Method for producing and screening monoclonal plant calli and suspension cell lines

Abstract

The present invention relates to a method for producing monoclonal plant calli and / or monoclonal plant suspension cell lines and / or monoclonal plant embryos, wherein the method comprises culturing protoplasts or microspores in isolation from each other, wherein the protoplasts or microspores are encapsulated or cultured in microwells. The cultured protoplasts or microspores are regenerated into monoclonal calli, monoclonal suspension cell lines and / or embryos, which can be screened by one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses.

Description

[0001] METHOD FOR PRODUCING AND SCREENING MONOCLONAL PLANT CALLI AND SUSPENSION CELL LINES

[0002] FIELD OF THE INVENTION

[0003] [1] The present invention relates to the field of plant biology.

[0004] BACKGROUND

[0005] [2] Traditional approaches applying mutagenesis to “multi-cell” systems, such as shoots, meristems, callus culture, or seeds, result in chimerism, a condition wherein cells of different genotypes coexist within the same plant tissue. Chimerism makes an additional dissolution step necessary before any screening can be applied.

[0006] [3] In the case of ex-vitro “multi-cell” systems, such as seeds, chimerism dissolution is achieved by generating a M2 population by self-fertilization or crossing from M1 mutants. Indeed, only reproductive cells (gametes) transmit their genotype to the next generation. Since somatic cells do not contribute to gamete formation, any chimeric genetic variations present in them are not inherited, leading to the disappearance of chimerism in the progeny.

[0007] [4] In case of in vitro “multi-cell” systems, such as callus culture, shoots or meristems, chimerism dissolution can be achieved by generating a M2 population, as for ex-vitro “multi-cell” systems, but also by selecting and propagating genetically stable tissues of the M1 mutants.

[0008] [5] The two chimera dissolution processes are labor-intensive, time-consuming and require significant spaces of growing chambers, greenhouses or fields. Indeed, generating the M2 population by selffertilization or crossing is resource intensive, particularly for perennial species, requiring 2-4 years to produce the first seed generation, depending on the genotype and species. Same for the chimera dissolution by repeating multiple in vitro vegetative propagation steps, as 3 to 6 cycles are often necessary to eliminate chimerism.

[0009] [6] By contrast, mutagenesis performed on in vitro “multi-cell” systems with the capacity to regenerate plants from a single cell (e.g., regeneration by somatic embryogenesis of somatic embryo from embryogenic cell), making the dissolution of chimerism unnecessary and screenings applicable to the M1 generation, significantly reducing the overall timeline and labor associated with mutation breeding. Direct in vitro regeneration into plantlets is also an efficient way to isolate each mutant line from the others.

[0010] [7] The application of in vitro mutagenesis on embryogenic cells followed by direct plant regeneration facilitates direct screening of the M1 generation, enabling the identification of traits of interest without requiring an intermediate M2 generation. Screening may focus on phenotypic traits (e.g., yield, stress resistance, quality) observable in regenerated plants. Alternatively, omics-based approaches (e.g., genomics, metabolomics) can be employed on the regenerated plants.[8] For example, the TILLING (Targeting Induced Local Lesions IN Genomes) method enables the screening and identification of induced mutations in target genes. When mutagenesis is applied on ex-vitro “multi-cell” systems, or in-vitro “multi-cell” systems and not followed by direct regeneration into plants with single cell origin, TILLING is conducted on whole organs (e.g., leaves, roots) of the M2 population, or the M1 population after chimera dissolution by repeating in vitro vegetative propagation steps. When mutagenesis is carried out in vitro on “multi-cell” systems, followed by direct regeneration into plants of single-cell origin, TILLING can be carried out on the M1 population, but must always be carried out on the whole organs of the regenerated plants (e.g., leaves, roots). As a result, breeders have to wait for the M1 population to be regenerated in plants, requiring 3 to 12 months for perennial species, before applying the TILLING method.

[0011] [9] To speed up the screening process, to save time, space, and labour, it would be ideal to avoid the necessity of in vitro plant regeneration before applying the TILLING method. Thus, it would be ideal to be able to perform screening and selection at cellular level, at the cell culture stage. To achieve this, each cell, each with a unique genetic background, should be isolated and regenerated into a monoclonal line, so that sampling and genetic screening can be carried out without compromising the integrity and survival of the genotype. However, current technologies do not enable, from a heterogenous cell population, to isolate, regenerate and screen monoclonal cell lines.

[0012]

[0010] Being able to isolate monoclonal plant cell lines from a heterogeneous population of in vitro “multicells” systems (plant callus, meristem...) or “single-cells” systems (e.g. protoplasts, microspores) could also be an alternative to current chimera dissolution processes required without being dependent on direct plant regeneration with single cell origin. Indeed, as it is possible to obtain “single-cell” systems from “multi-cell” systems (e.g. isolation of protoplasts from callus culture or any other plant tissues), it would then make it possible to isolate monoclonal plant cell lines from an initial “multi-cell” chimeric systems. Once each monoclonal cell line is isolated, it is possible to perform screening in a non-destructive manner by sampling only a part of the cell culture, and further go into a regeneration process into plants, which will lead to a population M1 of plants that won’t be chimeric.

[0013]

[0011] Additional limitations exist to introduce recessive traits. For such traits, it is imperative for plant breeders to induce specific mutations across all alleles of a target gene. Similarly, for polygenic traits controlled by multiple genes or gene copies, comprehensive mutagenesis of all relevant loci is required to achieve desired breeding objectives. However, the stochastic nature of random mutagenesis results in polymorphisms distributed unpredictably across the genome. Consequently, the probability of obtaining the requisite mutations across all alleles or gene copies within the M1 generation of mutagenized lines is exceedingly low.

[0014]

[0012] To address this, breeders typically undertake successive breeding steps to introduce the desired mutations into all gene copies. This process may be facilitated by self-fertilization in self-compatible species, enabling the accumulation of mutations within progeny lines. However, for plants that are sterile, self-incompatible, or constrained by breeding goals (e.g., maintaining hybrid or clonal characteristics), or in perennial species requiring multiple years to produce seeds, self-fertilization is infeasible. In such cases, breeders must rely on subsequent rounds of mutagenesis. Whenemploying successive mutagenesis, breeders must first regenerate M1 plants, identify lines harboring mutations in one allele of the target gene, and then initiate a cell culture, such as embryogenic cell culture, from explants of the M1 plants for subsequent mutagenesis. The explants utilized for cell culture initiation vary among species and may include leaves, buds, shoots, meristems, anthers, embryos, or seeds. The process of obtaining explants suitable for cell culture initiation can take months or even years, particularly for species with extended generation times.

[0015]

[0013] Furthermore, cell culture initiation is a protracted procedure, often requiring several months to produce sufficient cell culture material for mutagenic treatments. Thus, the existing workflow for successive mutagenesis is labor-intensive, time-consuming, and resource-demanding, creating significant inefficiencies in breeding programs.

[0016]

[0014] The existing methods for protoplast-based or microspore-based gene editing also suffer from important drawbacks. Since the advent of CRISPR / Cas9 and related gene-editing technologies, site-directed mutagenesis in crop genomes has become a cornerstone of modern crop improvement strategies. Despite the transformative potential of these technologies, current new breeding techniques (NBTs) predominantly rely on the integration of transgenic CRISPR / Cas9 constructs into host genomes, often facilitated by Agrobacterium-mediated transformation. This approach necessitates subsequent breeding steps to segregate and eliminate the transgene, requiring multiple generations and extensive selection.

[0017]

[0015] ln contrast, transient transformation methods, such as ribonucleoprotein (RNP)-based delivery or transient CRISPR / Cas9 expression in protoplasts or microspores, enable the precise editing of plant genomes without integrating foreign DNA. These methods allow the desired genetic modifications to be achieved within a single clonal generation, accelerating the breeding process and facilitating regulatory compliance by avoiding the introduction of transgenic elements.

[0018]

[0016] The workflow for transient transformation of protoplasts generally involves:

[0019] Protoplast lsolation& Purification: plant tissues cell walls are enzymatically digested to release the protoplasts;

[0020] Protoplast Transfection: the CRISPR / Cas system is introduced into protoplasts via polyethylene glycol (PEG)-mediated transfection, lipofection, electroporation or nanoparticle-based transfection;

[0021] Culture and Regeneration: transfected protoplasts are cultured to regenerate the cell wall and undergo cell divisions, forming cell colonies and later microcalli;

[0022] Plant Regeneration: microcalli are regenerated into plants through organogenesis or somatic embryogenesis;

[0023] Screening: regenerated plants are screened for the desired mutations.

[0024] The workflow for transient transformation of microspores generally involves:

[0025] Stress Induction & Harvest: Immature anthers containing microspores are subjected to thermal (heat shock or cold temperature) or osmotic stress to redirect microspores from gametophytic to sporophytic developmental pathway.Microspore Isolation & Purification: immature anthers are dissected from immature spikes or florets and ground in an isolation buffer to trigger their release. Microspores are purified by staged filtration and centrifugation to obtain viable single cells.

[0026] Microspore Transfection: the CRISPR / Cas system is introduced into microspores via polyethylene glycol (PEG)-mediated delivery, electroporation, or nanoparticle-based transfection;

[0027] Androgenesis induction and maturation: The purified microspores are cultured in a nutrient-rich induction medium to trigger cell division, forming globular, heart, and torpedo-stage embryos for dicots and pro-embryo, transition and coleoptilar stage for monocots.

[0028] Plant Regeneration: microspore-derived embryos are regenerated into plants via germination. Screening: regenerated plants are screened for the desired mutations.

[0029]

[0017] Preservation of monoclonality throughout this process is critical to avoid chimerism, which could compromise the genetic uniformity of the regenerated plants. However, current technologies present several limitations. A limitation is the cell density-dependence of the regeneration: protoplasts require optimal cell densities for division and regeneration. Bulk cultures in liquid or semi-solid media risk cell aggregation, leading to non-homogeneous callus formation and potential chimerism. Moreover, the manual collection of microcalli from bulk cultures raises concerns about the fidelity of monoclonality. In addition, when editing multiple genomic sites using multiplexed guide RNAs or successive transfections, the risk of chimerism and off-target effects is amplified.

[0030]

[0018] The workflow for double haploid plants production from microspores generally involves:

[0031] Stress Induction & Harvest: Immature anthers containing microspores are subjected to thermal (heat shock or cold temperature) or osmotic stress to redirect microspores from gametophytic to sporophytic developmental pathway.

[0032] Isolation & Purification: Immature anthers are dissected from immature spikes or florets and ground in an isolation buffer to trigger their release. Microspores are purified by staged filtration and centrifugation to obtain viable single cells.

[0033] Genome Doubling: In early induction of androgenesis, spontaneous chromosome doubling may occur at single cell stage or following the first cell divisions. Such events are species and culture condition dependent. Alternatively microspores are treated with antimitotic agents (like colchicine) following their isolation to double the chromosome count, resulting in 100% homozygous diploid cells.

[0034] Androgenesis induction and maturation: The purified microspores are cultured in a nutrient-rich induction medium to trigger cell division, forming globular, heart, and torpedo-stage embryos for dicots and pro-embryo, transition and coleoptilar stage for monocots.

[0035] Plant Regeneration: microspore-derived embryos are regenerated into plants via germination. Screening: regenerated plants are screened for identifying the diploid ones, usually by genotyping or flow cytometry.

[0036]

[0019] A novel solution is thus required to enable cell sampling without requiring plant regeneration, allowing the preservation of each cell line’s integrity for future applications, such as gene editing and successive random mutagenesis.SUMMARY OF THE INVENTION

[0037]

[0020] ln some aspect, the invention relates to a method for producing monoclonal plant calli and / or monoclonal suspension plant cell lines, wherein the method comprises:

[0038] a) providing a plurality of plant protoplasts or microspores, wherein the plant protoplasts or microspores are optionally genetically modified;

[0039] b) optionally genetically modifying the plant protoplasts or microspores;

[0040] c) isolating the plant protoplasts or microspores from each other;

[0041] d) encapsulating each isolated protoplast or microspore in a gel capsule to obtain a plurality of gel capsules, each gel capsule containing a single plant protoplast or microspore and having a volume of from 0.25 nL to 1 pL;

[0042] e) co-culturing the encapsulated protoplasts or microspores in a culture vessel containing culture medium, at a density of from 100 capsules / mL to 500,000 capsules / mL, preferably from 10,000 capsules / mL to 500,000 capsules / mL, whereby the protoplasts or microspores within the gel capsules undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and

[0043] f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

[0044]

[0021] The invention further relates to a method for producing monoclonal plant calli and / or monoclonal plant suspension cell lines, wherein the method comprises:

[0045] a) providing a plurality of plant protoplasts or microspores, wherein the plant protoplasts or microspores are optionally genetically modified;

[0046] b) optionally genetically modifying the plant protoplasts or microspores;

[0047] c) isolating the plant protoplasts or microspores from each other;

[0048] d) seeding each isolated protoplast or microspores in a microwell of an array of microwells, at a density of from 1000 cells / mL to 200,000 cells / mL, in particular from 10,000 cells / mL to 100,000 cells / mL, wherein the array of microwells is arranged to physically separate the isolated protoplasts from each other and enable liquid exchanges between the microwells;

[0049] e) co-culturing the seeded protoplasts or microspores, whereby said protoplasts or microspores undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics,metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

[0050] LEGEND OF THE FIGURES

[0051]

[0022] Figure 1 shows a schematic representation of embodiments of the method of the invention.

[0052]

[0023] Figure 2 shows microscopy images of single protoplasts encapsulation using microfluidics (A), flow cytometer cell sorter (B), and cell picker (C).

[0053]

[0024] Figure 3 shows microscopy images of the first divisions of a single encapsulated protoplast of BY-2 (A) into a cell colony (B) and (C).

[0054]

[0025] Figure 4 shows microscopy images of monoclonal colonies that have reached the limits of the capsule after bulk co-culture of capsules. Each capsule can be transferred either manually or automatically into liquid multiplication medium in 96-well format separately (A) and / or onto a semisolid multiplication medium in a Petri dish (B) for establishment of the cell line in an individual vessel, assuring monoclonality of each line. (C) Cell line overgrowing after the cells reach spatial limits of the capsule (on semi-solid multiplication medium).

[0055]

[0026] Figure 5 illustrates the picking up of monoclonal cell colonies using automated colony picker: (A) before (left) and after (right) picking of the colonies - the monoclonality is preserved by optimizing the size of microcapillary, picking volume, and speed of aspiration, allowing only transfer of a single colony present in a single well. (B) example of cell colonies transferred into a 96-well plate filled with a liquid medium for multiplication. (C) example of a single cell colony deposited onto the surface of a semi-solid multiplication medium. (D) Example of colony embedded in an alginate microdisc.

[0056]

[0027] Figure 6 illustrates the growth of a Robusta coffee (Coffea canephora) cell colony in a 50pM well starting from a single cell (Sievewell). The well is first seeded with a single cell (A) - and shows division and development into a cell colony after 3 weeks (B). Empty 50pM well, after the colony has been picked by colony picker (C).

[0057]

[0028] Figure 7 shows an example of monoclonality assessment of the protoplasts seeded into microwells.

[0058] First, the microwells are screened in a CellCellector (A) to further detect the wells (B) and single cells inside (C).

[0059]

[0029] Figure 8 shows a schematic representation of various approaches for growth scaling-up of cell colonies.

[0060]

[0030] Figure 9 shows the growth of encapsulated callus cultivated in semi-solid media. First panel is showing encapsulation of callus using a colony picker (A). Callus smaller than 500pm are picked with a methylcellulose module equipped with a 500pm disposable tip. The callus were collected together with sodium alginate and deposited over Ca-agar gel to induce polymerization. The distance between deposits in the grid was 3mm. The development of callus is measured by monitoring the growth parameters (length, width, area) after 7 weeks (B). Percentages of growth for each callus are calculated between WO and W7 (C).

[0031] Figure 10 illustrates the scaling-up of encapsulated callus growth cultivated in the liquid coculture system. Single callus of different sizes are encapsulated at week 0 (WO, start of the experiment), and co-cultured together (week 1 (W1) and week 4 (W4)) (A). The development of callus are measured by monitoring the length, width (B) and the area (C) after 2 weeks and 4 weeks of liquid coculture system.

[0061]

[0032] Figure 11 shows a schematic representation of various embodiments of culture with nursing cells.

[0062] Definitions

[0063]

[0033] As used herein, the term monoclonal, as in monoclonal cell colony, monoclonal cell aggregate, monoclonal callus or monoclonal suspension cell line, refers to a group of cells that have been derived from a single clonal origin, e.g. a single protoplast or a single cell, such as a single microspore.

[0064]

[0034] As used herein, a cell aggregate, or monoclonal cell aggregate refers to a compact cluster of cells.

[0065] A cell aggregate may be a callus.

[0066]

[0035] As used herein, a monoclonal callus refers to an undifferentiated, dedifferentiated, primary or embryogenic mass of cells capable of proliferating on solid or gelified media, or in a liquid medium as aggregated forms, wherein said cells are derived from a single parent cell, such as a single microspore, or protoplast. A callus can be a compact cluster of cells or a crumbly cluster of cells. As used herein, a callus or microcallus is generally a bigger structure than a cell colony, and typically has a size higher than 100 pm. Microcalli are calli of small size, visible under a microscope.

[0067]

[0036] As used herein, a cell colony refers to a cluster of cells which can further develop into a callus or a suspension cell line. A cell colony typically comprises less than 50 cells, in particular from 10 to 20 cells and typically has a size smaller than 100 pm, in particular from 25 to 100 pm.

[0068]

[0037] As used herein, a cell line is a population of cells able to renew themselves for extended periods of times in vitro under appropriate culture conditions. A suspension cell line is a single group of cells suspended in a liquid culture.

[0069]

[0038] As used herein, an embryo is a bipolar structure — including somatic embryos derived from protoplasts or gametic embryos derived from microspores — comprising organized shoot and root primordia. The term encompasses all developmental stages, including but not limited to, globular, heart, and torpedo stages.

[0070]

[0039] As used herein, an established plant cell line refers to a population of plant cells that has been successfully cultivated from an initial minimal input of material, such as plant microcalli or cell clusters. Established plant cell lines are characterized by their ability to grow autonomously and continuously under controlled in vitro conditions. Established plant cell lines are stable and resilient, allowing researchers to repeatedly sample cells without jeopardizing the integrity or viability of the culture.

[0071]

[0040] As used herein, the term microwell refers to any of the small depressions in a microplate (i.e.

[0072] microarray) used to hold samples for scientific experiments. The microplate is typically made of synthetic or natural polymers or hydrogels. The wells within these microplate or microarrays may befluidically connected wells, or otherwise interconnected via liquid pathways located above, below, or between the wells, thereby facilitating nutrient and signal exchange while maintaining physical isolation of the individual cells. The wells are for instance connected by pores or by a liquid layer located above or below the wells. A microwell, as used in the present disclosure, can have a micrometric (i.e. from 1pm to 1mm) size. The term microwell, in the present disclosure, includes arrays with volumes in the nanoliter-range. In some embodiments, a microwell has a size of from 10 pm to 1mm, in particular from 25 pm to 1mm, e.g. of about 25 pm, 50pm, 100pm, 200pm, 300pm, 400pm, 500pm, 600pm, 700pm, 800pm, 900pm or 1mm.

[0073]

[0041] As used herein, the term “capsule” as in “gel capsule” refers to a bead made of a gel material, e.g.

[0074] a hydrogel, generally of spherical or near-spherical form. In the invention, the capsule surrounds a single protoplast, a single microspore, a cell colony, a monoclonal callus, or an embryo separating it from the other encapsulated protoplasts, microspores, cell colonies, calli and / or embryos in the culture vessel.

[0075]

[0042] As used herein, the terms “genetically modified” or “genetic modification” refer to a cell, tissue, or organism whose genetic material has been altered in a way that does not occur naturally through mating or natural recombination. This modification is typically achieved through genetic engineering, a process that involves directly manipulating an organism’s DNA using biotechnological techniques. The goal of genetic modification is often to introduce new traits or characteristics to an organism, such as enhanced resistance to pests, improved nutritional content, or increased tolerance to environmental stress (e.g., drought or salinity). A genetic modification, as used herein, includes any type of modification in the genome of a cell, e.g. microspore, or protoplast, including modifications made by transgenesis, mutagenesis, such as random mutagenesis gene editing or epigenetic modifications.

[0076]

[0043] As used herein, RNA-Directed DNA Methylation is a technique which involves the use of RNA molecules to guide the addition of methyl groups to specific regions of the DNA. DNA methylation can modify gene expression without altering the underlying DNA sequence. By directing methylation to particular genomic sites, RNA molecules can effectively silence genes or regulate their activity in a stable manner. This modification is often used in epigenetic studies and plant breeding to control gene expression in a heritable way without changes to the genetic code itself. It is also possible to modulate epigenetic changes by application of a chemical treatment, such as 5-aza-2’- desoxycytidine (5-aza-dC).

[0077]

[0044] As used herein, genomic imprinting is an epigenetic phenomenon where certain genes are expressed in a parent-of-origin-specific manner. In this process, the expression of a gene depends on whether it is inherited from the mother or the father. Modifications such as DNA methylation or histone modifications can lead to the silencing of one allele (either the maternal or paternal) of a gene, thereby influencing the gene's expression. This technique can be used to modulate gene function in a precise manner, especially for genes that are subject to imprinted inheritance.

[0078]

[0045] As used herein, paramutation is an epigenetic phenomenon where one allele can induce heritable changes in the expression of another allele, typically without altering the DNA sequence. This occurs when one allele "changes" the activity of another allele in a way that can be passed down throughgenerations. Inducing paramutation through RNA-based methods is a way to control gene expression in plants or animals without altering the genetic code directly, and it can lead to stable and heritable modifications.

[0079]

[0046] As used herein, RNA interference (RNAi) is a process by which small RNA molecules (such as small interfering RNAs or siRNAs) bind to messenger RNAs (mRNAs) and trigger their degradation or prevent their translation into proteins. By introducing specific RNA molecules that match a target gene’s sequence, the expression of particular genes can be selectively silenced or reduced. RNAi is widely used for gene knockdown and to study gene function. This method is also used in crop improvement and biotechnology to control undesirable traits or enhance beneficial ones.

[0080]

[0047] As used herein, microRNA (miRNA) are small non-coding RNA molecules that regulate gene expression by binding to complementary sequences in mRNA molecules, typically leading to gene silencing. miRNAs play crucial roles in regulating various biological processes, including development, stress responses, and disease resistance. By manipulating miRNA pathways, gene expression can be modulated in a precise and controlled manner. This approach can be used to either silence genes or fine-tune gene expression levels.

[0081]

[0048] As used herein, piwi-lnteracting RNA (piRNA) refers to small RNA molecules that interact with Piwi proteins and are involved in the regulation of transposons and maintaining genome stability, particularly in germline cells. piRNAs can help silence transposable elements and other genetic sequences that could be harmful if left active. piRNA pathways can be harnessed to target specific genomic regions and modulate gene expression, providing another method of genetic modification that is distinct from traditional CRISPR-based techniques.

[0082]

[0049] As used herein, random mutagenesis is a technique that induces genetic mutations in a nontargeted manner using physical agents such as UV radiation, X-rays, or ionizing radiation; chemical agents like EMS (ethyl methanesulfonate), MNU (N-methyl-N-nitrosourea), or NG (N-methyl-N'- nitro-N-nitrosoguanidine); and / or biological factors including replication errors, transposons, or error-prone PCR. It can be applied to seeds, cells, or in vitro tissues and is widely used in crop improvement, microbial strain development, and functional genomics to study gene functions and enhance desirable traits.

[0083]

[0050] As used herein, genomics refers to the comprehensive study of an organism’s entire genome, including sequencing, mapping, annotation, and functional analysis. Genomic analysis encompasses the identification of genetic variations, structural features, and their biological implications. Genomics techniques are described, e.g. in Lesk, 2017.

[0084]

[0051] As used herein, proteomics refers to the large-scale study of proteins, including their structure, function, interactions, and post-translational modifications. Proteomic analysis enables the systematic identification and quantification of proteins to investigate cellular mechanisms, phenotype-related cellular processes and biomarker discovery. Proteomics techniques are described e.g. in Twyman et al., 2004

[0085]

[0052] As used herein, transcriptomics refers to the study of the complete set of RNA transcripts expressed by the genome under specific conditions, including messenger RNA (mRNA), non-coding RNA, and small RNA molecules. Transcriptomic analysis provides insights into gene regulation, cellularfunction, and phenotype related-associated transcriptional alterations. Transcriptomics techniques are described e.g. in Wang et al., 2009.

[0086]

[0053] As used herein, metabolomics refers to the comprehensive analysis of small-molecule metabolites within a biological system. Metabolomic analysis facilitates the characterization of metabolic pathways, biochemical networks, and physiological states, contributing to biomarker discovery and phenotypic diagnostics. Metabolomics techniques are described e.g. in Dettmer et al., 2007.

[0087]

[0054] As used herein, epigenomics refers to the study of epigenetic modifications that regulate gene expression without altering the DNA sequence. Epigenomic analysis includes DNA methylation, histone modifications, chromatin accessibility, and non-coding RNA-mediated regulation, providing insights into gene regulation and disease mechanisms. Epigenomics techniques are described e.g. in Allis et al., 2007.

[0088]

[0055] As used herein, lipidomics refers to the large-scale analysis of lipid molecules, including their structural diversity, metabolic pathways, and functional roles. Lipidomic analysis is employed to investigate lipid-mediated signaling, metabolic pathways, and phenotype-related lipidomic changes. Lipidomics techniques are described e.g. in Han and Gross 2014.

[0089]

[0056] As used herein, glycomics refers to the systematic study of glycans (carbohydrate structures) and their interactions with proteins, lipids, and other biomolecules. Glycomics analysis provides insights into glycosylation patterns, their biological significance, and their implications in phenotypes. Glycomics techniques are described e.g. in Varki et al., 2015.

[0090]

[0057] As used herein, phenomics refers to the large-scale investigation of phenotypic traits in relation to genetic and environmental factors. Phenomics analysis may include high-throughput imaging, physiological assessments, and computational modeling to establish genotype-phenotype relationships. Phenomics techniques are described, e.g. in Robinson et al. (2002).

[0091]

[0058] As used herein, the term successive mutagenesis refers to the iterative application of mutagenic treatments to a population of cells or organisms, aimed at generating genetic diversity over multiple cycles. This process enables the targeted selection of variants with enhanced or novel traits, facilitating advancements in biotechnology, genetic engineering, and cell line development. Each cycle of mutagenesis induces genetic alterations, which are followed by a period of selection to isolate the most desirable phenotypes and / or genotypes. The key advantage of successive mutagenesis lies in its ability to incrementally increase the diversity of genetic material within a cell population, thereby enabling the identification of specific variants that exhibit desired genotypes or improved properties, such as enhanced regeneration, growth rates, stress resistance, or production of target molecules.

[0092]

[0059] As used herein, the term culture vessel refers to any vessel that can provide a sterile environment for culturing cells. Exemplary culture vessels include a petri dish, flask, plate, multi-well plate, and the like.Detailed description of the invention

[0093]

[0060] In one aspect, the invention relates to a method for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, wherein the method comprises:

[0094] a) providing a plurality of plant cells, wherein the plant cells are optionally genetically modified; b) optionally genetically modifying the plant cells;

[0095] c) isolating the plant cells from each other;

[0096] d) encapsulating each isolated plant cell in a gel capsule to obtain a plurality of gel capsules, each gel capsule containing a single plant cell;

[0097] e) co-culturing the encapsulated plant cells in a culture vessel containing culture medium, whereby the plant cells within the gel capsules undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and

[0098] f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

[0099]

[0061] ln certain embodiments, the plant cells are single-cell systems, in particular selected from protoplasts or microspores.

[0100]

[0062] ln one aspect, the invention relates to a method for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, wherein the method comprises:

[0101] a) providing a plurality of plant protoplasts or microspores, wherein the plant protoplasts or microspores are optionally genetically modified;

[0102] b) optionally genetically modifying the plant protoplasts or microspores;

[0103] c) isolating the plant protoplasts or microspores from each other;

[0104] d) encapsulating each isolated protoplast or microspore in a gel capsule to obtain a plurality of gel capsules, each gel capsule containing a single plant protoplast or microspore;

[0105] e) co-culturing the encapsulated protoplasts or microspores in a culture vessel containing culture medium, whereby the protoplasts or microspores within the gel capsules undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and

[0106] f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

[0107]

[0063] The plant protoplasts or microspores provided at step (a) are optionally genetically modified.

[0064] The gel capsule at step (d) particularly has a volume of from 0.25 nL to 1 pL, more particularly from 0.25 nL to 500 nL, still more particularly from 0.25 nL to 200 nL, most particularly from 0.25 nL to 100 nL. In some embodiments, the gel capsule has a volume of from 1 nL to 1 pL, more particularly from 1 nL to 500 nL, still more particularly from 1 nL to 200 nL, most particularly from 1 nL to 100 nL. In some embodiments, the gel capsule has a volume of from 10 nL to 1 pL, more particularly from 10 nL to 500 nL, still more particularly from 10 nL to 200 nL, most particularly from 10 nL to 100 nL. The encapsulated protoplasts or microspores are particularly co-cultured at a density of from 100 capsules / mL to 500,000 capsules / mL, more particularly from 10,000 capsules / mL to 500,000 capsules / mL, still more particularly from 20,000 capsules / mL to 500,000 capsules / mL. In some embodiments, The encapsulated protoplasts or microspores are co-cultured at a density of from 1000 capsules / mL to 100,000 capsules / mL, more particularly from 10,000 capsules / mL to 100,000 capsules / mL, still more particularly from 20,000 capsules / mL to 100,000 capsules / mL.

[0108]

[0065] The monoclonal calli, monoclonal suspension cell lines and / or embryos formed at step (e) are preferably encapsulated monoclonal calli, monoclonal suspension cell lines and / or embryos.

[0109]

[0066] In another aspect of the invention, the invention relates to a method for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, wherein the method comprises: a) providing a plurality of plant cells, wherein the plant cells are optionally genetically modified; b) optionally genetically modifying the plant cells;

[0110] c) isolating the plant cells from each other;

[0111] d) seeding each isolated cell in a microwell of an array of microwells, wherein the array of microwells is arranged to physically separate the isolated cells from each other and enable liquid exchanges between the microwells;

[0112] e) co-culturing the seeded cells, whereby said cells undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and

[0113] f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

[0114]

[0067] ln certain embodiments, the plant cells are single-cell systems, in particular selected from protoplasts or microspores.

[0115]

[0068] In another aspect of the invention, the invention relates to a method for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, wherein the method comprises: a) providing a plurality of plant protoplasts or microspores, wherein the plant protoplasts or microspores are optionally genetically modified;

[0116] b) optionally genetically modifying the plant protoplasts or microspores;

[0117] c) isolating the plant protoplasts or microspores from each other;d) seeding each isolated protoplast and / or microspore in a microwell of an array of microwells, wherein the array of microwells is arranged to physically separate the isolated protoplasts or microspores from each other and enable liquid exchanges between the microwells;

[0118] e) co-culturing the seeded protoplasts or microspores, whereby said protoplasts or microspores undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

[0119]

[0069] The plant protoplasts or microspores provided at step (a) are optionally genetically modified.

[0120]

[0070] The isolated protoplasts or microspores are particularly seeded at step (d) at a density of from 1000 cells / mL to 200,000 cells / mL, in particular from 10,000 cells / mL to 200,000 cells / mL, more particularly from 20,000 to 200,000 cells / mL. In some embodiments, the isolated protoplasts or microspores are seeded at step (d) at a density of from 1000 cells / mL to 100,000 cells / mL, in particular from 10,000 cells / mL to 100,000 cells / mL, more particularly from 20,000 cells / mL to 100,000 cells / mL.

[0121]

[0071] The plurality of protoplasts, in the methods of the invention, may comprise a high number of protoplasts or microspores such as more than 100,000 protoplasts or microspores, in particular, more than 500,000 protoplasts or microspores, or more than 1 ,000,000 protoplasts or microspores. In some embodiments the plurality of protoplasts comprises from 100,000 protoplasts or microspores to 10,000,000 protoplasts or microspores, in particular from 500,000 protoplasts or microspores to 5,000,000 protoplasts or microspores.

[0122]

[0072] The disclosed invention addresses the issues discussed herein by enabling the isolation and establishment of monoclonal cell lines from populations which may be diverse genetically modified, such as mutagenized populations, facilitating direct screening while ensuring non-chimeric regenerated plants.

[0123]

[0073] The invention provides plant breeders with a platform that significantly accelerates the acquisition of screenable monoclonal plant cell lines while ensuring their non-chimeric character.

[0124]

[0074] The invention also provides improved processes of plant cell regeneration protoplasts-derived cell line or plant regeneration of a species / genotype relying on cultivation at higher densities with certainty of monoclonality of these lines.

[0125]

[0075] Plant cells or tissues, used as input, can result from random mutagenesis, genome editing technologies as well as other transformations such as RNAi, miRNA, piRNA, RNA-directed DNA methylation, paramutagenic, genomic imprinting research / breeding programs. Each cell line can then be used independently as starting material to regenerate improved plants or to apply other transformations. Breeders can thus perform early screenings directly on monoclonal cell cultures without regenerating the plants. Screening may be performed to analyze the function of a gene,production of metabolites of interest, phytotoxicity analyses, high biomass producing cell lines, haploidization and polyploidization, for instance. Subsequent mutagenesis can be performed on cell culture while saving the regeneration and cell culture induction steps.

[0126] Starting material

[0127]

[0076] ln certain embodiments, the present invention involves providing plant material and isolating plant protoplasts from the plant material. Plant samples are often in aggregated forms, such as tissues, organs, plant cells, including plant cell cultures, which can serve as the starting material for protoplast isolation. The plant material may be derived from a wide variety of plant parts, including but not limited to leaves, fruit, meristems, cotyledons, hypocotyls, anthers, stems, petioles, flowers, roots, root tips, rootstocks, pollen, ovules, embryos, seeds, and reproductive tissues such as scions or cuttings. Alternatively, plant cells, such as plant cell suspensions, clusters, or calli in either liquid or solid culture media may be used.

[0128]

[0077] I n some embodiments, the protoplasts are derived specifically from embryogenic cells. These cells may be cultured in vitro, either as suspended embryogenic cells or as embryogenic calli.

[0129]

[0078] Embryogenic cells are typically obtained through a sequence of tissue culture steps, including the formation of primary callus (callogenesis) followed by the induction of embryogenic callus (embryogenic callogenesis), where the primary callus cells are further stimulated to differentiate into embryogenic cells capable of forming somatic embryos. Leaf tissues are a particularly efficient and widely used source for initiating callus formation and subsequent embryogenic development. Furthermore, various other plant tissues, such as embryo axes, cotyledons, hypocotyls, or root explants, can also serve as viable starting material depending on the plant species and the specific tissue culture protocols.

[0130]

[0079] The starting material, e.g. plant cell cultures, plant tissues or plant calli, may be genetically diverse, i.e. heterogenous in the genetic makeup of the cells. For instance, the starting material is chimeric, comprising cells having different genomes. In particular embodiments, the starting material is a chimeric plant tissue or plant callus, and / or a heterogenous plant cell culture.

[0131]

[0080] In certain embodiments, the present invention also employs microspores as single-cell starting material. Microspores can be isolated from intact flower buds or spikes by dissecting immature anthers and releasing their locular contents, from isolated anther sacs, preferably at the mid- to late- uninucleate stage, at which the cells retain totipotency and can be reprogrammed toward embryogenesis. The donor plant material is selected to ensure high microspore yield and viability, taking into account species- and genotype-dependence of androgenic responses. Donor plants are preferably grown under controlled conditions to minimize abiotic stress and pathogen load, with balanced nutrition, adequate light intensity and photoperiod, and stable temperatures adapted to the species. In particular embodiments, donor plants are cultivated in greenhouses or growth chambers and monitored to harvest flower buds or immature spikes synchronously at the optimal stage window for microspore isolation. Preconditioning of donor plants, such as cold pretreatment of excised inflorescences or whole plants for 24h to one month at 4-10°C, may be applied toenhance embryogenic switch of microspores in Brassicaceae, Solanaceae, cereals, and other responsive taxa.

[0132]

[0081] ln some embodiments, microspores are induced toward gametic embryogenesis (androgenesis) by an abiotic stress cue compatible with monoclonal culture. Exemplary induction regimens include transient heat shocks, for example 32-35°C for 24-120 hours, or cold pretreatments of inflorescences followed by culture at permissive temperatures. Androgenesis-supporting media, such as NLN, N6, B medium, or species-optimized formulations, can be employed with defined osmoticum, vitamins, basal salts, and, where appropriate, low levels of auxins or cytokinins. Under these conditions, single isolated microspores undergo symmetrical divisions and transition through embryogenic stages, forming globular, heart, and torpedo-stage embryos for dicots and pro-embryo, transition and coleoptilar stage for monocots, which are preserved as monoclonal entities within their capsules or wells. In certain embodiments, nursing cells or organs (e.g. ovaries) are co-cultured in physically separated compartments, enabling exchange of soluble signals while preventing mixing with microspore-derived structures, thereby improving androgenesis efficiency without jeopardizing monoclonality.

[0133]

[0082] Microspores may be genetically modified prior to or after isolation, depending on the breeding objective. In particular embodiments, donor plants used for microspore isolation are previously subjected to mutagenic treatments ex vitro, such as EMS or irradiation, so that harvested microspores carry diverse, segregating alleles. Alternatively, isolated microspores are transiently transfected with gene-editing ribonucleoproteins by electroporation or PEG-mediated delivery under osmotically balanced conditions, achieving transgene-free edits in haploid genomes. When genome edits or random mutations are introduced at the microspore stage, the resulting embryogenic structures, calli, or suspension cell lines are monoclonal by construction, facilitating early nondestructive screening and downstream doubling of haploids to produce doubled haploid lines, if desired. In some embodiments, the microspores, or the embryos and cell lines derived therefrom, undergo chromosome doubling. This is preferably achieved through treatment with a doubling agent, such as colchicine.

[0134] Protoplast obtention

[0135]

[0083] In some embodiments of the invention, protoplasts are obtained from plant cells, or plant material containing plant cells, through cell wall breakdown. The cell wall can be disaggregated using enzymatic, chemical and / or mechanical means.

[0136]

[0084] ln some embodiments, step (a) comprises: (a1) providing a plurality of plant cells, particularly embryogenic cells, particularly wherein the plant cells are comprised in cell aggregates, plant tissues, and / or plant parts; and (a2) disaggregating the cell wall of the plant cells to obtain plant protoplasts.

[0137]

[0085] The process of protoplast isolation typically includes a step in which plant material may be harvested and subjected to enzymatic digestion using cellulases, pectinases, and / or hemicellulases to break down the cell wall and release the protoplasts into the surrounding medium. The choice of enzymes,their concentration, and incubation conditions (temperature, time) can vary depending on the plant species and tissue type. Additionally, osmotic stabilizers such as mannitol or sorbitol are often used in the isolation medium to maintain the integrity of the protoplasts during the enzymatic digestion.

[0138]

[0086] Once produced, the protoplasts can be purified by differential centrifugation or filtration to remove cell debris, undigested tissue, and other contaminants.

[0139] Genetic modification

[0140]

[0087] ln some embodiments, the protoplasts or microspores used for the method of the invention are genetically modified, such as by random mutagenesis. Step (a) may thus comprise providing a plurality of genetically-modified plant protoplasts or microspores.

[0141]

[0088] ln some embodiments, the method comprises a step of genetically modifying plant cells, and obtaining protoplasts from said plant cells. This step can enhance the efficiency of protoplast production by introducing desired traits at an early stage, ensuring uniformity in the subsequent processes. As a result, said plant cells are genetically modified and are subsequently used for producing protoplasts.

[0142]

[0089] Alternatively or in addition, the method can comprise a step of genetically modifying the protoplasts or microspores. Genetically modifying protoplasts allows a more homogeneous mutagenesis or gene edition than modifying plant cells with a cell wall. Genetically modifying protoplasts rather than (or in addition to) plant cells with a cell wall, also allows transgene-free gene editing, alleviating the need for using transgenes, which are often extraneous genes added in the plant genome. In particular, the genetic modification step is carried out before encapsulation of the protoplasts, ensuring that the modifications are stabilized in the cells before further processing.

[0143]

[0090] In some embodiments, the cells, e.g. microspores, and protoplasts used for the method of the invention are genetically modified prior to the first step of the method.

[0144]

[0091] Genetic modifications may include transfection with genes enhancing rates of cell divisions. Genetic modifications can also include transfection with reporter genes that can be used to help screening of protoplasts or microspores underwent successful transfection.

[0145]

[0092] Alternatively, or in addition, the method may involve a step of genetically modifying the protoplasts or microspores themselves. This step is particularly performed before encapsulation of the protoplasts or microspores.

[0146]

[0093] These various approaches share the objective of producing genetically modified protoplasts or microspores which serve as versatile tools for the subsequent steps of the process.

[0147]

[0094] In some aspects of the method of the invention, the protoplasts or microspores are not genetically modified. The method indeed comprises various applications for non-genetically modified plant cells, including the screening of native genes and native traits, wherein the screening is practiced on plant calli or plant cells lines and allows to analyze the cells by various omics techniques.

[0148]

[0095] Various techniques can be used to induce genetic modification.

[0149]

[0096] These techniques are selected based on the specific requirements of the modification, such as target accuracy, efficiency, or the type of genetic change desired. In some embodiments, the stepof genetically modifying comprises one or more of random mutagenesis, targeted genome editing, RNA-directed DNA methylation, genomic imprinting, paramutation induction, RNA interference (RNAi), microRNA (miRNA), and piRNA transformation. Each of these methods offers unique advantages, such as targeting specific genes or inducing broad-spectrum genetic variability.

[0150]

[0097] I n some embodiments, the protoplasts or microspores may be transformed using various methods such as particle bombardment, Agrobacterium-mediated transformation, electroporation, PEG- mediated transformation or nanoparticle-based transfection to introduce foreign genes or modify the plant genome.

[0151]

[0098] In some embodiments, the protoplasts or microspores may be genetically modified by mutagenesis.

[0152] Mutagenesis techniques range from chemical methods to advanced genome editing technologies and may include methods such as ethyl methanesulfonate (EMS) mutagenesis, oligonucleotide- directed mutagenesis (ODM), Zinc Finger Nuclease (ZFN) technology, Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR / Cas system, the CRISPR / Cpf system, engineered meganucleases, re-engineered homing endonucleases, and DNA-guided genome editing.

[0153]

[0099] ln specific embodiments, genetic modification(s) may be induced using random mutagenesis techniques. This approach can involve chemical mutagens, such as EMS, or exposure to UV irradiation, X-rays, or radioactive irradiation. Chemical mutagenesis can include treatment with a variety of mutagenic chemicals, such as sodium azide (NaN3), N-ethyl-N-nitrosourea (ENU), methyl nitrosoguanidine (MNNG), sulfonates like EMS or diethylsulfonate (DES), sulfur mustards (e.g., ethyl-2-chloroethylsulfide), nitrogen mustards (e.g., 2-chloroethyl-dimethylamine), and alkylating agents (e.g., ethylene oxide). ENU and EMS are commonly used to generate random mutants in plant breeding.

[0154]

[0100] Ethyl methanesulfonate (EMS) has shown significant potential in inducing mutations in embryogenic cells. EMS primarily functions by alkylating guanine bases, leading to point mutations that can result in a variety of genetic changes. In embryogenic cells, these mutations can be leveraged to create a broad spectrum of genetic diversity, which is essential for studying developmental pathways and improving desirable traits in plants. The application of EMS in embryogenic cells is particularly advantageous due to their totipotent nature, allowing the mutations to propagate throughout regenerated plants. This approach has been instrumental in developing crops with enhanced stress tolerance, improved yields, and novel characteristics, making EMS a valuable tool in both fundamental research and practical plant breeding.

[0155]

[0101] In some embodiments, the genetic modification(s) is (are) induced by means of genetic engineering. This approach includes the integration of new sequences, substitution of existing ones, or precise deletions. Genetic engineering techniques have enabled breakthroughs in developing crops with improved resistance to pests, enhanced nutritional value, and better adaptability to environmental changes.

[0156]

[0102] In certain embodiments, a CRISPR-Cas system, such as the Type II variant, may be utilized.

[0157] This system comprises a guide RNA, for instance, a single guide RNA (sgRNA) or separate crRNA and tracrRNA components, which specifically target genomic regions for modification. Accompanying the guide RNA is a nuclease that interacts with it to bind or cleave the target genomicsequence. Established protocols can be used for designing these guide RNAs, drawing insights from relevant studies such as those by Cui et al. (2018), Bauer et al. (2018), and Mohr et al. (2016), which provide comprehensive guidelines for optimal CRISPR design.

[0158]

[0103] Various nucleases, including Cas9, Cas12a / Cpf1 , and Cas3, from diverse sources like Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), can be employed in these methods. Functional applications may involve employing two guide RNAs to excise undesired genetic elements or a single guide RNA to induce nucleotide alterations at the cleavage site, potentially integrating new sequences via homologous recombination if auxiliary polynucleotides are introduced. These modifications can be achieved through vectors, including viral, plasmid, or Agrobacterium-based systems, or by directly introducing ribonucleoproteins (RNPs) into the protoplasts or microspores.

[0159]

[0104] Methods for introducing CRISPR components into protoplasts or microspores are versatile, including polyethylene glycol-mediated transfection, electroporation, and advanced techniques like nanoparticle-based delivery or biolistic bombardment. Importantly, CRISPR components can be designed to transiently modify protoplasts or microspores without integrating into their genomes, ensuring stability of the genetic changes while preventing persistent activity of the CRISPR system in regenerated plants.

[0160]

[0105] In some embodiments of the disclosed methods, a CRISPR-Cas system, such as the Type II CRISPR-Cas system, is employed. This system comprises a guide RNA, such as a single-guide RNA (sgRNA), which targets a specific genomic sequence for modification. The guide RNA works in conjunction with a nuclease that cleaves or binds to the target sequence. The guide RNA can take various forms, including a single RNA molecule combining both crRNA and tracrRNA elements, or as separate crRNA and tracrRNA components. Guide RNAs can be designed using standard methods, including those outlined in Cui et al. (2018) Interdisciplinary Sciences: Computational Life Sciences, 10(2):455-465; Bauer et al. (2018) Frontiers in Pharmacology, 12 Jul. 2018, doi:10.3389 / fphar.2018.00749; and Mohr et al. (2016) FEBS Journal, doi:10.1111 / febs.13777, all of which are incorporated by reference herein.

[0161]

[0106] The CRISPR system can utilize a variety of nucleases, including but not limited to Cas9, Cas12a / Cpf1 , or Cas3, sourced from organisms such as Streptococcus pyogenes (e.g., SpCas9), Staphylococcus aureus (SaCas9), Streptococcus thermophilus (StCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), or Campylobacter jejuni (CjCas9). The guide RNA and nuclease can be applied in multiple strategies to modify genomic sequences within protoplasts or microspores. For instance, two guide RNAs may target adjacent regions flanking an undesirable gene, resulting in the deletion of the targeted sequence. Alternatively, a single guide RNA can target a specific gene, and the nuclease cleavage followed by cellular repair can induce insertions, deletions, or mutations at the cleavage site. In some cases, additional polynucleotides are introduced into the protoplast or microspores alongside the guide RNA and nuclease. These polynucleotides may contain sequences homologous to the target genomic region, enabling deletion, insertion, or modification through homologous recombination (also known as homology- directed repair).

[0107] In specific embodiments, polynucleotides encoding both the guide RNA and the RNA-guided nuclease (such as Cas9) are introduced into the plant cells, microspores or protoplasts. This can be accomplished by introducing vectors (e.g., viral vectors, plasmid vectors, or Agrobacterium vectors) that express one or more guide RNAs and the RNA-guided nuclease. The introduction is often achieved via transfection, resulting in the expression of the guide RNA and nuclease in the protoplasts or microspores. In other scenarios, guide RNAs and nucleases may be preassembled into ribonucleoprotein complexes (RNPs), which are then delivered into the protoplasts or microspores.

[0162]

[0108] Various techniques can be used to introduce the CRISPR-Cas components into protoplasts or microspores. Methods such as polyethylene glycol (PEG)-mediated transformation (including PEG- calcium), electroporation, microinjection, DEAE-dextran treatment, lipofection, nanoparticle-based transfection, protein transduction domain-mediated transfection, and biolistic bombardment have been described in the literature, including in studies by Toda et al. (2019) Nature Plants, 5(4):363- 368; Osakabe et al. (2018) Nature Protocols, 13(12):2844-2863; Soda et al. (2018) Plant Physiology and Biochemistry, 131 :2-11 ; and WO2017061806A1 , all incorporated by reference.

[0163]

[0109] In certain embodiments, the polynucleotides encoding the CRISPR-Cas system (such as the guide RNA and nuclease) are transiently expressed within the plant cells, microspores or protoplasts but do not integrate into the plant cells, microspores or protoplast’s genome. As a result, the genomic changes induced by the CRISPR-Cas system are maintained in the protoplast or microspore and in any plants regenerated from it, but the CRISPR-Cas components themselves are not persistent and do not cause additional genomic modifications.

[0164]

[0110] In some embodiments, said genetic modification is performed by RNA-directed DNA methylation, genomic imprinting, paramutation induction, RNA interference (RNAi), microRNA (miRNA), or piRNA transformation. Each of these methods relies on RNA molecules to mediate changes in the genome or gene expression without altering the DNA sequence itself. These techniques represent a powerful toolbox for researchers aiming to control gene function in a targeted and reversible manner. They offer a wide range of applications in plant breeding, particularly in contexts where precise control of gene activity is required without permanent alterations to the genetic material.

[0165]

[0111] The genetic modifications described can lead to a broad range of desirable changes in plants.

[0166] These modifications can be used to regulate plant growth, either by inhibiting or enhancing it. For example, by modifying specific genes, the plant's growth rate could be slowed for applications where smaller plants are preferable, or accelerated for faster harvest cycles. Nutrient requirements of the plant can also be altered, making the plant more efficient in terms of nutrient uptake or reducing its dependence on certain fertilizers, thus enhancing its sustainability in various growing conditions.

[0167]

[0112] Furthermore, genetic modifications can increase or decrease the production of specific plant products, such as oils, fibers, or secondary metabolites. This could include altering the plant's natural pathways for producing compounds of economic or medicinal value. In addition, protein and saccharide content can be modified to enhance nutritional value or improve the plant’s suitability for use in bioengineering and biorefining processes.

[0113] The plants can also be adapted to survive in challenging environments, where typical crops may struggle. This includes stress resistance to conditions such as reduced light (e.g., low light levels under a canopy), lower temperatures (e.g., for crops grown in cooler climates), or brackish water (allowing plants to grow in soils with higher salt content, which could otherwise inhibit growth). Genetic modifications could also make the plant more resistant to microbial and pest infections, reducing the need for chemical pesticides and enhancing crop yield and quality.

[0168]

[0114] These types of changes can be brought about through the introduction of structural genes, which encode proteins responsible for specific traits (e.g., proteins involved in stress tolerance or resistance to pests), or regulatory genes, which control the expression of these proteins or other genes. By fine-tuning the expression of these genes, plants can be engineered to exhibit the desired traits.

[0169]

[0115] .As a consequence, introducing and screening genetic changes in species such as coffea, traditionally recalcitrant to protoplast culture, is of utmost importance for breeding purposes.

[0170]

[0116] Genetically modifying the protoplasts or microspores by techniques such as random mutagenesis may result into the production of a genetically-diverse population of protoplasts or microspores. Accordingly, in some embodiments, said plurality of protoplasts or microspores is a genetically diverse population of protoplasts. Typically, one or more protoplasts or microspores within the plurality of protoplasts or microspores used in the method of the invention may comprise a genome which differs from the genome of one or more other protoplasts or microspores within the plurality of protoplasts. In some embodiments, the genome of each protoplast and / or microspore differs from the genome of other protoplasts or microspores within the plurality of protoplasts or microspores, by at least one genetic modification, e.g. at least one mutation. In particular, said genetic diversity is the result of random mutagenesis. In some embodiments, the protoplasts or microspores comprise a genome which differs from other protoplasts or microspores by the presence of at least one mutation. In some embodiments, at least 2 protoplasts or microspores, in particular at least 3, 5, 10, 20, 50, 100, 500, 1000, 10000 or 100,000 protoplasts or microspores, within the population of protoplast and / or microspore comprise a unique genome. In some embodiments, each protoplast and / or each microspore has a unique genome. This may be the result of random mutagenesis applied to the protoplasts, microspores and / or to plant cells from which the protoplasts originate and / or from chimerism of the starting material, e.g. plant cell culture, plant tissue and / or calli from which the protoplasts originate

[0171]

[0117] When protoplasts or microspores having different genomes are used e.g. protoplasts or microspores issued from random mutagenesis, co-culturing the protoplasts or microspores in isolation from each other, as described in the present invention, is of utmost importance to prevent chimerism of regenerated plants,

[0172] Isolating the protoplasts and microspores

[0173]

[0118] In the method described in the present invention, protoplasts or microspores are isolated from one another to facilitate their co-cultivation and subsequent development into individual microcalli, suspension cell lines, or embryos. This isolation step reduces the risks typically associated withtraditional co-culture techniques, where protoplasts or microspores are not isolated. In non-isolated co-cultures, cross-contamination is a significant concern, which could lead to the formation of chimeric plants with genetically distinct cells that result from the fusion of protoplasts from different sources, or merging of growing microcalli together. Furthermore, traditional co-culturing requires maintaining very low cell densities (typically below 10,000 cells / mL), which can be problematic for certain recalcitrant plant species or genotypes that are not susceptible to regeneration under such conditions.

[0174]

[0119] The isolation of protoplasts or microspores in this method can be accomplished through a variety of approaches, as detailed in the invention. In certain embodiments, protoplasts or microspores are isolated and encapsulated in individual gel capsules, leading to the formation of a multitude of isolated gel capsules. This process of isolation and encapsulation can be performed either simultaneously or in successive steps.

[0175]

[0120] In some embodiments, microfluidics devices, flow cytometry devices, or micromanipulation devices are employed to achieve the isolation of protoplasts. Specifically, the protoplast or microspore isolation step can be carried out using techniques such as cell picking and / or cell dispensing.

[0176]

[0121] Single-cell picking is a method of isolation in which individual protoplasts or microspores are separated from a larger population. This process involves placing the protoplasts in a suitable culture medium and then isolating them one by one using soft aspiration with a cell picking device. Protoplasts or microspores can be initially provided in suspension or placed on a substrate, such as a gel plate, or arranged in arrays of microwells. The picking process is typically carried out using a micromanipulation device, which includes a micromanipulation arm capable of gently aspirating and picking individual cells. Examples of cell picking devices include automated microcapillary sampling devices, such as the Cellcellector (Sartorius) which is specifically designed to handle small volumes down to the nanoliter range.

[0177]

[0122] The single-cell picking process can be combined with a monoclonality detection step. This involves using an optical device equipped with cell recognition software to visually identify and verify the isolation of a single cell. Optical devices such as inverted microscopes (including brightfield, phase contrast, or fluorescence microscopes) are commonly employed. The recognition software uses algorithms that detect contrasts in the image, such as changes in the cell’s outline or the boundaries of the microwell, to confirm the presence of a single protoplast or a single microspore.

[0178]

[0123] Alternatively, single-cell dispensing can be used as another method for protoplast or microspore isolation. This technique involves the passage of protoplasts or microspores through a microfluidics device, where each protoplast or microspore is detected by a sensor. Various methods, such as impedance detection, brightfield detection, or fluorescence detection, can be used to identify the presence of individual cells. Once detected, protoplasts or microspores are dispensed as droplets into individual wells, each containing a single cell. To maintain the integrity of the protoplasts and avoid rupturing, the osmolality of the sheath fluid in the microfluidics device can be controlled, typically between 0.5 atm and 0.9 atm, with a preferred range of 0.6 atm to 0.8 atm. An example of sheath fluid mixture includes 1xPBS combined with 0.3M-0.5M sorbitol to prevent osmotic stress.An example of cell dispensing device which can be used in the present invention is the SH800S (Sony)

[0179]

[0124] In some embodiments, flow cytometry is employed for the single-cell dispensing process.

[0180]

[0125] In alternative embodiments, the method may not include a monoclonality detection step prior to encapsulation using microfluidics. Instead, protoplasts or microspores can be dispensed into droplets containing single cells by diluting the cell solution. The distribution of protoplasts or microspores in droplets follows a Poisson distribution, which means some droplets may contain more than one protoplast or microspore. In this case, an additional enrichment step is required to ensure that only capsules with a single protoplast or with a single microspore are used for cocultivation. This enrichment process involves distributing the gel capsules into a multiwell array, e.g. a microwell array, where one capsule is placed in each well. Monoclonality screening is then performed on the distributed capsules using automated or manual microscopy techniques (e.g., inverted microscopy, brightfield microscopy, or fluorescence microscopy) to identify capsules containing a single protoplast or a single microspore. Finally, the gel capsules containing single protoplasts or single microspores are sampled and subsequently co-cultured to foster the development of microcalli.

[0181] Encapsulation

[0182]

[0126] In some embodiments, the method of the invention involves encapsulating isolated protoplasts or isolated microspores in gel capsules, with each capsule containing a single protoplast or a single microspore. This step of encapsulation may comprise depositing a droplet composed of at least one gelling agent and a single protoplast or a single microspore onto a solid, semi-solid, or liquid surface. The droplet is then solidified into a gel capsule. To enhance the environment for protoplast growth or microspore growth, the droplet preferably includes a culture medium to maintain the protoplasts or microspores in optimal conditions during the encapsulation process.

[0183]

[0127] The deposition of the droplet can be accomplished using a microcapillary device, which is capable of both picking and dispensing individual cells through soft aspiration. This precise method allows for the careful handling of each protoplast or microspore to ensure their isolation and proper encapsulation.

[0184]

[0128] The gelling agent used for capsule formation is typically a hydrogel-forming agent, chosen from a variety of suitable materials such as alginate, agarose, pectin, or synthetic biomimetic hydrogels, or combinations thereof. These gelling agents are dissolved in the droplet at a concentration ranging from approximately 0.1% to 5% by weight, with a preferred concentration typically between 0.2% and 2%. In some embodiments, alginate is the preferred gelling agent. Low-viscosity forms of sodium alginate are particularly useful for creating stable, yet flexible capsules. The droplet containing the protoplast or microspore and gelling agent is solidified by contacting it with a calcium salt, such as calcium chloride, which induces the gelling process through ionic interaction with the alginate. The droplet may be deposited onto a solid surface, such as CaCh-agar plates. Once the droplet is deposited onto a surface or into a pre-established well, the next step involves solidifying the droplet into a gel capsule. This process occurs when the droplet, containing sodium alginateand the protoplast or microspore, is exposed to a calcium salt, typically calcium chloride (CaCI2), coated on the solid surface. Calcium ions from the calcium chloride interact with the alginate molecules, specifically with the guluronic acid blocks in the alginate chain, leading to the formation of a cross-linked network. This cross-linking induces the formation of a gel, effectively encapsulating the protoplast or microspore inside a protective, yet semi-permeable, capsule.

[0185]

[0129] The gel capsule provides a controlled environment for the protoplast or microspore. The gel is flexible enough to allow for gas and nutrient exchange, which is essential for cell survival and growth, while simultaneously providing protection from external stresses, such as mechanical damage or contamination. The thickness and rigidity of the gel capsule can be modulated by adjusting the concentration of calcium salt or the alginate concentration in the droplet.

[0186]

[0130] Alternatively, in some embodiments, the gelling agent can be agarose. Agarose is another gelling agent that can be used in protoplast or microspore encapsulation due to its ability to form a stable, non-toxic gel at relatively low concentrations. Agarose is a linear polysaccharide derived from agar, which is obtained from red algae. It is commonly used in molecular biology and cell culture applications due to its biocompatibility, ease of gelation, and the flexibility in controlling the physical properties of the gel. Agarose gels, unlike alginate gels, do not require ionic cross-linking for gel formation. Instead, agarose forms a gel upon cooling. For instance, freshly autoclaved solutions of agarose can be cooled down to form a gel.

[0187]

[0131] In the context of protoplast or microspore encapsulation, low-melting agarose is advantageous for its ability to gel at relatively low temperatures, typically around 24-30°C, while regular agarose requires a higher temperature (around 32-45°C) and they stay liquid above. This makes low-melting agarose ideal for encapsulating sensitive protoplasts or microspores without subjecting them to harsh or high-temperature conditions. This property allows for the gentle encapsulation of protoplasts or microspores, ensuring minimal damage to the cells during the solidification process.

[0188]

[0132] Where agarose is used, solidification of the droplet is achieved by applying reduced temperature conditions to the droplet, which causes the agarose to gel.

[0189]

[0133] The resulting gel capsules can take two forms: one where the gelling agent forms a thin, flexible envelope surrounding a regeneration medium that supports the protoplast's or microspore’s growth, or a more uniform structure where the capsule is entirely composed of the gelling agent itself.

[0190]

[0134] The volume of the droplet directly determines the size of the resulting capsule and thus the cell density within each capsule. Key factors that influence droplet volume include the capillary size and the dispensing pressure of the microfluidics devices. A well-chosen capillary size or chip nozzle allows for precise handling of plant protoplasts, whose diameters typically range between 50 pm to 120 pm, although certain cell types may be smaller, measuring around 10 pm to 15 pm. Capillary size or chip nozzle can also be chosen for precise handling of plant microspores, whose diameters typically range between 15 pm to 40 pm depending on species and developmental stage, although certain taxa or stages may present smaller microspores measuring around 10 pm to 15 pm or larger pollen grains approaching 50 pm. The preferred diameter of the capillary or chip nozzle typically ranges from 10 pm to 1000 pm, in particular 50 pm to 1000 pm to accommodate the full spectrum of protoplast sizes. The preferred diameter of the capillary or chip nozzle typically also ranges from10 pm to 1000 pm, in particular 15 pm to 100 pm to accommodate the full spectrum of microspores sizes A device such as the Sartorius Cellcellector® and Sony SH800S® are examples of microfluidics devices used for protoplast or microspore encapsulation. These devices can also be used for both the single-cell picking or cell dispensing, and encapsulation steps, ensuring streamlined workflow and consistency.

[0191]

[0135] The size of the droplets produced by the microcapillary device is directly proportional to the diameter of the capillary / chip nozzle. For optimal capsule formation and further protoplast and / or microspore culture, gel capsules generally have a volume ranging from 0.25 nL to 1 pL, in particular from 0.25 nL to 200 nL, more particularly from 0.5 nL to 150 nL, and even more particularly between 1 nLto 100 nL. The diameter of the capsules typically falls between 50 pm and 1250 pm, in particular from 75 pm to 650 pm, more particularly from 100 pm to 500 pm.

[0192]

[0136] The size of the gel capsule surrounding the protoplast or microspore is critical for providing an environment that supports the development and division of the protoplasts or microspores. If the capsule is too small, it may restrict the growth of the protoplast or microspore, preventing optimal cellular development. Conversely, excessively large capsules may result in low culture density, which can hinder the necessary intercellular exchanges essential for protoplast or microspore proliferation and overall cell growth. Achieving the right capsule size is therefore a key factor in promoting the successful regeneration of the protoplasts or microspores into viable plant cells, especially for recalcitrant plant species. The sizes of gel capsules used in the present invention promote efficient cell growth at the first stages of cell divisions of the encapsulated protoplasts or microspores.

[0193]

[0137] In preferred embodiments, the size of the bead is constantly adapted to the size of the cell colony, callus or suspension cell line contained within the bead. As described below, this is carried out by reencapsulating the cell colonies, calli or suspension cell lines, whenever necessary, during the cell culture.

[0194] Culture of the capsules

[0195]

[0138] The encapsulated protoplasts or microspores are co-cultured in a regeneration medium, where cell density influences the protoplasts' or microspores’ regenerative capacity into cell aggregates like calli or suspension cell lines. In certain embodiments, the co-culture density of capsules ranges from as low as 100 capsules / mL to as high as 50,000 capsules / mL. Other embodiments specify densities no higher than 500,000, 250,000, or 100,000 capsules / mL. The preferred density for coculturing protoplasts or microspores typically falls between 100 and 500,000 capsules / mL, with the optimal range being 10,000 to 500,000 capsules / mL. Even more precisely, some preferred densities lie between 20,000 and 500,000 capsules / mL or between 100,000 and 500,000 capsules / mL, while for specific applications, densities of 100 to 100,000 capsules / mL (or more ideally 10,000 to 100,000 capsules / mL) are selected, with 20,000 to 100,000 capsules / mL being the most preferred range.The density used in the present invention provides efficient growth with the gel capsules, in combination with appropriate capsule sizes, as described herein.

[0196]

[0139] This method introduces a versatile framework for co-culturing protoplasts or microspores at adjustable densities within a shared liquid medium. Unlike traditional regeneration techniques that utilize bulk alginate matrices or free-floating liquid cultures — methods that often fall short in maintaining monoclonality — this approach ensures high cell densities while preserving the distinct separation of individual regenerated cell lines. This separation is crucial for achieving monoclonality, a feature that guarantees both the reproducibility and reliability of experimental outcomes. Classical regeneration media used for protoplast or microspores cultures and known to the skilled person, can be used as regeneration media for co-culture of encapsulated protoplasts, or co-culture of protoplasts in microwell arrays, or co-culture of encapsulated microspores, or co-culture of microspores in microwell arrays. Upon culture, protoplasts or microspores within the gel capsules or microwells can undergo cell divisions to form monoclonal cell colonies, and then monoclonal calli and / or monoclonal suspension cell lines. Microspores within the gel capsules or microwells can undergo cell divisions to form haploid or double haploid embryos. In some embodiments, at step (e) of the method, the protoplast undergo cell divisions to form monoclonal cell colonies, and undergo further cell divisions to form monoclonal calli and / or monoclonal suspension cell lines. In some embodiments, at step (e) of the method, the microspores undergo cell divisions to form haploid or dihaploid embryos.

[0197]

[0140] Experimental evidence provided in the present disclosure confirms the advantages of this method, showing that co-culturing capsules, whether containing protoplasts, microspores or established cell lines, within a shared liquid medium enhances both regeneration and growth. Notably, as capsule density increases, the effective cell density within the medium also rises, directly contributing to more efficient regeneration. This increased capacity to culture at higher densities while still ensuring monoclonality represents a significant advantage over traditional regeneration methods.

[0198]

[0141] The disclosed method is highly adaptable, suitable for a range of culture vessels compatible with liquid media, including multiwell arrays such as microwell arrays, petri dishes, flasks, and bioreactors. Depending on the needs of the specific protoplasts, microspores or cell lines being cultured, the system can be maintained in a static state or subjected to controlled agitation. This flexibility allows researchers to fine-tune culture conditions for optimal regeneration.

[0199]

[0142] One of the standout advantages of this method is its ability to encapsulate protoplasts or microspores within a physical barrier. This encapsulation serves to mitigate the sensitivity of the protoplasts or microspores to fluctuations in environmental factors, such as osmolarity changes or mechanical agitation, which could otherwise compromise cell viability. By maintaining protoplasts or microspores within a gel capsule, the method allows for controlled agitation of the culture medium to improve oxygenation, without detrimental effects on protoplast or microspore growth or survival.

[0200]

[0143] The culture medium is preferably osmotically-controlled.

[0201]

[0144] In some embodiments, the culture medium comprises one or more of a basal salt mixture, vitamins (such as. thiamine, pyridoxine, nicotinic acid, folic acid, riboflavin, and / or calciumpantothenate), one or more carbon sources (such as sucrose, glucose, fructose, ribose, xylose, mannose, and / or cellobiose), a pH buffer (e.g. MES), antioxidants (such as polyvinylpyrrolidone and / or L-cysteine), a protein source (such as casein), plant growth regulators (e.g. myo-inositol, auxins such as 2,4-dichlorophenoxyacetic acid and / or naphthaleneacetic acid, and / or cytokinins such as benzylaminopurine (BAP)), and an organic supplement (such as coconut water) to support plant cell growth and differentiation. Osmolarity of the medium can be adjusted at required level for protoplast culture, e.g. to 600-800 mOsm / kg. For instance, the medium. In specific embodiments the culture medium comprises a basal salt mixture (e.g., Gamborg B5) at a concentration of 2.5-4.0 g / L; vitamins including thiamine (0.005-0.05 mg / L), pyridoxine (0.0005-0.005 mg / L), nicotinic acid (0.0005-0.005 mg / L), folic acid (0.0001-0.001 mg / L), riboflavin (0.00005-0.001 mg / L), and calcium pantothenate (0.00005-0.001 mg / L); carbon sources including sucrose (10-40 g / L), glucose (50- 150 g / L), fructose, ribose, xylose, mannose, and cellobiose (each 0.05-0.5 g / L); a pH buffer such as MES (0.2-1.0 g / L); antioxidants including polyvinylpyrrolidone (0.1-0.5 g / L) and L-cysteine (0.005-0.05 g / L); a protein source such as casein (0.1-0.5 g / L); plant growth regulators including myo-inositol (50-200 mg / L), auxins such as 2,4-dichlorophenoxyacetic acid (0.0002-0.001 mg / L) and naphthaleneacetic acid (0.0002-0.001 mg / L), and a cytokinin such as benzylaminopurine (0.0005-0.002 mg / L); and an organic supplement such as coconut water (5-20% v / v). In some embodiments, the medium additionally comprises 70-120g / L glucose, in particular 80-110g / L glucose as osmoticum, and / or equivalent amounts of carbohydrate compounds such as sucrose and / or mannitol. Further exemplary composition of regeneration medium, in particular for Coffea canephora, are described in the Examples. Protoplasts require cultivation in a high-osmotic- pressure medium to prevent rupture prior to the re-establishment of their cell walls. The media described in the present application are designed to promote protoplast development. Protoplast culture media are also described, e.g. in Smith, 2013, Fowke, 1985.

[0202]

[0145] The culture medium and conditions can also be optimized for microspore culture to ensure embryo generation and subsequent development while preserving monoclonality in capsules or microwells. In particular embodiments, microspores are cultured initially in high-osmotic, low-mineral media supplemented with defined carbohydrates and organic supplements, kept in darkness, and subjected to a transient stress treatment that triggers the embryogenic switch, followed by maintenance at permissive temperatures, e.g. 24-26°C.

[0203]

[0146] In specific embodiments, microspores are cultured in NLN medium adjusted for androgenesis (NLN-13), comprising a basal salt mixture (NLN salts) with macro- and micronutrients, iron-EDTA at 36-45 mg / L of Fe-EDTA, vitamins including thiamine (0.1-1 mg / L), pyridoxine (0.1-1 mg / L), nicotinic acid (0.1-1 mg / L), a carbon source of sucrose at 120-140 g / L (13% w / v) as osmoticum, optional casein hydrolysate at 0.25-1.0 g / L, L-glutamine at 0.5-2.0 g / L, and pH adjusted to 5.7-5.9. The culture is maintained at 32-35°C for 24-48 hours to induce androgenesis, then shifted to 24- 26°C. Cultures can be kept in complete darkness for the induction phase and early divisions, with optional low light introduced after globular-stage embryos appear. Osmolarity can be controlled at 450-650 mOsm / kg during induction and gradually reduced to 280-350 mOsm / kg during embryo enlargement and maturation by stepwise dilution of the carbon source. Antioxidants such aspolyvinylpyrrolidone (0.1 -0.5 g / L) and L-cysteine (0.005-0.05 g / L) can be added to mitigate phenolic oxidation. As embryos reach globular, heart, and torpedo stages within their capsules or wells, they can be reencapsulated into larger capsules or reseeded into larger microwells under reduced osmolarity and permissive temperatures, maintaining monoclonality and enabling timed, nondestructive sampling for genomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses.

[0204]

[0147] Furthermore, the culture conditions can be tailored to support specific regeneration pathways, such as callogenesis or somatic embryogenesis. Appropriate light exposure, along with other environmental parameters, can be adjusted to optimize the regeneration process, further enhancing the effectiveness and efficiency of the method. Exemplary callus multiplication medium are described, e.g. in Rezende et al., 2012.

[0205] Culture in microwells

[0206]

[0148] In certain embodiments, the methods of the present invention employ microwell arrays, e.g.

[0207] multiwell plates, for the culture and regeneration of protoplasts, wherein individual protoplasts or group of cells issued from said protoplasts (e.g. monoclonal cell colonies, monoclonal calli and / or monoclonal cell suspensions) are spatially separated yet share a common culture medium. In certain embodiments, the methods of the present invention employ microwell arrays, e.g. multiwell plates, for the culture and regeneration of microspores, wherein individual microspores or group of cells issued from said microspores (e.g. monoclonal cell colonies, monoclonal calli and / or embryos) are spatially separated yet share a common culture medium. The wells within these arrays may be fluidically connected wells, or otherwise interconnected via liquid pathways located above, below, or between the wells, thereby facilitating nutrient and signal exchange while maintaining physical isolation of the individual cells. The configuration of the wells may vary, for example, in shape (e.g., round, hexagonal, or square) and size (e.g., ranging from approximately 10 pm to 1 mm in diameter or length). The wells are typically separated by distances of a few micrometers, ensuring effective exchange of nutrients and signals between adjacent cells while preserving their physical isolation within the culture medium.

[0208]

[0149] A key aspect of the methods of the present invention is ensuring the achievement of monoclonality, wherein each well is populated with a single protoplast or a single microspore or, at a minimum, wells containing single cells are accurately identified at the onset of culture (day 0). This may be accomplished through the use of a cell picking device coupled with a monoclonality detection step as described herein. In particular, isolated protoplasts or isolated microspores are picked and subsequently detected utilizing an optical device integrated with image processing software, which enables visualization and recognition of the presence of a single protoplast or a single microspore within each well. These steps can be performed using inverted microscopy in conjunction with a micromanipulation robot, which facilitates precise seeding and subsequent verification of monoclonality. Furthermore, assessment of cell growth in individual wells can be performed through optical imaging coupled with image processing software, thus enabling the generation of growth curves. For more advanced analyses, fluorescence microscopy may be employed to identify specificfluorescently labeled cell populations. Protoplasts or microspores may, for instance, be transfected with reporter genes, e.g. fluorescent reporter genes such as GFP, which will serve to estimate editing efficiency and pick up only transfected cell colonies.

[0209]

[0150] A variety of microwell arrays with different well sizes and configurations are available for use in the present invention, offering flexibility and versatility for diverse experimental needs. Examples of microwell arrays suitable for use in the methods of the invention include, but are not limited to, Nanowell® plates which typically have wells of approximately 200 pm and Sievewell® plates which typically have wells of approximately 50 pm. The methods described herein apply this technology to successfully regenerate protoplasts or microspores from single plant cells, providing a robust platform for plant cell culture applications.

[0210]

[0151] In some embodiments, each microwell has a size of from 10 pm to 1mm, in particular from 20 pm to 1mm, more particularly from 25pm to 1mm. In some embodiments, each microwell has a size of from 10pm to 800pm, in particular from 10pm to 500pm. In some embodiments, each microwell has a size of from 25pm to 500pm, in particular from 25pm to 250pm, more particularly from 50pm to 250pm. By “size”, it is referred to the longer length of the well, i.e. the diameter of the well, for circular or hexagonal wells, or the longer side for rectangular / square wells.

[0211]

[0152] In some embodiments, each microwell has a volume of 0.5 nL to 100 nL, in particular from 1 nL to 50 nL, more particularly from 1 to 25 nL or from 2 nL to 25 nL.

[0212]

[0153] Protoplasts or microspores are typically seeded in the microwells at densities ranging from 1 ,000 cells / mL to 200,000 cells / mL, with more specific embodiments involving densities from 5,000 cells / mL to 150,000 cells / mL or from 5,000 cells / mL to 200,000 cells / mL, and even more particularly from 10,000 cells / mL to 100,000 cells / mL or from 10,000 cells / mL to 200,000 cells / mL. In other embodiments, higher densities of up to 500,000 cells / mL may be used, in particular from 1 ,000 cells / mLto 500,000 cells, mL, in particular from 5,000 cells / mLto 500,000 cells / mL, more particularly from 10,000 cells / mL to 500,000 cells / mL. The optimal seeding density will depend on the specific size and geometry of the wells used in the culture system. Upon culture, protoplasts or microspores within the microwells undergo cell divisions to form monoclonal cell colonies, and then monoclonal calli, monoclonal suspension cell lines and / or embryos. In some embodiments, at step (e) of the method, the protoplast or microspore undergo cell divisions to form monoclonal cell colonies, and undergo further cell divisions to form monoclonal calli and / or monoclonal suspension cell lines. The regeneration of monoclonal cell colonies, calli and cell suspension cell lines, has been successfully demonstrated by the inventors, as evidenced by the data presented in the examples and appended drawings.

[0213]

[0154] The methods of the present invention enable the generation of monoclonal cell lines that fully occupy the wells, providing a scalable, reproducible, and efficient solution for protoplast or microspore regeneration, with broad applications across the field of plant biotechnology.

[0214] Scalinq-up the cultures

[0215]

[0155] The calli or suspension cell line cultures, obtained by protoplast culture or by microwell culture, can be scaled-up to allow cell colonies, calli, embryos or suspension cell lines to further grow. Inthis way, microcalli, calli or embryos can be obtained that can be used for plant regeneration. Scaling-up of the cultures may involve scaling-up the supports in which the monoclonal calli or embryos are cultured, such as the capsules and / or the microwells utilized in the process.

[0216]

[0156] In some embodiments, step (e) comprises obtaining monoclonal cell colonies from the cocultured protoplasts, further co-culturing the monoclonal cell colonies and obtaining monoclonal calli and / or suspension cell lines.

[0217]

[0157] In some embodiments, step (e) comprises obtaining embryos from the microspores, e.g. haploid ordihaploid embryos.

[0218]

[0158] In some embodiments, the method may comprise one or more scaling-up steps, in particular within step (e) of the method. In particular, each scaling-up step may comprise any one of steps (i), (ii) and / or (iii), or their combination wherein:

[0219]

[0159] step (i) comprises encapsulating or reencapsulating, in a gel capsule, the monoclonal cell colonies, calli, suspension cell lines or embryos obtained from protoplast division or microspore division wherein each gel capsule comprises a single monoclonal cell colony, callus, suspension cell line or embryo, and co-culturing the gel capsules in a culture vessel containing culture medium, whereby the monoclonal cell colonies, calli, suspension cell lines or embryos are allowed to further divide; and

[0220]

[0160] step (ii) comprises seeding or reseeding the monoclonal cell colonies, calli, suspension cell lines or embryos obtained from protoplast division or microspore division, into microwells of an array of microwells, wherein the array is arranged to enable liquid exchanges between the microwells, and co-culturing the monoclonal cell colonies, calli, suspension cell lines or embryos in the array of microwells, whereby the monoclonal cell colonies, calli or suspension cell lines are allowed to further divide; and

[0221]

[0161] step (iii) comprises seeding the encapsulated monoclonal cell colonies, calli, suspension cell lines or embryos on a semi-solid or solid medium, and culturing to allow the monoclonal cell colonies, calli or suspension cell lines to further divide.

[0222]

[0162] In the case where the monoclonal cell colonies, calli, suspension cell lines or embryos are initially encapsulated, steps (i), (ii) and (iii) may comprise an initial step of dissolving the gel capsule of the encapsulated monoclonal cell colonies, calli, suspension cell lines or embryos, before a further step of (re)encapsulating or (re)seeding the monoclonal cell colonies, calli, suspension cell lines or embryos.

[0223]

[0163] In certain embodiments, the culture of monoclonal cell colonies, calli, suspension cell lines or embryos is scaled-up by encapsulation or reencapsulation, which comprises encapsulating or reencapsulating the monoclonal cell colonies, calli, suspension cell lines or embryos in gel capsules. This encapsulation or reencapsulation step can be applied to monoclonal cell colonies, calli, suspension cell lines or embryos derived from protoplast co-culture in gel capsules orthose obtained from co-culture in microwells. The size of the capsules in which the monoclonal cell colonies, calli, suspension cell lines or embryos are (re)encapsulated is chosen to allow further growth of the monoclonal cell colonies, calli, suspension cell lines or embryos. In some embodiments, the monoclonal cell colonies, calli, suspension cell lines or embryos are reencapsulated in gel capsuleswith an increased volume compared to the previous step, where the monoclonal cell colonies, calli, suspension cell lines or embryos were encapsulated in gel capsules at the previous step. The volume of the gel capsules may be increased by a factor of at least 20%, in particular at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the previous cycle. More specifically, the increase in volume may range from 20% to 1000%, preferably from 25% to 500%, and more preferably from 50% to 250% at each scaling-up cycle.

[0224]

[0164] In some embodiments, the scaling-up of encapsulation is achieved by dissolution of the gel capsules of the previous step, comprising a monoclonal cell colony, monoclonal callus, monoclonal suspension cell line, or embryo and reencapsulation of the monoclonal cell colony, callus, monoclonal suspension cell line or embryo. In some instances, the scaling-up of encapsulation may be achieved by spreading the capsules, or calli released from their capsules via dissolution, e.g. onto a transparent container. Colonies are subsequently isolated using a colony picker with capillary dimensions optimized according to the size of the capsules. The drop size for deposition is adjusted to produce capsules that are slightly larger than those previously picked. In certain embodiments, calli may be pre-released from their capsules by dissolution, facilitating their redistribution. The efficiency of this co-culture system for encapsulated calli has been demonstrated by the inventors (Fig. 10), illustrating the scalability of calli cultures from microcalli formation to the establishment of fully developed cell lines.

[0225]

[0165] The scaling-up process is typically carried out when the cells occupy at least 50%, and preferably at least 70%, 80%, or 90% of the capsule volume, e.g. from 70% to 100% or from 70% to 90% of the capsule volume. The term “scaling-up” refers to the process of encapsulating the monoclonal cell colonies, calli, suspension cell lines or embryos in capsules of larger size, sufficient to sustain their continued growth, or reseeding the monoclonal cell colonies, calli, suspension cell lines or embryos into microwells of an appropriate size to allow for further development.

[0226]

[0166] In certain embodiments, the method comprises one or more steps of encapsulating monoclonal colonies, calli, suspension cell lines and / or embryos in gel capsules having a diameter ranging from 200 pm to 500 pm. In other embodiments, the method comprises one or more steps of encapsulating monoclonal cell colonies, calli, suspension cell lines and / or embryos in gel capsules having a diameter ranging from 500 pm to 1000 pm. In other embodiments, the method comprises one or more steps of encapsulating monoclonal cell colonies, calli, suspension cell lines and / or embryos in gel capsules having a diameter higher than 1mm, in particular from 1mm to 5mm, more particularly from 1 mm to 2mm. For example, the method may include a step of encapsulation with a diameter of 200 pm to 500 pm, followed by a step with a diameter of 500 pm to 1000 pm, and a further encapsulation in capsules with diameters greater than 1 mm.

[0227]

[0167] In some embodiments, the method comprises at least 1 step of reencapsulation. In other embodiments, the method comprises more than 1 step of encapsulation, in particular at least 2 steps of encapsulation, more particularly at least 3, 4, 5, 6, 7, 8, 9 or 10 steps of encapsulation. In some embodiments, the method comprises from 1 to 10 steps of encapsulation, in particular from 1 to 5 steps of encapsulation. In other embodiments, the method comprises from 1 to 10, in particular from1 to 5 more particularly from 1 to 3 steps of encapsulation. In some embodiments the method comprises from 2 to 10 steps of encapsulation, in particular from 2 to 5 steps of encapsulation, more particularly from 2 to 3 steps of encapsulation. By “steps of encapsulation”, it is herein meant to encompass the initial step of encapsulation of protoplasts or microspores and any further step of encapsulation of cell clusters, including monoclonal calli, monoclonal cell colonies, suspension cell lines or embryos.

[0228]

[0168] Alternatively, scaling-up of the culture can be achieved through co-culturing monoclonal cell colonies, calli, suspension cell lines and / or embryos within microwells.

[0229]

[0169] Microwells can be seeded by various methods. In certain embodiments, colonies, calli, suspension cell lines or embryos are first released from their capsules via dissolution of the gelling agent. Dissolution can be accomplished using methods such as increasing the temperature for low- melting agarose, lowering the temperature for polyisocyanide hydrogels, or applying chelating agents like EDTA or sodium citrate to dissolve alginate or pectin gels. In other embodiments, encapsulated colonies, calli, suspension cell lines or embryos are directly introduced into the microwells, followed by in situ dissolution of the gelling agent constituting the capsule. This method offers the advantage of maintaining physical separation between individual cell lines throughout the scaling-up process, thus ensuring monoclonality. This approach is applicable to all types of cell lines, whether in the form of compact calli, suspensions or embryos. In both cases, colonies, calli, embryos or suspension cell lines can be seeded into the microwells by picking using a cell picking device, such as a microcapillary device, where the capillary size is adjusted according to the colony, callus, embryo or suspension cell line size.

[0230]

[0170] Once the monoclonal colonies, calli, suspension cell lines or embryos are seeded into the microwells, co-culturing is maintained until the calli or suspension cell lines reach the maximum permissible size for the well.

[0231]

[0171] In some embodiments, the culture is scaled-up when the colonies, calli, suspension cell lines or embryos occupy at least 50%, and more preferably at least 70%, 80%, or 90% of the microwell surface area. “Scaled-up” refers to the reseeding of monoclonal cell colonies, calli, suspension cell lines or embryos into larger microwells or their encapsulation in capsules of sufficient size to sustain their growth.

[0232]

[0172] The size of the microwells in which the monoclonal cell colonies, calli, suspension cell lines or embryos are seeded is chosen to allow further growth of the monoclonal cell colonies, calli, suspension cell lines or embryos. In some embodiments, the monoclonal cell colonies, calli, suspension cell lines or embryos seeded in microwells are reseeded in microwells with an increased volume compared to the previous step, where the colonies, calli, suspension cell lines or embryos were seeded in microwells at the previous step. The volume of the microwells may be increased by a factor of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the previous cycle. More specifically, the increase in volume may range from 20% to 1000%, preferably from 25% to 500%, and more preferably from 50% to 250% at each scaling-up cycle.

[0173] In some embodiments, the method comprises at least 1 step of seeding. In other embodiments, the method comprises more than 1 step of seeding in particular at least 2 steps of seeding, more particularly at least 3, 4, 5, 6, 7, 8, 9 or 10 steps of seeding. In other embodiments, the method comprises from 1 to 10, in particular from 1 to 5 more particularly from 1 to 3 steps of seeding. By “steps of seeding”, it is herein meant to encompass the initial step of seeding of protoplasts and any further step of seeding of monoclonal calli.

[0233]

[0174] In further embodiments, scaling-up of the culture may be achieved by seeding monoclonal cell colonies, calli, suspension cell lines or embryos at a low density onto semi-solid or solid medium, such as an agar plate, and culturing the colonies, calli, suspension cell lines or embryos to support their growth. Monoclonal colonies, calli, suspension cell lines or embryos can be obtained by dissolving encapsulated colonies, calli, suspension cell lines or embryos or by picking colonies, calli or suspension cell lines co-cultured in microwells. The colonies, calli, suspension cell lines or embryos are seeded at a sufficiently low density to allow their culture in isolation from one another.

[0234]

[0175] In some embodiments, monoclonal colonies, calli, suspension cell lines or embryos are cultured until reaching a size of from 1 to 10mm, which may involve several steps of scaling-up. The method may thus comprise one scaling-up cycle or more than one scaling-up cycle, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more scaling up cycle, in particular from 1 to 10 scaling-up cycles.

[0235]

[0176] In some embodiments, the scaling-up process may involve the combination of different scaling- up steps, such as step (i) followed by step (ii), and then step (iii), with various combinations being possible and understood by one skilled in the art. For instance, cells which were encapsulated at the previous step can be seeded in microwells at the next step. Similarly, cells which were seeded in microwells at the previous step can be encapsulated at the next step. Where the method comprises mixing encapsulation and microwell seeding, the scaling-up process may comprise encapsulating monoclonal colonies, calli, suspension cell lines and / or embryos in a capsule volume increased in comparison to the microwell volume in which the monoclonal colonies, calli and / or suspension cell lines were seeded at the previous cycle, e.g. by a factor of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the previous cycle. More specifically, the increase in volume may range from 20% to 1000%, preferably from 25% to 500%, and more preferably from 50% to 250% at each scaling-up cycle.

[0236]

[0177] In some embodiments, the scaling-up process may comprise seeding monoclonal colonies, calli, suspension cell lines and / or embryos in a microwell volume increased in comparison to the capsule volume in the monoclonal colonies, calli, suspension cell lines and / or embryos were seeded at the previous cycle were encapsulated at the previous cycle, e.g. by a factor of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% compared to the previous cycle. More specifically, the increase in volume may range from 20% to 1000%, preferably from 25% to 500%, and more preferably from 50% to 250% at each scaling-up cycle.

[0237]

[0178] Scaled-up cultures can then be cultivated to yield cell colonies, microcalli and / or embryos.

[0238] Furthermore, scaled-up cultures may also be grown to establish monoclonal cell lines, which canbe further cultured or utilized for downstream applications. As shown in the Figures (Figure 9), the method of the invention provides a sustained growth of the calli area, with an area increase by more than 80% in 7 weeks, i.e. more than 11% per week. The growth of the calli width has increased by more than 60% in 7 weeks, i.e. more than 8.5% per week. The growth of the calli length has increased by more than 35% in 7 weeks, i.e. more than 5% per week.

[0239] Automation

[0240]

[0179] One of the aspects of this method is the degree of automation achievable in key steps, significantly enhancing precision, efficiency, and reproducibility. Automated cell picking devices, such as micromanipulators equipped with image recognition software, ensure accurate isolation of single protoplasts, microspores or cell colonies, minimizing human error. Similarly, automated cell dispensing systems enable precise distribution of individual protoplasts or microspores into gel capsules or microwells, ensuring uniformity across experimental replicates. Encapsulation processes can also be automated using microfluidics platforms that control droplet size and composition, enabling high-throughput production of gel capsules. These systems not only standardize encapsulation parameters such as size and volume but also allow real-time monitoring of encapsulated cells to ensure viability and monoclonality. Automation extends to downstream processes such as culture monitoring and colony picking, where automated systems equipped with fluorescence-based imaging or other detection technologies can identify and isolate colonies with desired characteristics. By integrating automation at these critical stages, the method enhances throughput, and ensures greater consistency in generating monoclonal plant cell lines.

[0241]

[0180] In some embodiments, the step of isolating the plant protoplasts or microspores from each other is performed automatically, in particular with a cell picking device such as a microfluidics device, a flow cytometry device and / or a micromanipulation device. In some embodiments, the step of encapsulating each isolated protoplast or microspores in a gel capsule is performed automatically, in particular with a microfluidics device. In some embodiments, the step of seeding each isolated protoplast or microspores in a microwell of an array of microwells is performed automatically, in particular with a cell picking device such as a microfluidics device, a flow cytometry device and / or a micromanipulation device.

[0242] Successive Mutagenesis

[0243]

[0181] In certain embodiments, the method of the invention comprises performing multiple rounds of successive mutagenesis to introduce a series of genetic modifications within the target cells. Said rounds of successive mutagenesis may be carried out by random mutagenesis and / or targeted mutagenesis. For instance, multiple rounds of random mutagenesis may be carried out, or multiple rounds of targeted mutagenesis, or multiples rounds mixing random mutagenesis and targeted mutagenesis.

[0244]

[0182] This approach streamlines the traditional process of chain mutagenesis or successive mutagenesis, enabling the direct application and screening of mutagenic treatments to monoclonalcell lines without the need for extensive plant regeneration, explant preparation, or the induction of embryogenic cell lines. By eliminating these labor-intensive intermediate steps, the invention substantially reduces the time and effort typically required in conventional mutagenesis processes.

[0245]

[0183] The removal of these intermediary stages not only accelerates the breeding cycles but also provides a more efficient strategy for trait development, particularly in plant breeding programs. Mutagenesis and selection of monoclonal cell lines enable precise modifications, fostering the generation of desirable traits at a significantly faster pace than traditional methods. Furthermore, by utilizing monoclonal cell lines, the method inherently avoids the risks of chimerism that are often encountered in other cell culture systems. This eliminates the necessity for time-consuming and labor-intensive steps, thus streamlining the entire mutagenesis process and ensuring more reliable and uniform results.

[0246]

[0184] This advancement presents a transformative approach for plant breeding, as it accelerates genetic improvements while maintaining the integrity and purity of the desired traits across generations. By facilitating successive rounds of mutagenesis and selection within a single cell line, the method of the invention not only enhances efficiency but also provides a more controlled and systematic pathway for trait enhancement, significantly advancing the potential for precision breeding.

[0247]

[0185] In some embodiments, the method of the invention may comprise repeating steps (a) to (f) of the method using protoplasts derived from cells of the monoclonal calli and / or monoclonal suspension cell lines obtained according to the invention.

[0248]

[0186] In some embodiments, said repetition of steps (a) to (f) may be carried out to perform multiallelic editing of recessive mutations, wherein one allele of a mutation is edited at each cycle of step (a) to (f)

[0249]

[0187] In some embodiments, the method of the invention comprises one or more rounds of:

[0250] (i) obtaining plant cells from a monoclonal plant callus and / or monoclonal plant suspension cell line obtained at step (e) or (f),

[0251] (ii) optionally genetically modifying said plant cells;

[0252] (iii) obtaining protoplasts from said cells; and

[0253] (iv) performing steps (a) to (f) on the protoplasts, to produce monoclonal calli and / or monoclonal suspension cell lines from said protoplasts;

[0254]

[0188] In some embodiments, the method of the invention further comprises one or more rounds of: (i) obtaining plant cells from a monoclonal plant callus and / or monoclonal plant suspension cell line obtained at step (e) or (f);

[0255] (ii) obtaining protoplasts from said cells;

[0256] (iii) optionally genetically modifying said protoplasts; and

[0257] (iv) performing steps (a) to (f) on the protoplasts to produce monoclonal calli and / or monoclonal suspension cell lines from said protoplasts.

[0189] Several rounds of genetic modifications can be performed in this way, for instance to perform multiallelic editing of recessive mutations, or to perform editing of several genes. Multiallelic editing can be employed to ensure the correction or enhancement of recessive mutations, which may require alterations at multiple allelic sites to achieve the desired phenotypic outcome. Additionally, the method allows for the concurrent editing of multiple genes, facilitating the development of plants with multiple, potentially synergistic, traits in a single generation.

[0258] Nursing cells

[0259]

[0190] The growth and regeneration of cultured cells, whether encapsulated or not, can be significantly enhanced through the inclusion of nursing cells. In particular, some embodiments of the present method involve co-culturing gel capsules containing target cells with nursing cells. Nursing cells, as defined herein, are actively proliferating cells that promote the growth, survival, and regeneration of isolated protoplasts, isolated microspores or other target cells. Nursing cells may provide essential factors, nutrients, or structural support to the culture cells. These cells often create a microenvironment that mimics natural conditions, making it suitable for the target cells to thrive. Nursing cells can be employed to stimulate protoplast division or microspore division, facilitating the formation of calli even at low protoplast or microspores culture densities.

[0260]

[0191] Nursing cells may be derived from the same species as the target cells, for example from the same species with a different genotype. The nursing cells may also be from a different plant species, depending on the requirements of the culture.

[0261]

[0192] The nursing cells utilized in the disclosed methods may possess either a wild-type (WT) genotype or a genetically modified genotype. In some embodiments, the nursing cells are engineered to express a detectable marker, facilitating their subsequent identification and analysis. Additionally, these cells may be modified to incorporate transgenes that promote the overexpression of growth factors, cytokines, or cell-signaling molecules, thereby enhancing the growth and regenerative potential of the target cells. Non-limiting examples of suitable nursing cells include embryogenic cells or suspension cell lines, from the same species or closely related species as well as from unrelated species. Suitable nursing cells include callus-inducing cells, and cells modified to secrete specific proteins or hormones conducive to target cell proliferation and regeneration, for instance highly proliferating suspension cell lines genetically-modified to express a reporter gene. Nursing cells must also be adapted to a culture in the same medium as the protoplasts or microspores. Nursing cells are preferably adapted to the higher osmolarity of the regeneration medium, to promote successful regeneration of protoplasts or microspores.

[0262]

[0193] Examples of regeneration media which can be used with nursing cells are described in the Examples. In some embodiments, the regeneration medium comprises 0.1-1 M glucose. The medium can be further supplemented with hormones and appropriate growth factors.

[0263]

[0194] While nursing cells are commonly employed in traditional cell culture systems, one of the challenges is the potential for cross-contamination or mixing with the target cells. The methods described herein address this issue by physically segregating protoplasts from nursing cells, e.g. using gel capsules, thereby preserving the integrity of the monoclonal target cell cultures. In someembodiments, if the nursing cells are encapsulated, they are similarly isolated within their respective capsules, thus ensuring effective segregation and preventing undesired interactions.

[0264]

[0195] The following co-culture configurations are exemplified for promoting regeneration and growth of protoplasts or other isolated cells, such as microspores (Fig. 11):

[0265] Liquid Co-Culture with Nursing Cells in a Separate Compartment: In this configuration, the nursing cells are placed in a physically distinct compartment, such as a specialized plate insert, while sharing the same liquid medium with the target cells. This arrangement enables metabolic and biochemical exchanges between the nursing cells and target cells through the shared medium, while maintaining physical separation, which minimizes the risk of direct mixing.

[0266] Co-Culture of Nursing Cells in the Liquid Medium: In this embodiment, nursing cells are introduced directly into the liquid culture medium, either as free-floating cells or encapsulated within a matrix or gel. This setup facilitates close proximity between the nursing cells and target cells, allowing for a flexible degree of interaction while offering dynamic control over the co-culture environment.

[0267] Co-Culture on an Immobilized Nursing Layer: In this embodiment, nursing cells are immobilized onto a solid substrate or support material, forming a stable, fixed layer. The target cells are cultured near this immobilized layer, allowing for nutrient and growth factor exchange while preventing direct physical contact between the target cells and nursing cells. This configuration helps ensure that the growth and regeneration of the target cells are enhanced without compromising their separation.

[0268]

[0196] For protoplasts cultured in microwells, nursing cells may also be included in selected wells to promote the growth of neighboring protoplasts or microspores, thereby separating the nursing cells and neighboring protoplasts or microspores in different wells. The ratio of protoplasts or microspores to nursing cells can be adjusted and optimized for different species or genotypes, enabling the fine- tuning of the culture conditions to enhance cell growth in a species-specific or genotype-specific manner. In some embodiments, the nursing cells are seeded in at least 1%, in particular at least 5%, more particularly at least 10% of the microwells. In some instances, the nursing cells are seeded in from 1 to 50%, in, particular from 1 to 25%, more particularly from 1 to 10% of the microwells.

[0269]

[0197] While nursing cells are particularly well suited for promoting the growth and regeneration of isolated protoplasts, alternative co-culture strategies are preferred in certain embodiments involving microspores. In particular, for microspore culture, co-culture with plant organs has been found to provide a highly efficient system for stimulating microspore viability, division, and subsequent embryogenic development.

[0270]

[0198] In some embodiments, isolated microspores are co-cultured with plant organs, e.g. excised plant organs, such as ovaries, ovules, anthers, or other reproductive tissues, which act as biological nurse tissues. These organs are capable of releasing diffusible factors, including hormones, signaling molecules, metabolites, and developmental cues, that promote microspore embryogenesis. Such organ-based co-culture systems are known to more closely mimic the natural physiological environment encountered by microspores during development, thereby enhancing regeneration efficiency compared to co-culture with dispersed nursing cells.

[0199] The plant organs used for microspore co-culture may be derived from the same species as the microspores, for example from donor plants of the same genotype or a different genotype, or from a closely related species. In some embodiments, the plant organs are obtained from unrelated species, provided that the organs are compatible with the culture medium and capable of supporting microspore development. The organs may be freshly isolated prior to co-culture or maintained in vitro before use.

[0271]

[0200] In certain embodiments, the microspores are physically separated from the plant organs while sharing the same culture medium. For example, microspores may be encapsulated in gel capsules or cultured in microwells, while the plant organs are placed in the same culture vessel or in a separate compartment that allows exchange of soluble factors without direct physical contact. This physical separation facilitates monoclonal microspore culture while retaining the beneficial effects of organ-derived signals.

[0272]

[0201] In other embodiments, the plant organs are immobilized on a support or confined within a defined region of the culture system, such as a mesh, insert, or dedicated chamber, while microspores are cultured in an adjacent region. This configuration allows controlled exposure of microspores to organ-derived factors and minimizes the risk of mechanical interference or overgrowth.

[0273]

[0202] The ratio of microspores to plant organs, as well as the spatial arrangement between them, can be adjusted and optimized depending on the plant species, genotype, and developmental stage of the microspores. In some embodiments, a single organ or a fragment thereof is sufficient to support the development of microspores in multiple neighboring culture units, such as microwells or capsules.

[0274] High-throughput screening

[0275]

[0203] In certain embodiments, the method of the invention further comprises a screening step for assessing the genotype and / or phenotype of the monoclonal calli, suspension cultures and / or embryos. Such screening is preferably carried out utilizing one or more advanced analytical techniques selected from a group consisting of genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, among others. These analytical methods allow for a comprehensive evaluation of the genetic and phenotypic characteristics of the monoclonal calli, thereby facilitating the identification of desired traits or genetic modifications.

[0276]

[0204] In particular, the screening may involve the analysis of the genome of the cells within the monoclonal calli, suspension cell cultures and / or embryos to identify and characterize genetic modifications. Such genomic analysis is especially useful when the cells have undergone genetic modifications. In some embodiments, the genomic analysis is performed using the TILLING (Targeting Induced Local Lesions IN Genomes) technique, as detailed herein.

[0205] TILLING is a well-established reverse genetics technique that utilizes chemical mutagenesis methods to induce mutations in plant genomes, followed by high-throughput screening for the identification of mutations in specific genes. In this process, mutagenic agents such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MNU), or other mutagenic treatments (e.g., UV radiation) are employed to generate a pool of mutagenized individuals. Subsequently, high- throughput PCR-based screening of the pooled DNA samples is conducted to identify missense or nonsense mutations in the target genes of interest. This screening is facilitated by the use of S1 nucleases on the amplicons produced by PCR, such as CEL1 or ENDO1 , which cleave heteroduplexes formed between mutant and wild-type DNA, followed by detection of the cleavage products via electrophoresis, for example, utilizing a LI-COR gel analyzer system (Henikoff et al., Plant Physiology, 2004, 135: 630-636).

[0277]

[0206] Amplicons may also be sequenced, for instance by New Generation Sequencing (NGS) to identify SNP by comparison with the reference sequence of the wild-type. TILLING by NGS sequencing is a highly efficient and cost-effective screening technique, enabling the analysis of thousands of mutants in a single sequencing run (e.g., up to 5000 mutants). This method has been successfully applied to a wide range of plant species. Briefly, Tilling by NGS sequencing can be performed by DNA extraction followed by mixing the extracted DNA in equimolar proportions to produce a pool of gDNA, e.g. ranging from 16 to 96 individuals. This step may use a 3D pooling model, wherein samples are pooled along 3 axes: in rows (Z pools), in columns (Y pools) and by equivalent coordinates of different plates (X pools). This pool of DNA is used as a template for amplification by PCR of the genes of interest (amplicons), which are then prepared (indexing, purification) to produce an amplicon library. Several indexed libraries are then pooled together (in equimolar proportions) for the sequencing step. Examples of NGS technologies useful for TILLING by NGS include Illumina technology, and in particular the MiSeq device, which can sequence amplicons of between 250 and 600 base pairs. Subsequent bioinformatics analysis is used to identify mutations by comparing the sequences with the reference sequence, and to find the corresponding cell line.

[0278]

[0207] Furthermore, the TILLING method has been extended to EcoTILLING, wherein mutations present in natural populations are also identified (Till et al., Nat Protoc, 2006, 1 : 2465-77; Comai et al., Plant J, 2004, 37: 778-86). Traditionally, TILLING is performed on regenerated plantlets, where leaf samples are taken for DNA extraction, followed by sequencing for mutation detection.

[0279]

[0208] In contrast to traditional TILLING, which is typically conducted on regenerated plantlets, the present invention introduces the concept of Cell TILLING. In this novel approach, the genetic and phenotypic analysis is performed on cultured monoclonal cell colonies, calli, microcalli, suspension cell lines or embryos ratherthan on fully regenerated plantlets or plants. In this method, mutagenesis is first applied to plant cells, e.g. microspores, or protoplasts, which are subsequently cultured in microvolumes to form monoclonal calli. The TILLING process, including the screening for induced genetic modifications, is carried out on the monoclonal cell colonies, calli, suspension cell lines or embryos before they undergo any further regeneration into plantlets or mature plants.

[0209] The Cell TILLING technique described herein provides significant advantages over traditional methods. It enables high-throughput analysis of mutants with specific genotypic alterations, such as single nucleotide polymorphisms (SNPs) at targeted loci. By identifying desirable genotypic or phenotypic traits at an earlier stage in the process, Cell TILLING reduces the time and resources required for plant regeneration. This eliminates the need for regenerating non-targeted plants, thereby accelerating the selection process. For instance, in coffee plant breeding, traditional TILLING requires approximately 8 months to generate plantlets at the 3-leaf stage, which are then subjected to DNA extraction and analysis. In contrast, using Cell TILLING, genetic analysis can be conducted within approximately 2 months.

[0280]

[0210] In some embodiments, said screening comprises sampling cell material from the monoclonal calli and / or monoclonal suspension cell lines and performing one or more cell analysis on the sampled cell material. In some embodiments, the sampled cell material comprises DNA and / or RNA. In some embodiments, the sampled cell material comprises one or more cells.

[0281]

[0211] In some embodiments, the cell analysis comprises collecting a DNA and / or RNA sample from the monoclonal calli, monoclonal suspension cell line, and / or embryo and analyzing said DNA and / or RNA sample. In some embodiments, the cell analysis comprises collecting one or more cells from the monoclonal calli, monoclonal suspension cell line and / or embryo and analyzing said cell.

[0282]

[0212] Preferably, the cell analysis is performed in a non-destructive manner. Specifically, the analysis is carried out without impairing the continued growth and development of the monoclonal calli, monoclonal suspension cell line and / or embryo. This non-destructive approach ensures that the monoclonal calli, monoclonal suspension cell line and / or embryo, can continue their growth, regeneration, and differentiation into mature plant tissues, even after the analysis.

[0283]

[0213] Performing a non-destructive analysis with techniques may require sampling of cell material, i.e. DNA or other cellular molecules at a sufficiently advanced stage of growth of the calli and / or suspension cell lines, such that the sampling does not impair their further growth and regeneration. In this way, the analysis is not the endpoint of the process, and will be a tool for further breeding of plant lines.

[0284]

[0214] Accordingly, in one aspect, the non-destructive cell sampling is performed at a stage where the monoclonal calli, suspension cell lines and embryos have reached a growth stage allowing the collection of an adequate amount of cellular material for analysis. In some embodiments, cell material sampling is carried out when the calli, suspension cell lines and embryos have a size of 500 pm or more, in particular when the calli, suspension cell lines or embryos have a size of 1 mm or more. In particular, cell material sampling is carried out when the calli have reached a size of 500 pm to 10mm, more particularly 1mm to 10mm. In some embodiments, cell material sampling is carried out when the calli have reached a size of 500 pm to 5mm, more particularly 1 mm to 5mm. In some embodiments, cell material sampling is carried out when the calli have reached a size of 500 pm to 2.5mm, more particularly 1mm to 2.5mm. This sampled material can include one or more cells of the monoclonal calli, cell lines or embryos or any other relevant cell material. In some embodiments, cell material sampling is carried out on cell debris collected within the culture medium. The remainder, i.e. non-sampled part of the monoclonal callus, suspension cell lines or embryos isleft intact, allowing for continued growth and regeneration into a microcallus and / or ultimately into a mature plant. In some embodiments, cell-sampling is carried out with a cell picking device, as described in the present disclosure.

[0285]

[0215] Cell sampling can also be carried out on monoclonal suspension cell lines obtained according to the methods of the invention, in particular established cell lines. Non-destructive sampling on monoclonal suspension cell lines allows to preserve the monoclonal suspension cell line which can be further maintained for further screening assays, further genetic modifications and / or for plant regeneration.

[0286]

[0216] The method of the invention facilitates early-stage identification of valuable genetic traits while preserving the potential for further plant development.

[0287]

[0217] Accordingly, in some embodiments, the method of the invention comprises:

[0288] obtaining monoclonal calli, suspension cell lines and / or embryos, each monoclonal callus, suspension cell line or embryo being derived from a single protoplast or a single microspore, as described in the invention;

[0289] screening the monoclonal calli, suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, suspension cell lines and / or embryos such that the monoclonal calli, suspension cell lines and / or embryos remain able to undergo further cycles of cell divisions and / or to regenerate into a plant after the screening; further culturing the monoclonal calli, suspension cell lines and / or embryos after the analysis, to obtain calli, suspension cell lines and / or embryos able to regenerate into plants; and optionally regenerating said calli, suspension cell line and / or embryo into a plant.

[0290]

[0218] In some embodiments, the screening is carried out on monoclonal suspension cell lines, in particular established monoclonal cell lines, each monoclonal suspension cell line being derived from a single protoplast, as described in the invention;

[0291]

[0219] The method of the invention may thus comprise:

[0292] (i) obtaining monoclonal suspension cell lines, in particular established suspension monoclonal cell lines, each monoclonal cell line being derived from a single protoplast, as described in the invention;

[0293] (ii) screening the monoclonal suspension cell lines, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal suspension cell lines such that the monoclonal cell lines remain able to further undergo cell divisions and / or to regenerate into a plant after the screening;

[0294] (iii) further culturing the monoclonal suspension cell line to obtain calli and / or suspension cell line able to regenerate into plants; and

[0295] (iv) optionally regenerating said calli and / or suspension cell line into a plant.

[0220] In some embodiments, said screening step comprises a step of selecting one or more monoclonal calli, monoclonal suspension cell lines and / or embryos. Said selection is preferably carried out on the basis of the cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses.

[0296]

[0221] The selected monoclonal calli, monoclonal suspension cell lines and / or embryos can then be further used various purposes, e.g. : further culture to obtain further division of the cells; production of established cell line(s) for further storage, regeneration into a plant and / or induction of genetic modification(s).

[0297] Regenerating a plant

[0298]

[0222] In some embodiments, the method of the invention further comprises regenerating plants from the monoclonal calli, monoclonal suspension cell lines and / or embryos in particular the selected monoclonal calli, monoclonal suspension cell lines and / or embryos, further to the screening step.

[0299]

[0223] Said step of regenerating plants may comprise transferring the calli, suspension cell lines and / or embryos to an agar-solidified regeneration medium adapted to facilitate the development of the callus, suspension cell line or embryo into differentiated organ as a shoot, or a somatic embryo; and transferring the shoot or the somatic embryo to an agar-solidified plant medium adapted to support rooting, or germination of the somatic embryo, respectively. Appropriate media and conditions for regenerating a plant line are described, e.g. in Smith et al., 2013 and Germana et al.

[0300] 2016.

[0301]

[0224] In some embodiments, the gel capsule encapsulating the monoclonal calli, monoclonal suspension cell line or embryo is dissolved before the calli or suspension cell lines are transferred to an agar-solidified regeneration medium

[0302]

[0225] In some embodiments , monoclonal calli, suspension cell lines, or embryos, e.g. embryos obtained therefrom are preserved or stored for an indefinite period of time. This property allows to store the cell material, e.g. in libraries of cell lines or calli, for subsequent analysis as well as germination and plant production.

[0303]

[0226] In some embodiments, the method of the invention further comprises crossing a regenerated plant with another plant, e.g. a plant of the same genotype or of another genotype and collecting seeds from said cross. By such crossing step, the genetic modification(s) identified during the screening step, and the associated trait(s), can be transferred to another genetic background.

[0304] Plant species

[0305]

[0227] The methods described herein are adaptable to a wide range of plant species.

[0306]

[0228] Plants that may be useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophilatricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi / losa, Pagopyrum spp., Feijoa sellowl ana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, banana, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canadensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbel lata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefUcillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees. Alternatively algae and other non-Viridiplantae can be used for the methods of some embodiments of the invention.

[0307]

[0229] According to a specific embodiment, the plant is a woody plant species e.g., Actinidia chinensis (Actinidiaceae), Manihotesculenta (Euphorbiaceae), Firiodendron tulipifera (Magnoliaceae), Populus (Salicaceae), Santalum album (Santalaceae), Ulrnus (Ulmaceae) and different species of the Rosaceae (Malus, Prunus, Pyrus ) and the Rutaceae (Citrus, Microcitrus), Gymnospermae e.g., Picea glauca and Pinus taeda, forest trees (e.g., Betulaceae, Fagaceae, Gymnospermae and tropical tree species), fruit trees, shrubs or herbs, e.g., (banana, cocoa, coconut, coffee, date, grape and tea) and oil palm.

[0230] According to a specific embodiment, the plant is of a tropical crop e.g., coffee, macadamia, banana, pineapple, taro, papaya, mango, barley, beans, cassava, chickpea, cocoa (chocolate), cowpea, maize (corn), millet, rice, sorghum, sugarcane, sweet potato, tobacco, taro, tea, yam.

[0308]

[0231] According to a specific embodiment, the plant is asexually propagated.

[0309]

[0232] According to a specific embodiment, the plant is banana. According to a specific embodiment, the plant is tomato (Solatium lycopersicum). According to a specific embodiment, the plant is tobacco (e.g. Nicotiana tabacum, Nicotiana benthamania). According to a specific embodiment, the plant is coffee (in particular Coffea arabica, or Coffea canephora). According to a specific embodiment, the plant is grapevine (Vitis vinifera).

[0310]

[0233] According to some aspects, the invention relates to monoclonal calli, monoclonal suspension cell lines and / or embryos produced according to a method of the invention. According to some aspects, the invention relates to a population of monoclonal calli, monoclonal suspension cell lines and / or embryos produced according to a method of the invention, wherein the population comprises at least 2, 5, 10, 50, 100, 500, 1000, 5000, 10000, 50000 or 100000 monoclonal calli, monoclonal suspension cell lines and / or embryos. According to some aspects, the invention relates to a plant, a part thereof and / or one or more cells thereof, produced according to a method of the invention. According to some aspects, the invention relates to a population of plant, part thereof and / or cells thereof, produced according to a method of the invention, wherein the population comprises at least 2, 5, 10, 50, 100, 500, 1000, 5000, 10000 plants.

[0311] Kits

[0312]

[0234] In another aspect, the present disclosure provides kits for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, in particular haploid and / or dihaploid embryos. In certain embodiments, the kit comprises one or more components for implementing the methods described herein. The kit may include, for example, a cell isolating device, e.g. a cell picking device and / or cell dispensing device as well as containers containing any media required for use with these devices. The kit may also comprise microwell arrays, as described herein. The microwell arrays are for instance Nanowell® plates or Sievewell® plates. The kit may also comprise one or more containers comprising culture media and / or components of the culture media used in the method of the invention. The kit may also comprise one or more containers comprising reagents for genetically modifying plant cells, e.g. microspores and / or protoplast. The kit may also comprise instructions for use of any of the devices, containers and compounds, and / or for use of any of the steps of the method of the invention. Any combination of the aforementioned is encompassed within the invention.

[0313]

[0235] The general principles of the invention are disclosed in more detail herein, particularly by way of examples, such as those shown in the drawings and described in detail. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. The invention is amenable to various modifications and alternative forms, specifics of which are shown for several embodiments. The intention is to cover all modifications, equivalents, and alternatives falling within the principles and scope of the invention.

[0236] All references cited herein, including patents, patent applications, and other publications, are hereby incorporated by reference in their entirety for all purposes

[0314] EXAMPLES

[0315] Example 1: Encapsulation and Regeneration of Single Protoplasts into Monoclonal Microcalli

[0237] Single protoplasts were encapsulated and co-cultured within capsules, enabling their development into microcalli. The encapsulated protoplasts were cultured until their growth reached the spatial limits of the capsules.

[0316] Encapsulation Techniques

[0317]

[0238] Three distinct encapsulation methods were employed to ensure the monoclonality and effective regeneration of protoplasts.

[0318] a. Microfluidics-Based Encapsulation

[0319]

[0239] Microfluidics enables the precise dispensing of individual cells into alginate capsules. Singlecell detection is performed as cells pass through a sensor on a microfluidic chip. Detection methods include impedance, brightfield, or fluorescence imaging.

[0320]

[0240] Using this technique, alginate capsules of approximately 300 pm in diameter were produced, each containing a single BY-2 protoplast (Fig. 2A). The capsules were bulk-cultured in a shared vessel, allowing protoplasts to divide and form cell microcalli within their capsules (Fig. 3). Culture was carried out in standard BY-2 protoplast regeneration medium (MS / 2, 3% Sucrose, 0.4M mannitol, 1mg / L NAA, 0.3mg / L Kinetin, pH 5.7). The media was refreshed weekly in the liquid cultures and cultivated in darkness at 26 + / - 1 °C at 60% humidity. Alternatively, capsules were cultured on solid media (solidified Linsmaier & Skoog Basal media, 3% sucrose, 200 mg / L KH2PO4, and 0.2mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D)) and were as well cultured in the darkness at 26 + / - 1 °C at 60% humidity.

[0321]

[0241] The capsule volume was calculated to be approximately 0.014 pL, corresponding to a density of approximately 35,000 capsules per mL

[0322]

[0242] Once calli expanded to the capsule's limits (Fig. 4), each capsule was transferred individually:

[0323] Into liquid multiplication medium in 96-well plates (Fig. 4A).

[0324] Onto semi-solid multiplication medium in Petri dishes (Fig. 4B).

[0325]

[0243] This ensured the establishment of monoclonal cell lines. Capsules exceeding their spatial limits demonstrated overgrowth on semi-solid media (Fig. 4C). Alternatively, cells reaching capsule limits could be:

[0326] Dispensed (manually or automatically) into individual wells of a 96-well plate or semi-solid media for line establishment (Figs. 5B and 5C).Encapsulated into larger capsules, facilitating continued colony growth and individual culture establishment (Fig. 5D and Fig. 10).

[0327] b. Flow Cytometry-Based Encapsulation

[0328]

[0244] Flow cytometry cell sorting separated protoplasts based on size, granularity, and viability.

[0329] Protoplasts were dispensed into sodium alginate solutions, producing capsules approximately 200 pm in diameter (Fig. 2B). Efficient encapsulation required sheath fluid optimized with calcium chloride (CaCI2) and osmotic agents. Osmotic conditions can be adapted to the species, genotype, and tissue source. This ensured cell survival and regeneration of cell walls.

[0330] c. Micromanipulation Robot coupled with Microscopy

[0331]

[0245] In this method, protoplasts were seeded into microwell arrays and assessed for monoclonality using an inverted microscope. A micromanipulation robot was then employed to pick single protoplasts from microwells. The capillary, prefilled with sodium alginate solution, transferred the selected protoplast (Fig. 7A-C).

[0332]

[0246] The protoplast and alginate were dispensed onto a calcium agar surface, forming a capsule of approximately 100 nL in total volume (Fig. 2C). Once solidified, capsules were collected and bulk co-cultured until the microcalli reached sufficient size for transfer. Capsules were then placed individually onto semi-solid medium or into wells containing liquid multiplication medium, ensuring monoclonality as described above.

[0333] Example 2: Co-Culture in Nanoscopic Wells

[0334]

[0247] In this Example, protoplasts were seeded into microwell arrays and co-cultured at high densities.

[0335]

[0248] Robusta (Coffea canephora) protoplasts were seeded in Nanowell S200 plates (size of each microwell of 200pm diameter), at a density of 100,000 protoplasts / mL. Seeding was performed by dispensing protoplast culture in the microwells array, and was followed by monoclonality detection using a cell recognition software. The protoplasts were seeded in regeneration medium comprising (per L) Gamborg B5 salts (3.16396 g / L), myo-inositol (0.1 g / L), thiamine HCI (0.01 g / L), pyridoxine HCI (0.001 g / L), nicotinic acid (0.001 g / L), calcium pantothenate (0.0001 g / L), folic acid (0.0002 g / L), riboflavin (0.0001 g / L), citric acid (0.01 g / L), fructose (0.125 g / L), ribose (0.125 g / L), xylose (0.125 g / L), mannose (0.125 g / L), cellobiose (0.125 g / L), polyvinylpyrrolidone (PVP10, 0.2 g / L), sucrose (20 g / L), L-cysteine (0.01 g / L), casein (0.25 g / L), MES (0.5 g / L), 2,4-dichlorophenoxyacetic acid (2,4-D, 0.0005 g / L), naphthaleneacetic acid (NAA, 0.0005 g / L), benzylaminopurine (BAP, 0.001 g / L), coconut water (100 mL / L), and D-glucose monohydrate (99.1 g / L) and cultured in appropriate conditions. The medium was changed weekly with 0.1 M decrease of glucose concentration. The conditions of the culture were darkness, 26°C, and 60% humidity.

[0249] Calli were obtained after 3 weeks of culture in microwells (Fig. 6B). When the cells reach the limits of the microwell, calli can be collected individually by cell picking using a CellCellector micromanipulation robot, to scale-up the culture (Fig. 6C). The collected cells can be re-seeded in larger microwells, encapsulated in alginate gel capsules, or transferred to a semi-solid or solid medium, as described in Example 3.

[0336] Example 3: Culture scaling-up

[0337]

[0250] Cell colonies were transferred using an automated colony picker (Fig. 5). Two scaling options were employed for colony expansion:

[0338] 1. Transfer to Semi-Solid or Liquid Medium: Colonies were moved to semi-solid medium (Fig. 5C) or liquid medium in multi-well plates (Fig. 5B) for further regeneration (Fig. 9).

[0339] 2. Encapsulation and Bulk Co-Culture: Colonies were encapsulated and co-cultured in bulk, allowing spatial separation and ensuring monoclonality even in shared culture vessels (Fig. 10). Upon reaching sufficient size, the microcalli were distributed into individual wells of 96- or 384-well plates for continued growth.

[0340] Example 4: Isolation of Single Microspores and Culture in Microwell Arrays to Generate Monoclonal Embryos and Calli

[0341]

[0251] Donor Arabica (Coffea arabica) plants were conditioned in a greenhouse or growth chamber .

[0342] Floral buds were harvested a few days before flowering when microspores were at the mid- to late- uninucleate stage. To enhance embryogenic competence, excised inflorescences or whole plants were pretreated at 4-10°C for 24-72 hours prior to isolation.

[0343]

[0252] Floral material was sterilized and anthers were dissected from buds and released into a cold isolation buffer containing mannitol at 0.3-0.5 M to set osmolarity between approximately 450 and 650 mOsm / kg. Anthers were gently triturated using a sterile pestle or tapped to liberate microspores without disrupting the plasma membrane. The crude suspension was clarified by sequential filtration through sterile meshes of 100 pm and 40-70 pm to remove tapetai debris and intact tissues. Microspores were collected.

[0344]

[0253] Microspores were seeded in Nanowell S200 plates, at a density of 100,000 microspores / mL.

[0345] Seeding was performed by dispensing microspore suspension in the microwells array , and was followed by monoclonality detection using a cell recognition software.

[0346]

[0254] Following seeding, culture was initiated. A modified Gamborg’s B-5 formulation was used Gamborg’s B-5 with basal medium salts, 0.5x; glutamine, 250 mg 1-1 ; glycine, 2mg 1-1 ; HCI cysteine, 150 mg 1-1 ; myo-inositol, 100 mg 1-1 ; coconut water, 16% (v / v); MES, 600 mg 1-1 ; polyvinilpyrrolidone (PVP-10), 200 mg 1-1 ; maltose, 20% (w / v); naphthaleneacetic acid (NAA), 300 mg 1-1 ; 2,4-dichlorophenoxyacetic acid (2,4-D), 100 mg 1-1 ; kinetin, 100 mg 1-1 ; pH adjusted to 5.8 . Induction of androgenesis was achieved by transient heat shock at 32-35°C for 24-48 hours followed by maintenance at 24-26°C in darkness for early divisions. Osmolarity was maintained at450-650 mOsm / kg during induction and then reduced stepwise to 280-350 mOsm / kg as embryos enlarged from globular to heart and torpedo stages by diluting the carbohydrate osmoticum. Media exchanges were performed weekly by gentle supernatant replacement to avoid shear and preserve well integrity.

[0347]

[0255] Under these conditions, single microspores underwent symmetrical divisions and progressed to embryogenic structures within their individual wells, preserving monoclonality throughout development. Embryos were monitored by inverted microscopy to document stage transitions. When embryo size approached the spatial limit of the well, individual structures were harvested by micromanipulation. Harvested embryos were reseeded into larger microwells to continue development under reduced osmolarity, transferred to semi-solid medium for maturation and conversion, or reencapsulated in larger gel capsules to maintain physical isolation while permitting growth in shared liquid coculture. Monoclonality was confirmed at each handling step by imaging before and after picking and by ensuring that only single embryos or compact structures were transferred per operation. The workflow demonstrated that single microspores, isolated, dispensed, and picked with precise single-cell controls, reliably produced monoclonal embryos and calli suitable for scaling, non-destructive omics screening, and downstream plant regeneration.

[0348] Bibliographical references

[0349] Allis, C. D., Jenuwein, T., & Reinberg, D. (2007). Epigenetics. Cold Spring Harbor Laboratory Press. Bauer, S., et al. (2018). Frontiers in Pharmacology, 12 Jul. 2018.

[0350] https: / / doi.Org / 10.3389 / fphar.2018.00749

[0351] Comai, L., Young, K„ Till, B. J., et al. (2004). Plant J, 37, 778-786.

[0352] Cui, X., et al. (2018). Interdisciplinary Sciences: Computational Life Sciences, 10(2), 455-465.

[0353] Dettmer, K., Aronov, P. A., & Hammock, B. D. (2007). Metabolomics: A Powerful Tool in Systems Biology. Springer.

[0354] Fowke, L. C., & Constabel, F. (Eds.). (1985). Plant Protoplasts. CRC Press.

[0355] Germana, M. A., & Lambardi, M. (Eds.). (2016). In Vitro Embryogenesis in Higher Plants. Humana Press.

[0356] Han, X., & Gross, R. W. (2014). Lipidomics: Comprehensive Mass Spectrometry of Lipids. Wiley. Henikoff, S., et al. (2004). Plant Physiology, 135, 630-636.

[0357] International Patent Application WO2017 / 061806A1.

[0358] Lesk, A. M. (2017). Introduction to Genomics. Oxford University Press.

[0359] Mohr, T., et al. (2016). FEBS Journal, https: / / doi.org / 10.1111 / febs.13777

[0360] Osakabe, Y., et al. (2018). Nature Protocols, 13(12), 2844-2863.

[0361] Rezende, J. C. D. et al. (2012). Acta Scientiarum. Agronomy, 34, 93-98.

[0362] Robinson, S. H., et al. (2002). Phenomics: The Next Challenge. Nature.

[0363] Smith, R. H. (2013). Plant Tissue Culture: Techniques and Experiments. Academic Press.Soda, N., et al. (2018). Plant Physiology and Biochemistry, 131, 2-11.

[0364] Streit, W. R., & Daniel, R. (2010). Metagenomics: Theory, Methods, and Applications. Caister Academic Press.

[0365] Till, B. J., et al. (2006). Nature Protocols, 1 , 2465-2477.

[0366] Toda, E., et al. (2019). Nature Plants, 5(4), 363-368.

[0367] Twyman, R. M., et al. (2004). Proteomics: Principles, Techniques, and Applications. Garland Science. Varki, A., et al. (2015). Essentials of Glycobiology. Cold Spring Harbor Laboratory Press.

[0368] Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-Seq: A Revolutionary Tool for Transcriptomics. Nature Reviews Genetics, 10, 57-63.

Claims

CLAIMS

1. A method for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, wherein the method comprises:a) providing a plurality of plant protoplasts or microspores, wherein the plant protoplasts or microspores are optionally genetically modified;b) optionally genetically modifying the plant protoplasts or microspores;c) isolating the plant protoplasts or microspores from each other;d) encapsulating each isolated protoplast or microspore in a gel capsule to obtain a plurality of gel capsules, each gel capsule containing a single plant protoplast or microspore and having a volume of from 0.25 nL to 1 pL;e) co-culturing the encapsulated protoplasts or microspores in a culture vessel containing culture medium, at a density of from 100 capsules / mL to 500,000 capsules / mL, whereby the protoplasts or microspores within the gel capsules undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; andf) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screening is carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

2. A method for producing monoclonal plant calli, monoclonal plant suspension cell lines and / or embryos, wherein the method comprises:a) providing a plurality of plant protoplasts or microspores, wherein the plant protoplasts or microspores are optionally genetically modified;b) optionally genetically modifying the plant protoplasts or microspores;c) isolating the plant protoplasts or microspores from each other;d) seeding each isolated protoplast and / or microspore in a microwell of an array of microwells, at a density of from 1000 cells / mL to 200,000 cells / mL, wherein the array of microwells is arranged to physically separate the isolated protoplasts or microspores from each other and enable liquid exchanges between the microwells;e) co-culturing the seeded protoplasts or microspores, whereby said protoplasts or microspores undergo cell divisions to form monoclonal calli, monoclonal suspension cell lines and / or embryos; and f) screening the monoclonal calli, monoclonal suspension cell lines and / or embryos, wherein said screening includes one or more cell analysis selected from genomics, proteomics, transcriptomics, metabolomics, epigenomics, lipidomics, glycomics, and phenomics analyses, wherein said screeningis carried out without destructing the monoclonal calli, monoclonal suspension cell lines and / or embryos such that the monoclonal calli, monoclonal suspension cell lines and / or embryos remain able to undergo further cell divisions and / or to regenerate into a plant after the screening.

3. The method of claim 1 , wherein the encapsulated protoplasts or microspores are cocultured at a density of from 10,000 capsules / mL to 500,000 capsules / mL.

4. The method of claim 1 or 3, wherein each gel capsule has a diameter of from 200 pm to 500 pm.

5. The method of any one of claims 1 , 3 or 4, wherein the culture medium for capsule coculture is osmotically controlled at from 600 mOsm / kg to 800 mOsm / kg during early protoplast culture.

6. The method of claim 2, wherein the microwells have a size from 10 pm to 1 mm.

7. The method of any one of the preceding claims, wherein said plurality of protoplasts or microspores is a genetically diverse population of protoplasts or microspores, in particular wherein the genome of each protoplast or microspore differs from the genome of other protoplasts or microspore by at least one genetic modification such as a mutation, preferably as the result of random mutagenesis.

8. The method of any one of the preceding claims, wherein step (a) comprises: (a1) providing a plurality of plant cells, preferably embryogenic cells; and (a2) disaggregating the cell wall of the plant cells to obtain plant protoplasts, in particular wherein step (a1) comprises genetically modifying said plant cells.

9. The method of any one of the preceding claims, wherein said screening comprises sampling cell material from the monoclonal calli, monoclonal suspension cell lines and / or embryo and performing one or more cell analysis on the sampled cell material, preferably wherein the sampled cell material comprises DNA and / or RNA.

10. The method of any one of the preceding claims, wherein step (c) of isolating the plant protoplasts or microspores from each other is performed using a microfluidics device, a flow cytometry device, and / or a micromanipulation device, preferably wherein step (c) of isolating the plant protoplasts or microspores from each other comprises a step of cell monoclonality detection, in particular using microscopy imaging.

11. The method of claim 1, wherein step (d) of encapsulating each isolated protoplast or microspore in a gel capsule comprises depositing a droplet containing culture medium, at least one gelling agent, and a single cell on a solid, semi-solid, or liquid surface, and solidifying the droplet into a gel capsule, preferably wherein the gelling agent is a hydrogel-forming agent selected from alginate, agarose, pectin, synthetic biomimetic hydrogels, or their combination.

12. The method of any one of the preceding claims, wherein nursing cells are co-cultured in a physically separated compartment sharing the same liquid medium with the protoplasts or microspores.

13. The method of any one of the preceding claims, wherein plant organs are co-cultured in a physically separated compartment sharing the same liquid medium with the protoplasts or microspores.

14. The method of claim 1, wherein the gel capsules are co-cultured with nursing cells, preferably wherein:the nursing cells are separated from the gel capsules by a semi-permeable barrier permeable to the culture medium and providing a physical barrier between the cultured gel capsules encapsulating the isolated protoplasts or microspores and the nursing cells;the nursing cells are encapsulated in gel capsules and cultured in the same culture vessel as the gel capsules encapsulating the isolated protoplasts or microspores; orthe nursing cells are embedded in a gel layer in contact with the culture medium comprising the gel capsules encapsulating the isolated protoplasts or microspores.

15. The method of claim 2, wherein the seeded protoplasts or microspores are co-cultured with nursing cells, preferably wherein the nursing cells are seeded in selected wells to promote the growth of neighboring protoplasts or microspores, or wherein the nursing cells are co-cultured in a distinct compartment, sharing the same liquid medium with the cultured protoplasts or microspores, or wherein the nursing cells are co-cultured on an solid substrate, forming a stable, fixed layer.

16. The method of any one of the preceding claims, wherein step (e) comprises one or more scaling-up steps wherein each scaling-up step comprises:(i) encapsulating or reencapsulating, in a gel capsule, the monoclonal cell colonies, calli, suspension cell lines or embryos obtained from protoplast division or microspore division wherein each gel capsule comprises a single monoclonal cell colony, callus, suspension cell line or embryo, and co-culturing the gel capsules in a culture vessel containing culture medium, whereby the monoclonal cell colonies, calli, suspension cell lines or embryos are allowed to further divide(ii) seeding or reseeding the monoclonal cell colonies, calli, suspension cell lines or embryos obtained from protoplast division or microspore division, into microwells of an array of microwells, wherein the array is arranged to enable liquid exchanges between the microwells, and co-culturing the monoclonal cell colonies, calli, suspension cell lines or embryos in the array of microwells, whereby the monoclonal cell colonies, calli, suspension cell lines or embryos are allowed to further divide; or (iii) seeding the encapsulated monoclonal cell colonies, calli, suspension cell lines or embryos on a semi-solid or solid medium, and culturing to allow the monoclonal cell colonies, calli, suspension cell lines or embryos to further divide.

17. The method of claim 16, wherein at each scaling-up step, the monoclonal calli or embryos are seeded in a microwell of increased volume with respect to the previous step, wherein the volume of the microwells is increased by a factor of from 20% to 1000% at each cycle, preferably from 50% to 500% and / or wherein at each scaling-up step, the monoclonal calli are encapsulated in a gel capsule having an increased volume with respect to the previous step, wherein the volume of the gel capsules is increased by a factor of from 20% to 1000%, preferably from 50% to 500% at each cycle.

18. The method of any one of the preceding claims, wherein the step of genetically modifying comprises one or more of random mutagenesis, genome editing, epigenetic modification, RNA-directed DNA methylation, genomic imprinting, paramutation induction, RNA interference (RNAi), microRNA (miRNA), and piRNA transformation.

19. The method of any one of the preceding claims, further comprising one or more rounds of:(i) obtaining plant cells from a monoclonal plant callus, monoclonal plant suspension cell line and / or embryos obtained at step (e) or (f),(ii) optionally genetically modifying said plant cells;(iii) obtaining protoplasts from said cells; and(iv) performing steps (a) to (f) on the protoplasts, to produce monoclonal calli, monoclonal suspension cell lines and / or embryos from said protoplasts;and / or one or more rounds of:(i) obtaining plant cells from a monoclonal plant callus, monoclonal plant suspension cell line and / or embryo obtained at step (e) or (f);(ii) obtaining protoplasts from said cells;(iii) optionally genetically modifying said protoplasts; and(iv) performing steps (a) to (f) on the protoplasts to produce monoclonal calli, monoclonal suspension cell lines and / or embryos from said protoplasts,preferably wherein said one or more rounds are carried out to perform mu Itiallelic editing of one or more recessive mutations.

20. The method of any of the preceding claims, further comprising regenerating plants from the monoclonal calli, monoclonal suspension cell lines and / or embryos, optionally further comprising crossing a regenerated plant with another plant and collecting seeds from said cross.

21. The method of any one of the preceding claims, wherein the protoplasts or microspores are selected from Coffea Arabica, Coffea canephora, Vitis vinifera, Solanum lycopersicum, Nicotiana tabacum and Nicotiana benthamiana, protoplasts or microspores.

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