glyphosate, sulfonylureas and/or chlorsulfuron. More preferably, the herbicide is selected from chlorsulfuron and/or glufosinate-ammonium.

In another embodiment, the selection agent is an antibiotic. Antibiotics are well described in the art, and include those belonging to the penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfoamides, glycopeptides, aminoglycosides, carbapenems classes and the like. Preferably, the antibiotic is selected from hygromycin, bleomycin, streptomycin, spectinomycin, G418, kanamycin, ampicillin, amoxicillin, doxycycline, ciprofloxacin andior clindamycirt More preferably the antibiotic is selected from kanamycin and/or hygromycin.

In another embodiment, the selection agent is an anti-metabolite. Anti-metabolites are described elsewhere. Preferably the anti-metabolite is selected from methotrexate and/or sulphonamide.

In another embodiment, the selection agent is a plant elicitor. Plant elicitors are described elsewhere.

In one embodiment, applying a selection pressure comprises applying a selection agent. In another embodiment, the selection agent allows selection of gene edited or transformed plant stem cell or cells.

By target trait is meant a characteristic of interest. A target trait may be identified in a screening process, through the measurement of a marker indicative of said target trait. The marker may be a direct measure or indirect measure of a target trait. Thus, screening comprises the processes/methods applied to identify the presence, absence or level of a target trait, through the measurement of a marker indicative of said target trait.

In one embodiment, screening comprises screening for the phenotype of the target trait. For example, wherein the target trait is herbicide resistance to the herbicide glufosinate, screening may comprise observing which cells survive when exposed to the selection agent glufosinate.

In one embodiment, screening comprises screening for the expression of a target trait.

In one embodiment, screening for a target trait comprises screening for a marker associated with the target trait. For example, wherein the target trait is herbicide resistance to the herbicide glufosinate, screening may comprise screening for the expression of the Bar or Pat gene -a direct marker for resistance, as the encoded enzymes confers resistance -by transcript or protein analysis.

In a preferred embodiment, the target trait is screened for before and/or after the application of a selection pressure to the at least one stem cell of the plant stem cell culture.

In an additional embodiment, the target trait corresponds to a selection pressure applied to the at least one stem cell of the plant stem cell culture.

MARKERS AND TARGET TRAITS

A marker of a target trait may be at least one lipid, carbohydrate, glycolipid, protein, glycoprotein, lipoprotein or nucleic acid sequence. It is possible for any target trait to be identified providing there is an indicative marker and measurement. A target trait will be deemed to be present or absent when the associated marker is detected at higher/lower/altered levels relative to a control or wild-type plant cell or plant cell line.

The threshold of the marker that is indicative of a given target trait will differ according to the screening applied. The target trait may be a characteristic identifiable by multiple screening methods. Accordingly, the method preferably comprises selecting for the marker of target trait. In other words, selecting cell or cells that have the marker of the target trait.

For all aspects of this invention, a marker may be quantified as an increase or decrease in one of the screening measurements, for example, an increase or decrease of at least In a preferred embodiment, the target trait is detectable at the single cell level.

In one embodiment, the marker and/or target trait corresponds to a genetic modification introduced into at least one plant stem cell.

In one embodiment, the marker corresponds to a genetic modification and/or selection pressure for said marker.

1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% in comparison to a control or wild-type plant cell.

In a preferred embodiment, the target trait is screened for before and/or after, preferably after, the introduction of a genetic modification introduced to the at least one stem cell of the plant stem cell culture.

In one embodiment, the marker comprises a selectable marker. Selectable markers are conditionally dominant genes that confer an ability to grow in the presence of applied selection pressure agents that are normally toxic to plant cells or inhibitory to plant cell growth. Selectable markers and their screening processes are well-known to those skilled in the art. Selectable markers include antibiotic resistance genes, antimetabolite genes, herbicide resistance markers and fluorescent markers.

In one embodiment, the marker comprises an antibiotic resistance marker. Antibiotic resistance markers include the neo gene that encodes the enzyme neomycin phosphotransferase II (NPT II). This marker gene confers resistance to the antibiotic kanamycin and G418. The hpt/hyg gene from E.coli encodes Hygromycin phosphotransferase and confers resistance against and bleomycin. aadA from Shigella flexneri encodes Aminoglycoside adenyltransferase and confers resistance to streptomycin and spectinomycin; and Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus, Accordingly, in one embodiment, the antibiotic resistance marker is selected from kanamycin, G418, bleomycin, streptomycin, spectinomycin, hygromycin, rifampicin and/or a 13-lactam.

In one embodiment, the marker comprises an antimetabolite resistance marker. Antimetabolite marker genes are exemplified by the mutant Dihydrofolate reductase and Dihydrofolate synthase genes, which confer methotrexate and sulphonamide resistance, respectively. In one embodiment, the antimetabolite marker is selected from methotrexate and/or sulphonamide resistance.

In one embodiment, the marker comprises a herbicide resistance marker. A useful herbicide resistance marker is acetolactate synthase (ALS). The ALS gene catalyses the first step in the synthesis of branched-chain amino acids (valine, leucine, and isoleucine), and a mutation that causes a Pro/Ser amino acid substitution at residue 197 confers dominant resistance to the herbicide chlorsulfuron. Thus, in one embodiment, the marker is selected from a herbicide resistance marker, wherein preferably it is the mutant ALS gene containing Pro/Ser mutation at residue 197. Other exemplar herbicide resistance markers include: Phosphinothricin acetyltransferase (bar/pat) conferring resistance to glufosinate and L-phosphinothricin; bromoxynil nitrilase (bxn) conferring resistance to Bromoxynil; Glyphosphate oxidoreductase (gox), conferring resistance to glyphosate, and; enolpyruvyl shikimate phosphate synthase (aroA) conferring resistance against sulfonylureas.

In one embodiment, the marker comprises a reporter gene. A reporter gene may be regarded as the test gene whose expression can be quantified to determine if there has been successful genetic modification. A reporter gene is placed under the control of a promoter or regulator of a marker of interest, so that identification of the reporter gene is indicative of the marker of interest. In general, screening for a reporter gene comprises estimating the quantity of the protein it produces or the products formed downstream of a molecular pathway. The methods available to quantify a protein of a cell are well-known to those within the art, and discussed below.

In one embodiment, the marker comprises a fluorescent marker. Fluorescent markers comprise a marker that is or can be tagged with a fluorophore to emit visible light when stimulated by a laser light of specific wavelength, allowing for the detection of a target of interest it is associated with. In one embodiment, the fluorescent marker comprises an antibody-fluorophore conjugate designed to be complementary to a marker, for detection using fluorescent methods. In an alternative embodiment, the fluorescent marker comprises a fluorescent dye to stain intracellular components. Propidium Iodide is a popular red-fluorescent nuclear and chromosome counterstain suitable for live cells.

Alternatively, a marker may be operably linked to a fluorescent tag. In one embodiment, the fluorescent marker comprises an exogenous fluorescent marker, preferably Green Fluorescent Protein (GFP), which has been transformed into stem cells prior to screening. In one embodiment, a fluorescently tagged CRISPR enzyme is used.

In one embodiment, Cas9 is tagged with a fluorescent protein. In a preferred embodiment, Cas9 is tagged with GFP. In one embodiment, the construct is designed so that CRISPR/Cas9-mediated homology directed repair allows for fluorescent tagging of endogenous proteins, whereby a fluorescent protein such as GFP is attached to an endogenous protein of interest. Accordingly, in one embodiment, the marker comprises a reporter gene consisting of a GFP-tagged CRISPR-Cas9 construct product.

Alternative fluorescent proteins for use as tags include monomeric Infrared Fluorescent Protein (mIFP), Long Stokes Shift monomeric Orange (LssmOrange), Tag Red Fluorescent Protein 657 (TagRFP657), monomeric Orange2 (mOrange2), monomeric Apple(mApple), Sapphire, monomeric Tag Blue Fluorescent Protein (mTagBFP2), tdTomato, monomeric Cherry (mCherry) and Enhanced Yellow Fluorescent Protein (EYFP). Such fluorescent markers can be screened for using fluorescent technologies discussed below.

In one embodiment, the marker is the reporter gene luciferase. The system relies on two different reporter genes to evaluate regulated gene expression Luciferase is an enzyme used for bioluminescence by various organisms in nature, most famously the firefly. A construct in which the regulatory region of a target gene is fused with the DNA coding sequence for luciferase should be generated. A separate DNA construct containing the hypothetical regulator protein is also required. Both constructs should be transformed into cells. If the protein is able to upregulate transcription of the target gene, the cells will express luciferase. If the protein downregulates transcription, the cells will express less luciferase than normal. It is also possible to quantify the measure of the effect of the protein on expression of the target gene by lysing the cells, adding in an appropriate substrate (typically luciferin) and detecting any light produced on a luminometer.

The GUS reporter system is a useful transgenic fluorescent gene system within plant molecular biology. The GUS reporter system utilizes the uidA gene to produce pGlucuronidase, and convert specific colourless or non-fluorescent substrates into stable coloured or fluorescent products. GUS is susceptible to inhibition from some heavy metal ions, Cu2+, Zn2+ so is not appropriate as a marker for heavy metal-related target traits.

Quantitative estimation of the enzyme can be measured by a fluorometric method (using substrate 4-methylumbelliferryl P-D-glucuronide which is hydrolysed to 4-methylumbelliferone) and qualitative data about the enzyme by histochemical methods (enzyme localization can be detected by chromogenic substance such as substrate X-gluc).

In one embodiment of the invention, the marker is indicative of stem-cell morphology, cell longevity, high proliferative potential and/or a positive response to a plant immune elicitor. In one embodiment, the marker of stem-cell morphology is selected from small size and/or higher circularity and/or visible nucleoli. These markers can be analysed using imaging flow cytometers and/or mass cytometers technologies. In one embodiment, the marker of cell longevity is selected from telomerase activity and/or WOX4 and/or CLE41/44 and/or CRE1 and/or WOL and/or AHK. The high expression of any of these markers is indicative of stem cell identity, longevity and a non-differentiated state. In one embodiment, the marker of high proliferation potential is selected from; iodo-deoxyuridine incorporation (IdU, analogous to the fluorescent BrdU) to mark cells in S phase and/or antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. The detection of such markers can be achieved through flow cytometry, or combined in mass cytometry as reported in Behbehani et al., 2012.

In one embodiment, the marker for responsiveness to a plant immune elicitor is selected from phytohormones, including jasmonic acid (JA), jasmonates, salicylic acid (SA), ethylene (ET) and/or abscisic acid (ABA). Methods to screen for such products are well-known and have been described elsewhere. In an additional embodiment, a marker for positive response to plant immune elicitor is also selected from cellular calcium concentrations, protein kinase activation and/or oxidative signalling in a single stem cell or across a population.

SCREENING

Screening comprises the methods and technologies applied to identify the presence, absence or level of a target trait, through the measurement of a marker indicative of said target trait. Accordingly, in one embodiment, screening may comprise marker-assisted identification of plant stem cell(s).

In one embodiment, screening comprises a positive selection approach, wherein stem cells with the target trait are removed from a population and retained for downstream analysis. In another embodiment, screening comprises a depletion approach, wherein stem cells without the target trait are removed from the overall population. In a further embodiment, screening includes a negative selection approach wherein several stem cell types are removed to leave one stem cell type in the population.

Screening methods to detect herbicide resistance markers are known to those in the art, and described in the literature (Peterson JM, 2009). In one embodiment, the target trait is herbicide resistance and a selection agent comprising a herbicide is applied in a screening process. In a preferred embodiment, the herbicide is selected from at least one of chlorsulfuron, glufosinate-ammonium, L-phosphinothricin, glyphosate, and/or a sulfonylurea. In an alternative or additional embodiment, the herbicide target trait is screened for by gene transcript abundance, by the methods described herein.

Screening methods to detect fluorescent markers are known to those in the art. Flow cytometry is a technology that rapidly analyses fluorescently-labelled single cells as they flow past single or multiple lasers while suspended in a buffered salt-based solution.

Confocal microscopy analyses live cell samples by scanning a laser through a sample in a well or on a slide, receiving the fluorescent emission through a pinhole. Confocal microscopy can also be combined with high-resolution mass spectroscopy to achieve cell quantification of metabolites. In both fluorescence screening technologies, a sample is analysed for visible light scatter and one or multiple fluorescence parameters. The fluorescence data can be analysed for marker presence, quantity and distribution within the cell sample, thus allowing determination of a target trait. Imaging flow cytometers can be used to screen for both fluorescent markers and morphological markers. Imaging flow cytometers combine traditional flow cytometry with fluorescence microscopy for the rapid analysis of a sample for morphology and multi-parameter fluorescence at both a single cell and population level. Imaging flow cytometry can track protein distributions within individual cells like a confocal or fluorescence microscope but also process large numbers of cells like a flow cytometer.

In one embodiment, screening comprises mass cytometry. Mass cytometry enables the measurement of multiple features at the single-cell level, by combining the key principles of flow cytometry and elemental mass spectrometry. Mass cytometry utilises marker-specific antibodies conjugated with heavy metal labels, that are detected and quantified by time of flight (TOF) mass spectrometry. Mass cytometry allows for discrimination between multiple isotopes of different atomic mass, thereby allowing the analysis of multiple antibodies and, by extension, cellular features. Mass cytometry has successfully been used to evaluate the expression of cell-surface protein expression, transcription factors that drive gene expression programs and RNA transcript levels. Protocols for optimised mass cytometry protocols and the developments of the technology are well reported within the field (McCarthy et al. 2017; Spitzer et al. 2016).

In one embodiment, screening comprises the use of DNA-barcoded antibodies. DNAbarcoded antibodies recognise cell surface proteins and undergo split pool sequencing (SPLiT-seq) to measure protein abundance on the surface of cells.

In one embodiment, screening additionally comprises nucleic acid sequencing.

In one embodiment, screening additionally comprises DNA sequencing. In one embodiment, screening comprises DNA sequencing of at least one stem cell. Preferably sequencing occurs at the single-cell level. Sequencing may comprise whole genome sequencing or amplicon sequencing. Single cell genomic DNA sequencing allows the identification of stem cells within a population that harbour specific genetic markers, including single nucleotide polymorphisms, restriction fragment length polymorphisms, variable number tandem repeats, microsatellites and copy number variants. Single cell genomic DNA sequencing may comprise whole genome amplification (WGA). In one embodiment, single cell genomic DNA sequencing includes multiple displacement amplification, multiple annealing and looping based amplification cycles, and/or degenerate oligonucleotide-primed PCR. In further embodiments, single cell DNA sequencing includes bioinformatics analysis utilising SCcaller, Monovar, LiRA and/or Conbase computational packages.

In one embodiment, screening additionally comprises single cell RNA sequencing (scRNA-seq) of at least one stem cell. scRNA-seq identifies stem cells within a population that have adjusted levels of transcript abundance. In one embodiment, scRNA-seq comprises the whole-transcriptome amplification method Smart-se. Smart-se can be used for full length cDNA amplification with oligo-dT priming and template switching. In further embodiments, scRNA-seq comprises Smart-seq2, Quartz-Seq and/or CEL-seq methodology to measure single cell transcript abundance. In one embodiment, scRNA-seq measurements comprise the abundance of non-polyA transcripts, including long noncoding RNAs and enhancer RNAs, comprise RamDa-seq methodology.

scRNA-seq requires appropriate cDNA library construction from individual cells. Accordingly, in one embodiment, library construction for a single cell comprises the microdroplet methodology. In this, a cell or nucleus is isolated in an oil droplet that includes reverse transcription reaction solution and a barcoded bead. Reverse transcription is performed within the oil droplet with the associated droplet barcode. In one embodiment, library construction for a single cell comprises a microwell-plate methodology, in which both the cell and barcoded bead are isolated together in a well. Preferably, this comprises the Nx1-seq and/or Seq-Well protocols.

In one embodiment, high-throughput scRNA-seq comprises sci-RNA-seq and/or sci_RNA-seq3. In one embodiment, cell isolation, library construction and amplification are combined in one scRNA-seq protocol. Preferably, amplification is based on Smartseq through microfluids, such as within the C1 Single Cell Auto Prep system. In one embodiment, microdroplet based systems are combined with other functional steps for the purposes of scRNA-seq, preferably following Chromium, ddSEQ, Nadia and/or inDrop methodologies. In one embodiment, microwell based systems are combined with other functional steps for the purposes of scRNA-seq, preferably following Rhapsody and/or ICELL8 methodology.

In one embodiment, screening comprises measuring gene transcript abundance by means of reverse transcription polymerase chain reaction (RT-PCR). In RT-PCR, mRNA isolated from single cells is reverse transcribed to cDNA and subsequently amplified by PCR. Selection of stem cells can then be informed by adjustments in mRNA abundance relative to wild-type or control stem cells. RT-PCR can also be used to confirm the expression of an introduced transgene.

In one embodiment, screening comprises sequencing of the entire, or fragments of, the epigenome of single stem cells. Epigenetic features to be measured include DNA methylation and chromatin states. In one embodiment, stem cell DNA methylation features are analysed using single-cell bisulfite sequencing and/or single cell reduced representation bisulfite sequencing. In a further embodiment, epigenome sequencing comprises single cell chromatin status measurements using single cell ChIP-seq. In one embodiment, single cell ChIP comprises the microfluidic procedure Drop-ChIP. In one embodiment, epigenome sequencing comprises assays for transposase-accessible chromatin using sequencing (ATAC-seq) to measure open chromatin patterns in stem cells. In a further embodiment, C1 and Chromium cell isolation approaches are combined with ATAC-seq to achieve single cell epigenome sequencing.

In one embodiment, screening comprises measuring the abundance of metabolites within single stem cells, and selecting stem cells based on the abundance of specific metabolites. Fluorometric detection methods can be used to detect adjustments in the level of metabolites relative to wild-type stem cells.

In one embodiment, screening includes centrifugation to separate stem cells into layers based on cell density and/or cell size. Generation of a density gradient within a gradient medium would allow separation of stem cells based on density, whereas differential centrifugation could be used to separate stem cells based on size. Stem cells with altered properties relative to wild-type cells could be isolated in this way.

In one embodiment, screening includes separation by sedimentation. Denser stem cells within a population collect at the bottom of a solution more rapidly than less dense stem cells without the need for centrifugation.

In one embodiment, screening includes cell separation by adhesion, in which the adhesive properties of cells to each other or a surface provides a means to separate out cells from a population.

In one embodiment, screening comprises measuring the abundance of proteins within single stem cells, and selecting target stem cells based on the abundance or characteristics of certain proteins. Methods to measure protein abundance at the single cell level are well known to the art. In one embodiment, measuring the abundance of proteins within single stem cells comprises immunoassays. In immunoassays protein detection requires the use of antibodies complimentary to certain proteins of interest. In further embodiments, single-cell micro well arrays are required to provide single-cell resolution for protein analysis.

In one embodiment, measuring the abundance of proteins within single stem cells comprises proximity ligation assays, in which pairs of antibodies operably linked to complementary oligonucleotides are used to recognise certain proteins within a cell lysate. Recognition of a protein by both antibodies within the pair bring the complementary oligonucleotides into proximity, forming a template that can be extended by polymerisation and subsequently amplified to create a quantifiable reporter molecule using PCR. In one working of the method, antibodies operably linked to oligonucleotides are used to probe cell surface proteins, as oppose to internal proteins within the cell lysate.

Protein abundance may alternatively be determined by antibodies operably linked to DNA tags, wherein the antibodies recognise specific proteins of interest on target cells and the abundance of proteins is quantified by DNA sequencing. In one embodiment, multiple DNA-bound-antibodies can be used simultaneously in multiplexing reactions. In one embodiment, this technique is used to identify surface proteins. In one embodiment, this technique is used to identify intracellular proteins from a stem cell lysate. In one embodiment, screening comprises CITE-Seq. CITE-Seq combines single-cell RNA sequencing and multiplexed measurement of protein levels by using oligonucleotidelabelled antibodies to integrate cellular protein and transcriptome measurements (Stoeckius et al., 2017).

In one embodiment, measuring the abundance of proteins within single stem cells comprises use of enzyme-linked immunosorbent assays. In one embodiment, the enzyme-linked immunosorbent assays comprise the use of single-cell barcode chip, wherein single cells are segregated into nanolitre-volume microchambers, lysed or left to secrete proteins, and exposed to an array of immobilised antibody barcode strips.

Proteins bound to the array can be detected via secondary fluorescent antibodies, with the location of the fluorescence signal communicating the identity of the target protein to a reading device. In a further embodiment of this method, a nanowell array-based microengraving technique can be used to analyse thousands of cells in parallel.

In one embodiment, measuring the abundance of proteins within single stem cells comprises mass cytometry, as described above but for the purposes of quantifying the abundance of intracellular proteins found within the lysate of single stem cells.

In one embodiment, measuring the abundance of proteins within single stem cells comprises capillary electrophoresis-laser-induced fluorescence detection.

In one embodiment, measuring the abundance of proteins within single stem cells comprises single cell Western-blotting, wherein cell suspensions are preferably separated by gel electrophoresis, followed by probing with primary and secondary antibodies and staining to visualise the presence of target proteins. In one embodiment, the single cell Western-botting method includes in-gel sieving, immobilisation and/or probing of separated proteins.

In one embodiment, measuring the abundance of proteins within single stem cells comprises use of high-resolution accurate mass spectrometry (MS). In a further embodiment, MS comprises nanoliquid chromatography-electrospray ionisation-tandem MS (nano-LC-MS). In further embodiments, microfluidic devices and nanoscale separation techniques are used to accompany (nano-LC-MS). In further embodiments, MS comprises nanoPOTS, SCoPE-MS, iPAD, Capillary Electrophoresis-MS and/or the use OAD chip technologies.

In one embodiment, screening comprises multi-omic screening, wherein stem cells are screened using genomic, epigenomic and proteomic technologies.

In one embodiment, screening comprises multiple methods outlined above to identify, isolate and record the differences in single stem cells. Preferably, the multiple steps that comprise this approach are performed following Beacon System workflows designed by Berkeley Lights.

In one embodiment, the screening comprises cell sorting said stem cells or stem cell line according to the target trait.

In a preferred embodiment, the screening further comprises cell sorting. By cell sorting is meant grouping plant stem cells or a stem cell line using a marker indicative of a target trait. A cell sorter allows the user to select (gate) for a population of cells which is positive (or negative) for the desired parameters and then direct those cells into a collection vessel. In one embodiment, screening comprises cell sorting and another screening method.

In one embodiment, screening comprises florescence activated cell sorting (FAGS). In FAGS cells presenting specific fluorescent properties are isolated from the overall cell population. In this embodiment, the ratio between the wavelengths of fluorescence emitted by stem cells provide a means to identify and separate target stem cells. In one embodiment, the fluorescent signal generated by stem cells is the result of expression of an exogenous fluorescent marker, which has been transformed into stem cells prior to screening. In a preferred embodiment, the fluorescent marker is enhanced Green Fluorescent Protein (eGFP) and the screening method is FACs. In one embodiment, the exogenous fluorescent marker is operably linked to a gene editing protein, preferably Cas9, that is expressed along with the fluorescent marker in transformed stem cells. In an alternative embodiment, the fluorescent signal generated by stem cells is the result of an antibody or protein labelled with a fluorescent marker that has attached to a complementary ligand on the surface of the stem cell.

In one embodiment, screening comprises magnetic activated cell sorting (MACS).

MACS, also known as immunomagnetic cell separation, harnesses magnetism to separate target stem cells from an overall population through the recognition of surface molecules on the target stem cell by binding proteins, including antibodies, lectins or enzymes, which are operably linked to magnetic beads. Consequently, stem cells that have been recognised by a magnetic-bead linked protein can be withdrawn from the overall cell population by means of a magnetic field.

In one embodiment, screening includes buoyancy activated cell sorting (BAGS). In BAGS, antibodies operably linked to buoyant microbubbles recognise surface molecules on target stem cells, causing target stem cells to separate from the overall population.

Microbubbles are formed from a shell that can comprise lipids, proteins or other polymers, and a gaseous core that provides a means for the microbubble to float in a solution, thereby bringing the attached target cells to the surface.

In one embodiment, screening occurs on an opto-fluidic device, preferably an opto-fluidic chip. Microfluidic devices apply varying degrees of a given force to separate cells with different properties. Microfluidic devices can be used to perform both active (where separation requires an external source of power), and passive (where separation relies on inherent features of the device's microchannels) forms of cell sorting. In a preferred embodiment, the microfluidic device is a Beacon (Berkeley Lights) single-cell optofluidic system. In a most preferred embodiment, the microfluidic device is an OptoSelect chip.

SELECTION

Selection refers to the choosing of a stem cell or stem cell line with a target trait, as determined by screening, for further maintenance and/or growth and/or transformation and/or regeneration.

In all aspects of this invention, selection comprises selecting for a marker of a target trait.

REGENERATION / PLANTS Regeneration refers to the growth of a plant or plant part from a single plant stem cell or cells. In one embodiment, regeneration is followed by further screening said plant or plant part for the target trait.

In one embodiment of the high-throughput screening method presented herein, the method comprises regenerating a plant or plant part from said single plant stem cell or cells and further screening said plant for the target trait.

In another embodiment, method comprises selecting a plant or part thereof with the target trait for further breeding.

In one aspect of the invention, there is provided a method of producing a genetically altered plant, the method comprising a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; b. introducing at least one genetic modification to at least one stem cell of the plant stem cell culture then optionally applying at least one selection pressure to at least one stem cell of the plant stem cell culture wherein said genetic modification and/or selection pressure corresponds to the target trait; c. screening for said target trait in the at least one single plant stem cell or cells; d. selecting said single plant stem cell or cells with the target trait; and e. regenerating a plant or plant part from said single plant stem cell or cells.

The definitions and embodiments relating to parts (a to d) are equivalent to the embodiments for the transformation and high-throughput screening methods presented elsewhere in the application.

Regenerating a plant comprises the formation of in vitro shoot, multiple shooting, profuse in vitro shooting and plantlet regeneration from callus. Suitable methods and protocols for regeneration are known in the art, for example, see Mishra, Tanmayee & GOYAL, Arvind & Sen, Arnab. (2015). Somatic Embryogenesis and Genetic Fidelity Study of Micropropagated Medicinal Species, Canna indica. Horticulturae. 1. 3-13.

10.3390/horticulturae1010003.

In a further aspect of the invention there is provided a plant obtained or obtainable by the above-described methods.

Accordingly, in one embodiment, there is provided a transformed plant stem cell, plant part or plant obtained or obtainable by the above-described methods.

In one embodiment, there is provided a genetically edited plant stem cell, plant part or plant obtained or obtainable by the above-described methods.

A plant according to all aspects of the invention described herein may be a monocot, a dicot or gymnosperm plant.

In one embodiment, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a most preferred embodiment, the plant is a vascular plant.

In one embodiment, the plant may be selected from brassicas, legumes, cereals, citrus, root vegetables, tuber and rhizome crops, fruits including berries and soft fruits and fruit, nut and seed bearing trees.

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned carry at least one of the herein described mutations. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the mutations as described herein.

The invention also extends to products or metabolites or extracts that can be obtained from the plant stem cells or plant stem cell lines of the invention.

The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. In another aspect of the invention, there is provided a product derived from a plant as described herein or from a part thereof.

In a most preferred embodiment, the plant part or harvestable product is a seed or grain.

Therefore, in a further aspect of the invention, there is provided a seed or grain produced from a genetically altered plant as described herein. Accordingly, in one aspect of the invention there is provided seed, wherein the seed has been genetically altered by the methods described herein. Also provided is progeny plant obtained from the seed as well as seed obtained from that progeny.

In an alternative embodiment, the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny produced from a genetically altered plant as described herein.

A control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

In another aspect of this invention there is provided a method of high-throughput screening of candidate crop protection agents, the method comprising: a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; b. optionally introducing at least one genetic modification to at least one stem cell of the plant stem cell culture; c. applying at least one crop protection agent to at least one stem cell of the plant stem cell culture; d. screening for a target trait or phenotype in the at least one single plant stem cell or cells; e. selecting said single plant stem cell or cells with the target trait; and f. regenerating a plant or plant part from said single plant stem cell or cells.

Crop protection agents are chemical or biological substances used to control unwanted pests and pathogens. Pests are organisms that cause destruction by their physical interactions with a crop, and include insects, molluscs, birds and rodents. Pathogens cause destruction by infecting plants and propagating disease within them. Examples of pathogens include viruses; bacteria, fungi; nematodes and the like.

Crop protection agents include insecticides, fungicides, acaricides and herbicides, which target insects, fungi, ticks and mites and competing vegetation, respectively. In one embodiment, crop protection agent is selected from a pesticide, insecticide, fungicide, acaricide and/or herbicides. Crop protection agents may be natural and/or synthetic. Crop protection agents are well-known to the art and may be selected from the organophosphates, carbamates, pyrethroids, sulfonylurea herbicides and the like.

In the aspect above, a target trait may comprise a phenotype or expression of a measurable cellular component. For example, the phenotype may be cell death, cell growth inhibition/delay, altered cell morphology (such as cell size) or expression/absence of expression of cell product or metabolite. Methods and technologies to screen and detect a phenotypic and genotypic target trait have been described in depth elsewhere in this application.

The invention is now described in the following non-limiting example.

EXAMPLES

Example 1 -Genetic modification of cultured Taxus baccata plant stem cells.

Agrobacterium tumefaciens LBA4404 competent cells were transformed with either a NOSp::Hygromycin:NOSt; MASp::mkate2:HSPt; Ubiq11:NanoLuciferase:35St or NOSp:: Hygromycin: NOSt;MASp::mkate2: HSPOJ biq 10p::SXVE: U biq5t; LexAp::MYB3H A:Ubiq5t;Ubiq11p:NanoLuciferase:HSPt vector using a heat shock method. The resulting cells were spread on LB agar plates containing 10 mg/L rifampicin & spectinomycin 100mg/L or 50 mg/L Kanamycin for 2-3 days in incubator at 30 °C. One colony was selected and the cells incubated in 5 mL YEP liquid medium for 16-24 hours with respective antibiotics at 28 °C on a shaker. 1 mL overnight cell culture was added to 100 mL YEP liquid medium with respective antibiotics and the cells incubated for 2024 hours at 28 °C on a shaker until the Optical density at 600nm, OD600=2.0-2.5 and the cells were collected by centrifuge at 3000 g for 15mins. The cells were resuspended with mL TB liquid medium and then add 100-250 pM acetosyringone for additional 1 hours at 25 °C shaker (100 rpm/min).

Secondly, stem cells isolated from the leaves of Taxus baccata were prepared for transformation. Leaves of 7. baccata were collected and surface sterilized as follows: 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, leaves were washed 3 times with distilled water. On a hard, sterile surface a leaf was cut vertically along the midrib of the leaf to expose the inner vascular tissue, containing the target plant stem cells. Dissected leaves were laid on Murashige & Skoog medium, described in Literature, for cell growth and callus induction. A callus formed and expanded on the exposed leaf vein. Among the calli, cells of calli that had good growth rate were selected. Proliferating stem cells were transferred onto a petri-dish with cell line induction medium.

Plant stem cells were cultured in a flask containing liquid TB liquid, on a 120 rpm rotating shaker at 25±1 °C on 16h/8h light/dark cycle. Cells showing good growth rate were subcultured every 14 days to establish a cell suspension culture. 50 mL of stem cells from a 5-7 days' subculture were selected and placed in a 50 mL falcon tubes and 15 mins and allowed to settle. The liquid medium was discarded and the resulting cells used for agrobacterium inoculation.

The prepared Agrobacterium culture was added to 0.02% silwet 77 before inoculation.

50 mL agrobacterium culture was used to resuspend the stem cells (from 50 mL original stem cell culture). The inoculated cells were kept at 25 °C under dark conditions at 110 rpm/min for 30 to 60 mins. The inoculated stem cells were washed twice with 50 ml TB medium, and then resuspended in 50 mL TB medium grown under dark conditions at 25 °C at 110 rpm/min for 3 days.

After 3 days, plant stem cells in the 50 mL falcon tube were collected, washed 5-7 times with sterilized H2O, and placed on TB agar medium with 500 pM of Timentin for another 10-14 days for recovery. Cells were transferred to TB agar medium with 500 pM of Timentin and 25 pg/L hygromycin for 20-40 days, and then transferred to TB agar medium with 400 pM of Timentin and 25 pg/L hygromycin for 20-40 days. Hygromycin resistant stem cells were selected by a negative selection approach, by removing the black non-resistance stem cells, and transferred to TB agar medium with 300 pM of Timentin and 25 pg/L hygromycin. Selected stem cells were subcultured 3 or 4 times on TB agar medium with 25 pg/L hygromycin to establish a transformed stem cell line.

In order to confirm the transformation, genomic DNA was extracted from non-transformed and candidate transformed lines. The obtained genomic DNA was subjected to PCR using report gene NanoLuciferase's primers to confirm the presence of the insertion. The expected size of added transgene PCR band is 399 by and was amplified in candidate lines 3,4 and 5 similar to positive control (lane 7), but was absent from non-transformed lines (Fig. 1).

We further examined selected transformed lines from PCR by western blot. We chose candidate line 27, expressing MYB3-HA driven by estradiol inducible promoter (Lex), and line 3-4, expressing MYB3-HA driven by 35S CaMV promoter (355). Total protein was extracted from non-transformed and candidate transformed lines and then subjected to western blot against HA antibody, with an expected band size of 35 KD. Primary antibody was Anti-HA and secondary antibody, Anti-mouse. In the presence of 20 uM estradiol a -35 KD protein was detected (Fig. 2), suggesting that the application of estradiol can activate MYB3-HA expression. Further, we also examined the 35::MYB3-HA line; a 35 KD protein was detected in the transformed lines 3-5, but not in the mock line (non-transformed) (Fig. 2) Our data suggest the candidate transformed lines have been successfully transformed and further, the target protein was appropriately expressed.

Thus in this example, plant stem cells were transformed by A. tumefaciens and subjected to a high-throughput screening protocol to identify transformed cells containing the target trait. A selection agent (hygromycin), that corresponded to the target trait and genetic modification, was applied prior to the screening. Screening comprised screening for a phenotype (screening for survival on antibiotic exposure) and a reporter gene marker (luciferase activity) that were indicative of the target trait. Expression of the target trait marker was confirmed in transformed cells. Transformed cells were selected, subcultured and used to establish a transformed stem cell line.

Example 2 -Genetic modification of cultured G. glabra plant stem cells Initiation and culture of plant stem cell suspension culture. The cells derived from an exposed vein of G. glabra leaves were cultured in a flask containing the stem cell liquid media defined in materials. Cells were cultured on a 120 rpm rotating shaker at 25± 1 °C on 16h/8h light/dark cycle, with a two-week subculture interval.

Culture Agrobacterium tumefaciens with desired vector and preparation of inoculum. Agrobacterium tumefaciens strain C58C1 harbouring the vectors pRGB32.U3.F3H or pRGB32.U3.FNSI I were grown was grown in LB media with appropriate antibiotics at 28 -30 °C on 220 rpm shaking incubator until the optical density of A600 (0D600) reached 0.6.

Co-cultivation of plant stem cells and Agrobacterium. Cultured Agrobacterium was pelleted by centrifugation and suspended in plant suspension medium to a final OD600 = 0.1. 1 mL of resuspended Agrobacterium were added into 50 mL of co-cultivation medium and co-incubate on a 120 rpm rotating shaker at 25±1 °C for 1 hour.

Stem cells were plated with acetosyringone. Co-incubated plant stem cells were washed with sterilized dH2O to remove agrobacterium and settled by placing the flask on the stand while removing the liquid by pipette. Settled plant stem cells were further dried on sterilized filter paper and were subsequently placed on solid plate composed of co-cultivation media in supplement with 2 g/L gelrite. The plates were kept in dark at 25±1 °C for 3 days. Thus, transformed and untransformed G. glabra stem cells were incubated together on selection media. Untransformed G. glabra became necrotic after 7 days of incubation while G. glabra transformants remained healthy, thereby removing the untransformed population of plant stem cells (Fig. 4).

Plant stem cells were transferred to plate with appropriate antibiotic for selection. After incubation, plant stem cells were transferred to a solid plate with selection medium in standard plant stem cell growth condition for selection. G. galbra stem cells were confirmed to be hygromycin sensitive. G. glabra stem cells were shown to be sensitive to 5 mg/L of hygromycin. At this concentration cell lysis occurs and the media became cloudy and necrotic (Fig 3). Plant stem cells were transferred to new plate on a 2-week interval.

Initiation and cultivation of stable transgenic plant stem cells suspension culture. Successfully selected plant stem cells were cultured in a flask containing the liquid media, with a 2-week subculture interval. Validate the effect of transformation by various methods; The effect of transformation was assayed via various approaches including qPCR/PCR, western-blot and metabolism analysis. PCR analysis of extracted RNA from transformed G. glabra stem cells revealed a possible truncation of the F3H gene following agroinfiltration with pRGEB32.ubiq3.F3H.polyT carrying C58C1 (Fig.5) sgRNA pairs were designed to remove a 200 by section of the target gene (Fig 5).

MATERIALS

G. glabra plant stem cells Agrobacterium tumefaciens (C58C1) culture containing pRGB32.U3.FNSII/F3H.PolyT. Antibiotics (kanamycin for Agrobacterium selection and hygromycin for plant transformants selection) G. glabra plant stem cell medium (plant suspension medium): Composition Contents (mg/L) Inorganic salts NH4NO3 1650 H3B03 6.2 CaCl2 332.2 CoCl2 6H20 0.025 CuSO4.5H20 0.025 Na2-EDTA 37.26 FeSO4-7H20 27.8 MgSO4 180.7 MnSO4.H20 16.9 Na2Mo04.2H20 0.25 KI 0.83 KNO3 1900 KH2PO4 170 ZnSO4. 7H20 8.6 Vitamin Myo-inositol 100 Thiamine-HCI 10 Nicotinic acid 1 Pyridoxine-HCI 1 Amino acid Casein hydrosylate 500 Hormone 6-BA 1 Kinetin 0.1 NAA 0.3 2,4-D 3 Sucrose 30000 Co-cultivation medium: G. glabra plant stem cell medium supplemented with 50 pM acetosyringone.

Selection medium: G. glabra plant stem cell medium supplemented with 100 mg/L Activated charcoal, 2 g/L gelrite, 150 mg/L Cefalexin, 50 mg/L Timentin and 10 mg/L Hygromycin.

In one aspect of the invention, there is provided a cell culture medium comprising inorganic salts, vitamins, amino acids, hormones and sucrose.

In one embodiment, the inorganic salts comprise NH4NO3, H3B03, CaCl2, CoC12.6H20, CuSO4.5H20, Nat-EDTA, FeSO4.7H20, MgSO4, MnSO4.H20, Na2Mo04.2H20. KI, KNO3, KH2PO4, ZnSO4.7H20.

In one embodiment, the vitamin comprises myo-inositol, thiamine-HCL, Nicotinic acid, Pyridoxine-HCL.

In one embodiment, the amino acid comprises casein hydrosylate.

In one embodiment, the hormone comprises 6-BA, Kinetin, NAA and 2,4-D.

In one embodiment, the sucrose is at the concentration 3000 mg/L.

Thus, in one embodiment the cell culture medium comprises at least one of inorganic salts, vitaimins, amino acids, hormones and sucrose, wherein preferably the inorganic salts may be selected from NH4NO3, H3B03, CaCl2, CoC12.6H20, CuSO4.5H20, Na2-EDTA, FeSO4.7H20, MgSO4, MnSO4.H20, Na2Mo04.2H20. KI, KNO3 and KH2PO4, ZnSO4.7H20; and wherein preferably, the vitamin may be selected from myo-inositol, thiamine-HCL, Nicotinic acid and Pyridoxine-HCL; wherein preferably the amino acid may be casein hydrosylate; and wherein preferably the hormone may be selected from 6-BA, Kinetin, NAA and 2,4-D

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Claims (25)

1. CLAIMS: 1. A method of introducing at least one genetic modification to at least one plant stem cell of a stem cell culture, wherein the plant stem cell is a vascular stem cell(s), the method comprising a. isolating at least one stem cell or cells from a tissue of a meristem of a plant, preferably the primary meristem in a leaf; b. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; and c. introducing at least one genetic modification into the cell or cells.
2. 2. The method of claim 1, wherein the method further comprises selecting for at least one high-performance stem cell in the stem cell culture, the method comprising: a. optionally applying at least one selection pressure to at least one stem cell, wherein said selection pressure corresponds to a high-performance indicator or trait; b. screening for a high-performance trait in the at least one single plant stem cell; and c. selecting said single plant stem cell or cells with the high-performance trait.
3. 3. A plant stem cell(s), and/or product and/or or extract thereof, obtained or obtainable by the method of claim 1 or 2.
4. 4. A genetically altered plant or part thereof, and/or products of said plant or plant thereof, obtained or obtainable from the method of any of claims 1 to 3.
5. 5. A method of high-throughput screening of plant stem cells for a target trait, wherein the target trait is detectable at a single cell level, the method comprising a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture; b. optionally introducing at least one genetic modification to at least one stem cell of the plant stem cell culture and/or optionally applying at least one selection pressure to at least one stem cell of the plant stem cell culture wherein said genetic modification and/or selection pressure corresponds to the target trait; c. screening for said target trait in the at least one single plant stem cell or cells; d. selecting said single plant stem cell or cells with the target trait; and e. optionally regenerating a plant, plant part or plant cell line from said single plant stem cell or cells.
6. 6. The method of claim 5, wherein the plant stem cell(s) is a vascular stem cell(s), preferably from a primary or secondary meristematic tissue, more preferably obtained from primary meristem tissue in a leaf of a plant.
7. 7. The method of claim 5 or 6, wherein the stem cell culture does not or does not substantially comprise dedifferentiated cells and/or protoplasts. 15
8. 8. The method of any of claims 5 to 7, wherein the method comprises introducing a genetic modification and then a selection pressure to the at least one stem cell, wherein the selection pressure is preferably selected from an abiotic and/or biotic stress
9. 9. The method of claim 8, wherein the genetic modification comprises introduction of at least one exogenous nucleic sequence; or wherein the genetic modification is gene editing, preferably CRISPR to introduce at least one mutation into a least one target gene and/or promoter.
10. 10. The method of claim 9, wherein the method comprises introducing at least one CRISPR enzyme, wherein the CRISPR enzyme is fluorescently tagged and/or a base editor.
11. 11. The method of any of claims 8 to 10, wherein applying a selection pressure comprises applying a selection agent, wherein the selection agent allows selection of gene edited or transformed plant stem cell or cells.
12. 12. The method of claim 11, wherein the selection agent is selected from a herbicide, antibiotic and/or an antimetabolite.
13. 13. The method of any of claims 5 to 12, wherein the method comprises screening said plant stem cell or cells for expression of the target trait.
14. 14. The method of claim 13, wherein the method comprises screening said plant stem cell or cells for the phenotype of the target trait, and/or screening for a marker associated with the target trait, wherein preferably said phenotype is an improvement in plant performance.
15. 15. The method of any of claims 5 to 14, wherein the method comprises introducing into at least one stem cell of the plant stem cell culture at least one exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence further comprises a nucleic acid sequence encoding a marker, and wherein the method further comprises screening and/or selecting for said marker.
16. 16. The method of claim 15, wherein the marker is selected from an antibiotic-resistance gene, a reporter gene and/or a florescence marker.
17. 17. The method of any of claims 13 to 16, wherein screening comprises fluorescence-activated cell sorting and/or mass cytometry and/or marker-assisted identification and/or nucleic acid sequencing and/or metabolomics screening and/or transcriptome sequencing
18. 18. The method of any of claims 13 to 17, wherein screening is not carried out on a callus.
19. 19. The method of any of claims 13 to 18, wherein screening occurs on an opto-fluidic chip.
20. 20. The method of any of claims 5 to 19 wherein the plant is selected from a dicotyledon, a monocotyledon or a gymnosperm.
21. 21. The method of any of claims 5 to 20, wherein the method comprises regenerating a plant or plant part from said single plant stem cell or cells and further screening said plant for the target trait.
22. 22. The method of any of claims 5 to 21, wherein the method comprises selecting a plant or part thereof with the target trait for further breeding.
23. 23. A method of high-throughput screening of crop protection agents, the method comprising a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture, wherein preferably the stem cell line is obtained from primary meristem tissue in a leaf of a plant; b. optionally introducing at least one genetic modification to at least one stem cell of the plant stem cell culture; c. applying at least one crop protection agent to at least one stem cell of the plant stem cell culture; d. screening for at least one target trait or phenotype; e. selecting said single plant stem cell or cells with the target trait; and f. regenerating a plant or plant part from said single plant stem cell or cells.
24. 24. A method of producing a genetically altered plant, the method comprising a. culturing a plant stem cell line comprising a substantially genetically homogenous undifferentiated cell population to obtain a plant stem cell culture, wherein preferably the stem cell line is obtained from primary meristem tissue in a leaf of a plant; b. introducing at least one genetic modification to at least one stem cell of the plant stem cell culture then optionally applying at least one selection pressure to at least one stem cell of the plant stem cell culture wherein said genetic modification and/or selection pressure corresponds to the target trait; c. screening for said target trait in the at least one single plant stem cell or cells; d. selecting said single plant stem cell or cells with the target trait; and e. regenerating a plant or plant part from said single plant stem cell or cells.
25. 25. A plant stem cell(s), and products or extracts thereof, obtained or obtainable by the method of claims 5 to 24.