WO2016105185A1

WO2016105185A1 - Plant callus populations

Info

Publication number: WO2016105185A1
Application number: PCT/NL2015/050855
Authority: WO
Inventors: Franck George Paul LHUISSIER; Bernarda Gerharda Johanna Fierens-Onstenk; Robert Sevenier; Paul Bundock; Anita KETELAARS-BONNÉ
Original assignee: Keygene N.V.
Priority date: 2014-12-22
Filing date: 2015-12-11
Publication date: 2016-06-30

Abstract

The current disclosure relates to the field of plant biotechnology. More specifically, methods are provided to select a (sub)population of plant cells, in particular plant calli that are enriched for plant cells, in particular plant calli that comprise a genome-editing event and/or a genetic-modification event. The selected (sub) population has a high percentage of plant calli that comprise the desired genome-editing event and/or a genetic-modification event, for example relative to the total population from which the subpopulation is selected. Also provided are methods for obtaining at least one plant cell that comprises the genome-editing event and/or a genetic-modification event, the method involving the selection of the (sub)population, screening and regeneration of the at least one plant cell, for example into a plant shoot, plant tissues or plant.

Description

Plant callus populations Field of the Invention

[001] The current disclosure relates to the field of plant biotechnology. More specifically, methods are provided to select a (sub)population of plant cells, in particular plant calli that are enriched for plant cells, in particular plant calli that comprise a genome-editing event and/or a genetic-modification event. The selected (sub) population has a high percentage of plant calli that comprise the desired genome-editing event and/or a genetic-modification event, for example relative to the total population from which the subpopulation is selected. Also provided are methods for obtaining at least one plant cell that comprises the genome-editing event and/or a genetic-modification event, the method involving the selection of the (sub)population, screening and regeneration of the at least one plant cell, for example into a plant shoot, plant tissues or plant.

Background

[002] Plant breeding has been very successful over the last 100 years in domesticating a wide range of plant species and improving significantly the yield and quality of plant products. This has been achieved through the selection of allelic variation providing improvements in both plant cultivation and consumption traits.

[003] Typically, a cross is performed between two parents to produce the hybrid F1 progeny which are then selfed to create homozygous alleles and the resulting F2 progeny are screened for the phenotype of interest. Screening can be performed based solely on the phenotype, or as is more often the case, by using molecular markers tightly linked to the allele(s) of interest.

[004] The molecular assisted breeding (MAB) approach has the advantage that F2 populations can be screened at a very early growth stage so that large populations do not need to be maintained and also the progeny can be screened for many markers linked to many alleles so that the desired alleles can be identified and the undesired alleles can be eliminated.

[005] Conventional plant breeding has led to a reduction in the total amount of allelic variation, either through conscious selection against traits that were viewed to be deleterious or through unconscious loss due to the lack of selection pressure on variation that was considered neutral at the time. [006] The total world population is expected to increase significantly in the coming decades and there is a realization that the yield of many crops must also increase while at the same time utilizing the same area of arable land and using fewer resources such as water and fertilizer. Due to the reduced allelic variation in cultivated species, novel alleles that can confer such traits as novel biotic resistance (e.g. insects, bacteria, fungi and viruses) and adaptation to abiotic stresses (e.g. water limitation, heat, salt concentration) are unlikely to be present in the cultivated germplasm and must therefore either be integrated from wild germplasm or introduced in the cultivated germplasm through mutagenesis.

[007] Mutagenesis breeding is done by exposure of seeds or plant parts to a mutagenic treatment. This can done using chemicals (e.g ethyl-methane sulfonate, EMS) or radiation but both methods rely upon the DNA repair mechanisms of the plant repairing the DNA damage incorrectly, resulting in a change in the DNA sequence. The treated plants can be screened for phenotypic changes (termed a forward screen) or for sequence changes in a specific gene of interest (a reverse screen) using various genotyping techniques. Many useful mutations have been identified in this way but it is important to note that these mutagenesis techniques cause DNA changes throughout the genome and that once the interesting plants have been identified they must be extensively backcrossed to eliminate the additional deleterious mutations.

[008] It is for this reason that mutagenesis breeding is less applicable to crops in which crossing is not regularly performed and that are vegetatively propagated.

[009] Several agents are able to destroy the chemical bonds in the sugar-phosphate backbone of DNA. If this damage occurs in close proximity on both DNA strands then the chromosome becomes broken leading to two free DNA ends which must be joined back together to restore chromosome integrity. Such DNA damage is termed a double strand break (DSB) and is one of the most severe forms of DNA damage that the cell must deal with.

[010] The cell has evolved two different mechanisms that are able to repair DNA DSB's, non-homologous end joining (NHEJ) and homologous recombination (HR). The proteins involved in the HR pathway perform DSB repair by a copying mechanism using identical sequence information located elsewhere in the genome as a template, for instance on the homologous parental chromosome, to rejoin the DNA ends produced at the DSB. This is a precise DNA repair mechanism leading to the restoration of the original DNA sequence at the position of the DSB.

[01 1] The HR pathway is thought to be less active in plants cells where the majority of DSB's seem to be repaired by the NHEJ pathway. The NHEJ pathway includes many DNA damage sensing and processing enzymes that are able to recognize DNA DSB's and religate the DNA ends without the need for a template elsewhere in the genome. The NHEJ mechanism can be mutagenic because processing of the free DNA ends can lead to the loss of a few nucleotides which results in a small deletion or insertion (usually from 1-10 bps), an indel, upon religation.

[012] DNA DSB's can also be produced by proteins such as restriction endonucleases which recognize small palindromic DNA sequences which are present thousands of time in a complex genome. However, DNA binding proteins such as restriction endonucleases can be rationally designed to recognize and bind to a longer DNA sequence (>18 bps) that is likely to be present only once in a complex genome. By combining the DNA binding protein with a nuclease a DNA DSB can be targeted to a specific genomic position such as a gene of interest.

[013] As DNA DSBs are often mis-repaired by the cellular machinery the DSB is likely to result in an indel in, for example, a gene of interest, and if this is in the coding region then it may result in a null mutation and the complete loss of gene function. Producing DNA DSBs at defined genomic locations is termed targeted mutagenesis or site-directed/site- specific mutagenesis. The ability to create site-specific DNA DSB's is a powerful technology that has emerged in the past few years and currently there are four different protein based systems to achieve this, (1) meganucleases (Silva et al. 2011 Curr Gene Ther 1 1 (1): 1 1-27), (2) zinc finger nucleases (ZFN) (Urnov et al. 2010 Nature Rev. Genet. 1 1 : 636-646), (3) TAL effector nucleases (TALENs) (Joung et al. 2013 Nature Rev. Mol. Cell Biol. 14: 49-55) and (4) CRISPR's (Hsu et al. 2014 Cell 157(6) :1262-1278 or Doudna et al (2014) Science 346: DOI: 10.1 126/science.1258096).

[014] Another example of site-directed mutagenesis is oligonucleotide-directed mutagenesis using an oligonucleotide such as for example described in WO 2012/074385. /pet

[015] While there are currently several competing technologies in plants for introducing a genome-editing event and/or a genetic-modification event, one bottleneck limiting their application is how the agents applied in these technologies can be introduced into plant cells to create the desired change and subsequently how these cells can be identified and be regenerated via tissue culture into mutant plants.

[016] The current inventors have found new methods that may advantageously be applied in genome-editing events and/or a genetic-modification events in plant cells, using for example genome-editing events tools like meganucleases, zinc finger nucleases, TAL effector nucleases (TALENs), CRISPR's or oligonucleotide-directed mutagenesis using an oligonucleotide. Description

Definitions [017] In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references are incorporated herein in their entirety by reference. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

[018] As used herein, the terms "a", "an", and "the", in their singular forms, refer to plural referents unless the context clearly dictates otherwise. For example, a method for isolating "a" DNA molecule, includes isolating a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).

[019] As used herein, and unless specifically stated or obvious from context, the term "about" refers to being within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. The term "about" can be understood as within 50%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.5%, 0.1 %, 0.05%, or 0.01 % of the stated value.

[020] As used herein, the term "and/or" refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[021] As used herein, the term "at least" refers to a situation wherein a particular value is the same as said particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, ... , etc. At least part is to be understood as a certain fraction of the total, for example 0.1 , 1 , 5, 10, 20, 50,

80, 90 or 100% of the total, or any number in between.

[022] The term "callus" (plural "calli") is well-known to the skilled person in plant biology and refers to a mass of unorganized (and in general totipotent) cells derived from plant tissue They may be derived from any type of cell, for example mesophyll cells or meristematic cells. Within the context of the current disclosure a callus may be as small as consisting of an aggregate of two cells.

[023] As used herein, the terms "comprising" and "to comprise", and their conjugations, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb "to consist of. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". It is further understood that, when referring to "sequences" herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.

[024] As used herein, the term "genotyping" refers to the process of determining genetic variations among individuals in a species. The genotype of an organism is the inherited instructions it carries within its genetic code. For genotyping, many methods exist to determine genotype among individuals. The chosen method generally depends on the throughput needs, which is a function of both the number of individuals being genotyped and the number of genotypes being tested for each individual. The chosen method also depends on the amount of sample material available from each individual or sample. Well- established methods are, for example, PCR-based: digital PCR, quantitative PCR, High Resolution Melting curve analysis (HRM), KASPar assay; Sequence-based: SNPselect, Sanger sequencing etc ( see e.g. Hayward et al. 2015 Molecular Marker Applications in Plants in Plant Genotyping: Methods and Protocols (eds J. Batley), Methods I Mol.Biol. vol 1245).

[025] The term "clonal" as used herein in the context of plant calli is to be understood as indicating that all cells present in a certain callus are derived from one and the same protoplast; i.e. it originates from a single cell. In contrast, a chimeric callus consists of at least two types of cells; i.e. originates from more than one cells.

[026] The term "genome editing" refers to a process in which genomic DNA of a living cell is altered by inserting, replacing, or removing one or more bases using mutagenic molecules. The number of bases inserted, replaced or removed may for instance be 1 , 2, 5, 10, 15, 20, 25, 30, 50 or more. The mutagenic molecules used can for instance be gene repair oligonucleotides or endonucleases artificially engineered to create specific double-strand breaks (DSBs) or single-strand nicks at predefined locations in the genome. The breaks or nicks are repaired by the cell's own repair mechanisms using natural processes of mismatch repair, base excision repair, homologous recombination or non- homologous end-joining (NHEJ). There are currently four families of engineered nucleases being used: Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas9 system, and engineered homing endonucleases or meganucleases. Genome editing can also be achieved by oligonucleotide-directed mutagenesis using oligonucleotides (ODM), also referred to as targeted nucleotide exchange (TIME). Genome editing includes site-specific mutagenesis, site-specific gene addition and gene targeting. Genome editing excludes the stable introduction of exogenous DNA. [027] The term "genetic modification" refers to a process in which exogenous DNA fragments from outside the cell or organism are integrated in the genome of living cells. This integration may be stable. The exogenous DNA fragments may for instance be larger than 20, 50, 100, 500, 1000, 5000 or 10000 base pairs. Genetic modification includes recombinant nucleic acid (DNA or RNA) techniques. It includes transfer of genes or parts of genes across species boundaries and the creation of transgenic cells and organisms.

Brief description of the Drawings

[028] FIGURE 1 : Sequence of the Cas9 ORF optimized for Solanaceae codon usage

[029] FIGURE 2: sgRNA cassettes for mutagenesis of the tomato CENH3 gene; The A. thaliana U6 promoter sequences are underlined. The sgRNA sequences are shown in italics with the 20 bps region corresponding to the tomato CENH3 gene shown in bold.

[030] FIGURE 3: INDEL mutations found at the CENH3 locus of calli that had been sorted using the COPAS.

Detailed Description

[031] The invention is as described in the accompanying claims and claims.

[032] In a first aspect the current disclosure relates to a method for obtaining a (sub)population of plant calli that is enriched for plant calli that comprise a genome-editing event and/or a genetic-modification event. The population obtained by the method according to the invention has a high percentage of plant calli that comprise the genome- editing event and/or genetic-modification event, for example relative to the total or initial population from which the subpopulation is selected.

[033] In other words, with the method as disclosed herein, from a first population of plant calli a second population of plant calli can be selected, wherein said selected population is high(er) in the percentage of calli that can be positively identified as comprising a genome-editing event and/or a genetic-modification event.

[034] In addition, with the method disclosed herein a population of plant calli is obtained wherein the percentage of plant calli that are clonal is increased relative to the original population, i.e. the plant callus is derived from one original plant protoplast. In particular the method disclosed herein allows obtaining a population of plant calli that has a high(er) percentage of calli that can be positively identified or comprising a genome-editing event and/or a genetic-modification event (i.e. that comprise a desired change in the genome) and that, at the same time, are clonal.

[035] The method disclosed herein comprises providing a multitude of plant protoplasts, subjecting said multitude of plant protoplasts to a treatment to introduce a genome-editing event and/or a genetic-modification event, allowing the multitude of plant protoplasts to grow into plant calli, preferably until 0.5 - 5% (by number) of the plant calli is larger than 700 micrometer, and selecting at least part of the plant calli that have a size between 80 micrometer and 500 micrometer. Within the context of the current invention 0.5 - 5% is indicated by the number of calli of the total population of calli.

[036] The skilled person understand how to determine that 0.5 - 5% (by the number; ie of total calli) is larger than 700 micrometer, for example using common techniques available to the skilled person, including, for example FACS analysis and the like. It was surprisingly found that when 0.5 - 5% of the calli population reaches a size of 700 micrometer (or more), the population of calli that has a size of between 80 micrometer and 500 micrometer is a population on interest within the context of the current disclosure, and as explained below. The invention is thus based on the realization that first the population of plant calli need to be grown until a certain amount of the total calli has reached a size of larger than 700 micrometer, and secondly next, a second subpopulation, i.e those calli that have a size between 80 micrometer and 500 micrometer, is the population of interest and from which calli may be selected, as detailed herein.

[037] The method disclosed herein is applicable to any kind of plant from which plant protoplasts may be obtained, and, preferably, that can be regenerated into (whole) (fertile) plants. Examples include tomato, tobacco, canola, lettuce, and so on The skilled person is well aware of methods in the art to obtain such plant protoplasts and how to culture such protoplasts. In a preferred embodiment, the protoplasts are tomato protoplasts.

[038] In a step of the method, a multitude of plant protoplasts are provided. In a preferred embodiment the multitude of plant protoplasts are living plant protoplasts having retained the capability to develop into plant calli when subjected to the proper culture conditions. As will be understood by the skilled person, the exact number of protoplasts provided is not critical and depends on, for example, the type of plant from which the protoplasts are derived and the further set-up of the experiment/steps of the method. A non-binding example of the amount of cells is 5 million protoplasts (5x10⁶).

[039] In a further step, the multitude of plant protoplasts is subjected to a treatment to introduce, in the DNA present in the plant protoplasts, a genome-editing event and/or a genetic-modification event. The treatment that is applied to the multitude of plant protoplasts is thus a treatment that allows for introducing a genome-editing event and/or a genetic-modification event in the genome of the plant protoplast. In other words, the treatment to which the plant protoplasts are subjected is not a treatment that randomly introduces mutations throughout the genome of the plant protoplast, such as, for example, treatments using ethyl methane sulfonate (EMS) or radiation.

[040] It is to be noted that the treatment not necessarily has to introduce a modification at only one specific site, but may also be designed as such that it introduces a modification at more than one specific site in the genome. The treatment thus introduces a genome- editing event and/or a genetic-modification event at a predetermined site in the genome of the plant protoplast.

[041] At the same time it is to be noted that although the specific site where the genome- editing event and/or a genetic-modification event should be introduced may be predetermined, the type of modification to be introduced may not be. Examples of possible mutations include introducing point mutations, deletions of nucleotide(s), addition/insertion of nucleotides(s) and/or substitution of nucleotide(s). The modification may lead to, for example, disruption of a gene, reduction of expression of a gene, or modification of the properties of for example a promoter and/or an expressed gene product.

[042] The skilled person is well aware of methods, technology and conditions available in the art for treating the multitude of protoplasts so as to introduce genome-editing event and/or a genetic-modification event. At the same time the skilled person will understand that normally only part of the multitude of protoplasts are successfully treated, i.e. only in part of the treated multitude of protoplasts a genome-editing event and/or a genetic- modification event will be introduced.

[043] For example, there are several different methods for introducing the reagents, such as the CRISPR Cas9 and sgRNA, used to establish the genome-editing event, into plant cells. These include, for example, electroporation or bombardment of single cells or tissues and polyethylene glycol- (PEG) mediated transfection of protoplasts.

[044] Each method involves the introduction of the reagents into individual plant cells where the double-strand break (DSB) induction and subsequent repair takes place. These cells are then induced to divide to form a mass of undifferentiated plant cells, a callus, that is then able to regenerate into a complete plant.

[045] The bombardment transformation methods rely on the co-introduction of transgene(s) carrying the CRISPR reagents together with a selection marker that provides resistance to a compound such as an antibiotic, hormone or herbicide. A selective compound is then included in the plant cell growth medium and suppresses the growth of plant cells that have not been stably transformed, thus enriching for the cells carrying stably integrated copy(s) of the transgene. However, such transformation methods can be time consuming.

[046] PEG mediated transformation of protoplasts is a good alternative to stable transformation methods. Plant protoplasts can be isolated in large numbers from leaves and then incubated with PEG and DNA (or RNA) constructs expressing the CRISPR reagents. DNA based constructs such as plasmids are transported to the protoplast nucleus where the Cas9 mRNA and the sgRNA is produced, but in this case the DNA constructs do not integrate into the plant genome. As each protoplast takes up multiple DNA plasmids a very high (transient) expression level of the CRISPR reagents can be obtained which increases the efficiency of the mutagenesis process.

[047] As the DNA constructs are unable to replicate in the plant cells they are degraded and the CRISPR reagents disappear from the cells. It is also useful to determine the efficiency of the mutagenesis experiment by investigating how many of the protoplasts have indels at the target sequence after treatment. This is done by sampling a portion of the treated cells, isolating their genomic DNA and then using this to amplify the target sequence using PCR. While the majority of PCR products will not show any sequence changes, a subset of these will carry indels and the ratio between these two populations can be used to calculate a mutagenesis efficiency (%).

[048] It is important to realize that although PEG transformation of protoplasts is very efficient (up to 95% in tomato), modification/mutations are not induced in all of the treated protoplasts. Therefore, the protoplast population consists of mutated and wild type cells (heterogeneous population).

[049] Therefore, in a preferred embodiment, the treatment to introduce a genome-editing event in the multitude of protoplasts comprises PEG-mediated transformation of the multitude of plant protoplasts, for example, PEG-medicated transformation for introducing the agents (such as, for example, for the Cas/RNA CRISPR nuclease system or the Zinc Finger Nuclease) used to introduce the genome-editing event. For example, in this embodiment, the Cas protein and sgRNA itself are transfected into the protoplasts.

[050] As will be understood by the skilled person, in an embodiment and as an alternative, there might be provided a multitude of protoplasts that has already been treated to introduce a genome-editing event and/or a genetic-modification event, and thus comprising a heterologous population of protoplast in which part is mutated and part remains wild type.

[051] Under such conditions the step of (again) subjecting such protoplasts to a treatment to introduced a genome-editing event and/or a genetic-modification event may be omitted (although not necessarily).

[052] In a further step the treated multitude of plant protoplasts is allowed to grow into plant calli, i.e. into aggregates of two or more cells. A callus may thus consist of only two cell but may also consist of more cells, for example 10, 20, 100, 1000 or more cells.

[053] The skilled person is well aware of suitable growth conditions to allow the multitude of plant protoplast to develop into plant calli. For example, the efficiency of plant regeneration from single protoplasts depends on the species and genotype, but there are general approaches that apply to all protoplast systems. For example, the first protoplast divisions require a high protoplast density, for example 10,000 per ml_ to 500,000 per ml_ and therefore to achieve this the protoplasts are often embedded in a solid matrix such as alginate or agarose that maintains a high protoplast density and promotes (micro)calli formation.

[054] If the protoplast density is too low then the number of dividing protoplasts (also known as the plating efficiency) will also be low. Once a protoplast has undergone a number of divisions and formed a (micro)callus (for example, about 2, 4, 5, 6, 7 or more weeks after transfection) then the high density is no longer necessary and individual (micro)calli can be isolated and are mature enough to continue cell divisions to produce mature calli.

[055] In a further step of the method disclosed herein, and after the treated plant protoplasts are allowed to grow into plant calli until 0.5 - 5% of the plant calli are larger than 700 micrometer, the population that is enriched for plant calli that comprise a genome-editing event and/or a genetic-modification event is obtained by selecting (at least part of) those plant calli that developed and that are have a size between 80 micrometer and 500 micrometer (for example, as measured along the major axis of the callus; i.e the longest axis in the callus).

[056] As can be witnessed from the examples it was surprisingly found that when such calli were screened, a high frequency of calli that comprised the genome-editing event and/or a genetic-modification event was found in comparison to a selection method including calli of larger sizes (see example).

[057] In other words, by discarding plant calli that do not have a size between 80 micrometer and 500 micrometer (after all the plant calli were grown until 0.5 - 5% of the plant calli is larger than 700 micrometer), a population of plant calli is obtained with a higher frequency of calli comprising the genome-editing event and/or a genetic- modification event. Indeed, whereas in the plant calli that belong to the selected population frequencies up to 7% were obtained, no genome-editing event and/or a genetic-modification events were detected in the calli that do not have a size between 80 micrometer and 500 micrometer (see examples); i.e. the bigger calli.

[058] This is in strong contrast to the practice for selecting plant calli used in the prior art. In the prior art and in practice, the skilled person selects plant calli using tweezers. This however requires a minimal size of the plant callus of about 1 mm or more. In other words, the methods practiced by the skilled person select plant calli that do not have a size between 80 micrometer and 500 micrometer (after all the plant calli were grown until 0.5 - 5% of the plant calli is larger than 700 micrometer) but are larger than about 1 mm, which at the same time are plant calli that show a low(er) frequency of calli comprising the genome-editing event and/or a genetic-modification event.

[059] In addition it was found that calli that have a size between 80 micrometer and 500 micrometer (after all the plant calli were grown until 0.5 - 5% of the plant calli is larger than 700 micrometer) are with high frequency clonal plant calli, i.e. not chimeric calli, whereas, in contrast in the population of calli that are larger, for example larger than 1 mm, with higher frequency calli can be found that a not clonal but chimeric in nature.

[060] These chimeric calli can negatively influence the outcome of the genome-editing event and/or a genetic-modification event experiment as the presence of chimeric calli will complicate the subsequent genotyping and will give different results depending upon which part of the callus is sampled. Also regeneration of chimeric callus will be problematic because the regenerating shoots may be derived from wild type rather than mutant tissue. These problems can lead to (indel) mutations being identified in the callus but not being present in the shoots derived from that callus, or in missing mutations in the (chimeric) calli due to sampling a wild type cell in the chimeric calli.

[061] In a preferred embodiment of the method disclosed herein the plant calli that are selected in step d) are plant calli that can grow in the absence of other plant calli.

[062] As explained herein, the first protoplast divisions require a high protoplast density, for example from 10,000 per ml_ to 500,000 per ml_ If the protoplast density is too low then the number of dividing protoplasts (also known as the plating efficiency) will also be low. Once a protoplast has undergone a number of divisions and formed a (micro)callus (for example, about 5 weeks after transfection) then the high density is no longer necessary and individual (micro)calli can be isolated and are mature enough to continue cell divisions to produce mature calli in the absence of other plant calli. As will be understood by the skilled person, the exact number of division required depends on the plant species and genotype used in the experiment and/or the medium conditions used to allow the plant protoplast to grow into plant calli

[063] Therefore, in one embodiment, the plant protoplasts are allowed to grow into plant calli for a period to allow (the quickest growing calli) to divide for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 times. In an alternative embodiment, the protoplast are allowed to grow into plant calli for a period of at least 7 days, 14 days, 21 days, 30 days, 40 days, 60 days or more.

[064] In fact the skilled person will understand how, for a given plant protoplast, they can, for example, empirically determine without undue burden at what size a plant calli is able to develop further in the absence of other plant calli. The skilled person may, for example determine once, and using identical culture conditions, for different (groups of) sizes of plant calli if these are able to develop further into more mature plant calli (i.e. are able to continue to divide) in the absence of other plant calli. Therefore the skilled person will have no problem in selecting, from the plant calli that have a size between 80 micrometer and 500 micrometer (after all the plant calli were grown until 0.5 - 5% of the plant calli is larger than 700 micrometer) those calli that are able to grow in the absence of other plant calli. [065] Additionally or alternatively the skilled person may (pre)select for calli by staining with a dye that becomes more or less fluorescent depending on the fitness of the biological material. Fluorescein di-acetate (FDA) is such dye. FDA by itself is non-fluorescent but upon contact with metabolically active material will become fluorescent. The level of fluorescence is directly correlated to the esterase activity of the biological material which is indicative of degree of metabolic activity. Therefore, an actively dividing callus consisting of many cells will be brighter than a slow dividing callus consisting of a few, large vacuolated cells. Furthermore, debris and dead material will not stain and therefore FDA allows their separation from the valuable material. This fluorescence can be combined with for example size of the plant calli in order to only sort live material of the appropriate size.

[066] In another embodiment the plant calli that are selected in step d) are smaller than about 400 micrometer, or smaller than 300 micrometer, or smaller than 200 micrometer.

[067] In another embodiment the plant calli that are selected in step d) are larger than 100 micrometer but are smaller than about 500micrometer, or smaller than 400 micrometer, or smaller than 300 micrometer, or smaller than 200 micrometer.

[068] In general, the person skilled in the art will understand that, for example, and within the concept of the current invention selecting plant calli that are smaller than a certain size, for example 500 micrometer, indicates that a population is provided that is relatively enriched in plant calli that are smaller than said certain size. In one embodiment it does not exclude that part of the plant calli present are not smaller than said certain size, for example 500 micrometer. However, in a preferred embodiment the majority, preferably at least 90%, 95%, 99%, 100% of the selected cells have a size that is smaller than the said given size, for example 500 micrometer. This equally applies when selecting for plant calli that are larger than a certain size, for example larger than 80 micrometer, or when selecting cells that are within certain size ranges.

[069] The plant calli that are selected may have a size within the ranges as defined and disclosed herein. For example, the size of the selected plant calli may be within the range of 80 micrometer to 500 micrometer, for example about 100 micrometer and about 400.

[070] As explained above, the multitude of plant protoplasts provided in the method may either be protoplasts that have not yet been subjected to a treatment to introduce a genome-editing event and/or a genetic-modification event or may be protoplasts that have already been subjected to a treatment to introduce a genome-editing event and/or a genetic-modification event (and which population may be heterogeneous i.e. comprises modified and wild-type cells). In the latter situation the step of subjecting the multitude of plant protoplasts to a treatment to introduce a site genetic modification may be omitted, however it is also possible to subject said cells to a further treatment to introduce a (further) site specific genetic modification.

[071] In a further embodiment, and after selection of (at least) part of the plant calli that have a size between 80 micrometer and 500 micrometer (after all the plant calli were grown until 0.5 - 5% of the plant calli is larger than 700 micrometer), the method further comprises the step of

-screening at least part of the selected plant calli for the genome-editing event and/or a genetic-modification event and regeneration of at least one plant cell from a callus comprising the genome-editing event and/or a genetic-modification event; or

-regenerating of at least one plant cell from at least part of the selected plant calli and screening of at least part of the regenerated plant cells for the genome-editing event and/or a genetic-modification event.

[072] The skilled person is well aware of techniques and method to screen at least part of the selected plant calli for the presence of the genome-editing event and/or a genetic- modification event. Examples of such techniques are those provided in the examples, and/or include PCR followed by genotyping or any other genotyping assay Other examples include TaqMan®, KASPar™, SNPselect™, and AFLP®.

[073] Likewise, the person skilled in the art is well aware of available techniques and method for regenerating at least one plant cell from a calli. Such method are readily available in the prior art and depend on the plant species from which the plant calli is derived. Examples are described in Masani et al, 2013, Regeneration of viable oil plam plants from protoplasts by optimizing media components, growth regulators and cultivation procedures. Plant Sci. 210:1 18-127; Niedz et al, 1985, Plant Regeneration from leaf protoplasts of six tomato cultivars. Plant Sci. 39: 199-204; Shepard and Totten, 1975, Isolation and regeneration of tobacco mesophyll cell protoplasts under low osmotic conditions. Plant Physiol 55(4):689-694; Masuda et al, 1989, Callus formation and plant regeneration from rice protoplasts purified by density gradient centrifugation, Plant Sci. 62(2):237-246.

[074] In a preferred embodiment the regeneration of (or into) at least one plant cell comprises regeneration into an aggregate of at least two plant cells, a plant shoot, a plant embryo, a plant tissue or a whole (preferably fertile) plant.

[075] It is however important to note that after the selection of the plant calli that have a size between 80 micrometer and 500 micrometer (after all the plant calli were grown until 0.5 - 5% of the plant calli is larger than 700 micrometer), it may, before screening for the genome-editing event and/or a genetic-modification event followed by regeneration or before regeneration followed by screening, first be decided to further culture the selected calli that are selected individually and allow the selected calli to further grow and divide, even to sizes beyond 700 micrometer. In such circumstance the calli are allowed to continue to grow in the absence of other calli or plant cells. In other words, no plant cells or other calli are present in the compartment comprising the individual plant calli.

[076] As explained herein, in principal any treatment for genome-editing event and/or a genetic-modification event, e.g., that can be used to introduce a genome-editing event, may be used in the method disclosed herein. However in a preferred embodiment the modification is by

-introducing or expressing at least one (functional) site-specific nuclease in the multitude of plant protoplasts, preferably wherein said nuclease is selected from the group consisting of engineered nucleases, Cas/RNA CRISPR nuclease, zinc- finger nuclease, meganuclease and TAL-effector nuclease, preferably wherein said nuclease is Cas/RNA CRISPR nuclease, preferably wherein said Cas/RNA CRISPR nuclease comprises sgRNA and Cas9 protein and/or expression vectors therefore; and/or by

-oligonucleotide-directed mutagenesis using a oligonucleotide, preferably wherein the oligonucleotide is a single-stranded oligonucleotide.

[077] Engineered nucleases are nowadays an increasingly popular tool in techniques of targeted mutagenesis i.e. to introduce genome-editing events. When using nucleases these techniques are also referred to as genome editing with engineered nucleases (GEEN). With these techniques, DNA can be inserted, replaced, or removed from a genome using artificially engineered nucleases. The nucleases create specific double- stranded break (DSB) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break. The introduction of single-strand breaks is also possible. There are currently four families of engineered nucleases being used by the skilled person, and all four may be used within the context of the current invention: Zinc finger nucleases (ZFNs), Transcription Activator- Like Effector Nucleases (TALENs), the CRISPR/Cas system, and engineered meganuclease re-engineered homing endonucleases (meganucleases).

[078] The meganucleases, including such homing endonucleases as l-Scel, can be altered to confer an altered DNA sequence affinity and their activity has been reported in plants (Kirik el al. 2000,. EMBO J. 19: 5562-5566)

[079] Zinc finger nucleases consist of two domains, an array of zinc finger domains and a nuclease domain, usually derived from the restriction enzyme Fok\. Each zinc finger domain can be engineered to recognize a specific 3 bps triplet and by linking together a number of these, a longer DNA sequence can be specifically recognized. The Fok\ domain, for example, must dimerize before cutting DNA and so two ZFN proteins are designed to target sequences on the opposite DNA strands separated by a short spacer region of 5-6 bps. Binding of both of the ZFN proteins to their respective target sequence brings, for example, both Fok\ domains opposite each other on the DNA helix in the spacer region where a DNA DSB's is then induced. Many studies have shown that ZFN are effective in inducing small INDEL's at an endogenous target sequence in many different plant species (Curtin et al. 2012, Genome engineering of crops with designer nucleases, The Plant Genome, 5(2):42-50).

[080] TALEN's are site specific nucleases derived from TAL effectors which are produced by Xanthomonas species which cause a variety of different plant diseases. TAL effectors consist of a number of repeating protein domains, each of which is able to specifically recognize and bind to one of the 4 nucleotides. Different combinations of these DNA binding domains recognize a unique DNA sequence, often in plant gene promoters, which is targeted by the pathogen TAL effector which binds and influences plant gene expression to enhance pathogenicity. The domains specific for each nucleotide have been identified and arrays of these domains can be produced which have high binding affinity for any DNA sequence. These arrays are then fused to the nuclease domain of, for example, Fok\ to create a TALEN and, similar to ZFN, two TALEN proteins are used to induce a DNA DSB in a spacer region at the target sequence. Several papers have described the use of TALEN's to create mutations at the target sequence (Curtin et al. , Genome engineering of crops with designer nucleases, The Plant Genome, 5(2):42-50).

[081] The CRISPR technology is derived from bacteria where it is used as a system to defend against invading molecular pathogens such as plasmids and bacteriophages. Specific loci in the bacterial genome consist of arrays of short sequences derived from the genomes of molecular pathogens which are the result of previous infections. Small RNAs (crRNAs) are produced from these loci that interact with the tracrRNA and these RNA molecules together then target the Cas9 protein to the specific complementary sequence in the molecular pathogen's genome. The Cas9 protein has nuclease activity and is able to produce a specific DNA double strand break (DSB) at the target sequence in the pathogen genome which then becomes degraded. Expression of both the Cas9 protein, tracrRNA and crRNA (the components of the CRISPR system) targeting a genomic sequence in the cells of plants and animals creates targeted DSBs at the genomic target sequence that is often mis-repaired by the cellular DNA machinery, resulting in a small insertion or deletion (INDEL) (Feng et al. (2013) Cell Res. 1 : 4; Li et al. (2013) Nat. Biotech. 31 : 689-691 ; Nekrasov et al. (2013) Nat. Biotech. 31 : 691-693; Shan et al. (2013) Nat. Biotech. 31 : 686-688). In the context of the present invention, the terms Cas/RNA CRISPR nuclease, Cas9/sgRNA, Cas9/gRNA, Cas/RNA, CRISPR-Cas and CRISPR- Cas9 are used interchangeably, and all refer to the RNA-programmable CRISPR-Cas9 technology and its various embodiments (such as described for instance in Hsu et al. 2014 Cell 157(6) :1262-1278 or Doudna et al (2014) Science 346: DOI: 10.1126/science.1258096).

[082] An IN DEL in the coding sequence of a gene or even in an intron often leads to loss of gene function. For practical purposes, the tracrRNA and crRNA are usually combined into one chimeric guide RNA (sgRNA), which is another component of the CRISPR system. Site specific nucleases can induce targeted DSBs at a high efficiency and thus plants containing INDELs in the target sequence can be easily identified. Genome engineering through the use of site specific nucleases such as the CRISPR systems has many applications, especially in polyploid species, and is becoming increasingly important for crop improvement.

[083] As will be understood by the skilled person it is contemplated that the required elements of the nucleases may be introduced in the plant protoplasts directly or may be via the use of vectors that, when expressed in the plant protoplasts provide for the required elements of the nuclease systems described above, or combinations thereof. Preferably, the elements are transfected into the protoplast using polyethylene glycol- mediated transformation (PEG-mediated transformation), a technique, and varieties thereof, well-known to the skilled person.

[084] Also contemplated is that a site-specific modification is introduced by using oligonucleotide-directed mutagenesis using an oligonucleotide (with a length of for example, between 5 and 200 nucleotides, for example 10 - 150 nucleotides, preferably wherein the oligonucleotide is a single-stranded oligonucleotide.

[085] Oligonucleotide-directed mutagenesis is a site-directed mutagenesis method that is based on the delivery into the eukaryotic cell nucleus of synthetic mutagenic oligonucleotides (double- or single-stranded) that resemble DNA in their Watson-Crick base pairing properties, but may be chemically different from DNA. (Alexeev and Yoon, Nature Biotechnol. 16: 1343, 1998 ; Rice, Nature Biotechnol. 19: 321 , 2001 ; Kmiec, J. Clin. Invest. 112: 632, 2003 ). Once introduced into the cell, such oligonucleotides base pair with the complementary sequence at the target locus. By deliberately designing a mismatch in the oligonucleotide, the mismatch may induce a nucleotide conversion at the corresponding position in the target genomic sequence. This method allows the conversion of single or at most a few nucleotides in endogenous loci, and may, for example, be applied to create stop codons in existing loci, resulting in a disruption of their function, or to create codon changes, resulting in genes encoding proteins with altered amino acid composition (protein engineering). [086] Oligonucleotide-directed mutagenesis has been described in plant, animal and yeast cells. Two different classes of synthetic oligonucleotides have been used in these studies, the chimeric DNA:RNA oligonucleotides or single stranded oligonucleotides.

[087] The first examples of oligonucleotide-directed mutagenesis using chimeras came from animal cells (reviewed in Igoucheva et al. 2001 Gene Therapy 8, 391-399 ) and were then also later used to achieve targeted mutagenesis in plant cells (Beetham et al. 1999 Proc.Natl.Acad.Sci.USA 96: 8774-8778 ; Zhu et al. 1999 Proc. Natl. Acad. Sci. USA 96, 8768-8773 ; Zhu et al. 2000 Nature Biotech. 18, 555-558 ; Kochevenko et al. 2003 Plant Phys. 132: 174-184 ; Okuzaki et al. 2004 Plant Cell Rep. 22: 509-512 ).

[088] Due to the difficulties of working with chimeras, more reliable alternative oligonucleotide designs have been sought. Several laboratories have investigated the ability of single stranded (ss) oligonucleotides to perform targeted mutagenesis. These have been found to give more reproducible results, be simpler to synthesize, and can also include modified nucleotides to improve the performance of the mutagenic oligonucleotide in the cell (Liu et al. 2002 Nucl. Acids Res. 30: 2742-2750 ; review, Parekh-Olmedo et al.

2005 Gene Therapy 12: 639-646 ; Dong et al.2006 Plant Cell Rep. 25: 457-65 ; De Piedoue et al. 2007 Oligonucleotides 27: 258-263 ).

[089] Oligonucleotide-directed mutagenesis has been described in a variety of patent applications such as WO0173002 , WO03/027265 , WO01/87914 , WO99/58702 , W097/48714 , WO02/10364, WO2007/073166 and WO2007/073170, the latter two describing the use of modified nucleotides, such as Locked nucleic acids (LNA) and C5- propyne pyrimidines, in the oligonucleotide. In one embodiment it is contemplated that the oligonucleotide used in the oligonucleotide-directed mutagenesis comprises at least one modified nucleotide, such as a LNA.

[090] WO2009/082190 discloses that polyethylene glycol mediated introduction of the oligonucleotides is preferred and increases overall efficiency. It is therefore contemplated that, in a preferred embodiment, the oligonucleotide is introduced in the plant protoplast using polyethylene glycol, i.e. by polyethylene glycol mediated transfection.

[091] The skilled person is well aware of techniques to select for the plant calli with the desired size, as disclosed herein. He may for example select the protoplasts using an automated device, for example an automated sorter, even more preferably a flow cytometer, one non-binding example being a COPAS platform (Union Biometrica, Holliston, Massachusetts).

[092] In a further aspect there is provided for a method of obtaining at least one plant cell, wherein the method comprises providing a multitude of plant protoplasts; subjecting said multitude of plant protoplasts to a treatment to introduce a genome-editing event and/or a genetic-modification event; allowing the multitude of plant protoplasts to grow into plant calli until 0.5 - 5% (by number) of the plant calli is larger than 700 micrometer; selecting at least part of the plant calli that are smaller than have a size of between 80 micrometer and 500 micrometer; screening at least part of the selected plant calli for the genome-editing event and regeneration of at least one plant cell from a callus comprising the genome- editing event and/or a genetic-modification event; or regenerating of at least one plant cell from at least part of the selected plant calli and screening of at least part of the regenerated plant cells for the genome-editing event and/or a genetic-modification event. Embodiments and preferences for this method are as described above, and as described in the claims and examples.

[093] Having illustrated and described the principles of the invention and its preferred embodiments, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles.

Examples

[094] Several targeted mutagenesis (site-directed mutagenesis) experiments were performed using the tomato CENH3 gene as an example. This was done by introducing CRISPR reagents (for research purposes/proof on concept) into tomato protoplasts and then allowing these to develop further into (micro)calli. These were then isolated in two different ways, by manual picking or through sorting using a COPAS flow cytometer. Both of these populations of (micro)calli were then were then genotyped for the presence of indel mutations at the CENH3 locus. Surprisingly, we found that we were only able to detect indel mutations (in high frequency) in the (micro)calli that had been sorted using the COPAS apparatus. Therefore, we have identified and solved a bottleneck limiting the application of targeted mutagenesis technologies in plant cells. Results are comparable when another gene as the target in the method of the invention is used.

Example 1 : Mutagenesis of the tomato CENH3 gene using the CRISPR technology

Constructs

[095] The sequence of the Streptococcus pyogenes Cas9 ORF (Accession number NC_002737) was used to design a variant that had altered codon usage for optimal expression in tomato, Solanaceae lycopersicum. The resulting ORF is shown in figure 1. The ORF was then synthesized (www.geneart.com) flanked by both Xhol (5') and Sacl (3') sites and cloned into a plasmid. The Cas9 ORF fragment was then isolated from this plasmid after digestion with Xhol and Sacl. Plasmid pKG7381 carries a 6xHIS tagged version of green fluorescent protein (GFP) flanked by Xhol and Sacl sites. The GFP ORF in pKG7381 was replaced by the Cas9 ORF using the Xhol and Sacl sites, resulting in the construct pKG7230 that carries the Cas9 ORF with a nuclear localization sequence (NLS) and 6xHIS tag translationally fused at its N terminus. The constitutive cauliflower mosaic virus 35S promoter present on the vector pKG7230 was used to express Cas9 in tomato protoplasts.

[096] In addition two sgRNAs (targeting exonl and exon 4) were designed for the mutagenesis of the tomato CENH3 gene and were linked to the Arabidopsis thaliana U6 promoter for in planta expression. These cassettes were then synthesized (figure 2) and cloned into plasmid constructs to generate pKG9276 (exon 1 sgRNA) and pKG9286 (exon 4 sgRNA).

Tomato protoplasts isolation and transfection

[097] In vitro shoot cultures of Solanum lycopersicum var Moneyberg were maintained on MS20 medium with 0.8% agar in high plastic jars at 16/8 h photoperiod of 2000 lux at 25°C and 60-70% RH (Relative humidity). Young leaves (1 g) were gently sliced perpendicularly to the mid nerve to ease the penetration of the enzyme mixture. Sliced leaves were transferred to the enzyme mixture (2% Cellulase Onozuka RS, 0.4% Macerozyme Onozuka R10 in CPW9M (Frearson EM, Power JB and Cocking EC, 1973, The isolation, culture and regeneration of Petunia leaf protoplasts. Developmental Biology, 33: 130-137) and cell wall digestion was allowed to proceed overnight in the dark at 25°C. The protoplasts were filtered through a 50 μηι nylon sieve and were harvested by centrifugation for 5 minutes at 800 rpm. Protoplasts were resuspended in CPW9M (Frearson, 1973, supra) medium and 3 mL CPW18S (Frearson, 1973, supra) were added at the bottom of each tube using a long-neck glass Pasteur pipette. Live protoplasts were harvested by centrifugation for 10 minutes at 800 rpm as the cell fraction at the interface between the sucrose and CPW9M medium. Protoplasts were counted and resuspended in MaMg (Negrutiu I., Shillito R., Potrykus I, Biasini G. and Sala F., 1987, Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Molecular Biology, 8: 363-373.) medium at a final density of 10⁶ per mL. To 12 mL tubes, 500 (500,000 protoplasts) of the protoplast suspension plasmids as described below (transfection vectors) and 500 of PEG solution (40% PEG4000 (w/v), 7% Mannitol (w/v), 100 mM Ca(N0₃)₂) were added. After gentle but thorough mixing, transfection was allowed to take place for 20 minutes at room temperature. Then 10 mL of calcium nitrate 0.275 M solution was added and thoroughly, but gently mixed in. Transfected protoplasts were harvested by centrifugation for 5 minutes at 800 rpm and resuspended in 9M culture medium (Frearson EM, Power JB and Cocking EC, 1973, The isolation, culture and regeneration of Petunia leaf protoplasts. Developmental Biology, 33:130-137) at a density of 0.5 x 10⁶ per ml and transferred to a 4cm diameter petri dish and an equal volume of 2% alginate solution was added to the protoplasts in 9M medium directly after PEG treatment and 1 ml aliquots were spread over Ca-Agar plates and allowed to polymerize for 1 hour. Tomato protoplast transfection

[098] Tomato protoplasts were isolated and transfected as described above. For each mutagenesis experiment 20μg of pKG7230 and "^g of either pKG9276 or pKG9286 was transfected to 500,000 protoplasts which were then embedded in alginate. These were maintained for 12 days in liquid K8p medium (Kao K.N. and Michayluk M.R., 1975, Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta, 126: 105-1 10) and the alginate discs were then cut into 5 mm wide strips, layered over solid TM-DB medium (Shahin, 1985, Totipotency of tomato protoplasts. Theoretical and Applied Genetics, 69:235-240) and incubated further in the dark at 28°C. The alginate strips were transferred to fresh TM-DB medium (Tan M.-L- M.C., Rietveld E.M., van Marrewijk A.M. and Kool A.J., 1987, Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration. Plant Cell Reports, 6: 172-175) every 3 weeks and incubated under low light at 25°C until the (micro)calli had developed sufficiently for sorting by the different methods. Tomato plant regeneration from the individual calli occurred after successive transfer to GM medium (Tan M.-L- M.C., 1987, supra) supplemented with 1 mg.l-1 zeatin and 0.2 mg.l-1 GA3 and MS medium supplemented with 2 mg.l-1 zeatin and 0.1 mg.l-1 IAA media after which regenerated tomato plantlets were rooted on MS medium supplemented with 0.5 mg.l-1 IBA before transfer to the greenhouse. (Shahin E.A., 1985, Totipotency of tomato protoplasts, Theoretical and Applied Genetics, 69: 235-240; Tan M.-L- M.C., Rietveld E.M., van

Marrewijk A.M. and Kool A.J., 1987, Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration. Plant Cell Reports, 6: 172-175; Tan M.-L., 1987, Somatic Hybridisation and Cybridisation in some Solanaceaea (thesis); Frearson EM, Power JB and Cocking EC, 1973, The isolation, culture and regeneration of Petunia leaf protoplasts. Developmental Biology, 33: 130-137; Negrutiu I., Shillito R., Potrykus I, Biasini G. and Sala F., 1987, Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Molecular Biology, 8: 363-373; Kao K.N. and Michayluk M.R., 1975, Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta, 126: 105-1 10). Callus sampling

[099] Two different methods for sampling and genotyping the callus were compared, manual picking using tweezers and sorting using a COPAS (Holliston, Massachusetts, USA). For manual picking individual calli about 1-2mm in size in alginate strips were picked using sterile tweezers and transferred to fresh TM-DB plates for further growth. For COPAS sorting the alginate strips containing calli were incubated with 50 mM sodium citrate, 5% mannitol (w/v), without adjustment of the pH. Incubation is usually performed for 1 to 2 hours at room temperature after which the calli suspension is centrifuged for 5 minutes at 2000 xg in a tabletop centrifuge in swinging buckets. The supernatant is discarded and the calli are re-suspended in appropriate culture medium. The (micro)calli were then sorted by the COPAS (Parameters are in arbitrary units, the apparatus is a: COPASTM Plus HTS; Sorting parameters: extinction (EXT, Y axis) vs. time of flight (TOF, X axis) Scale 2048 for both parameters; Drop parameters: delay 14; width 5; Threshold parameters: Signal 100; TOF minimum 70; Pressure parameters: Sheath 1.93; Sample 0.26; Sorter 1.51 ; Sorting gate coordinates:27x34; 62x15, 102x256, 256x256, 256x82) and arrayed on TM-DB plates in a 96 well format.

Callus genotyping

[100] Calli that had been picked manually were maintained on TM-DB medium until they were about 5mm in size and then transferred to medium that promotes shoot regeneration. Once shoots were formed these were sampled for DNA isolation and a PCR was performed using primers that flank the target sites. For exon 1 these were 5'- CTGCTTTTGGTTCTTCTTCTC-3' and 5'- TCACCGCTTCTTATCTTCAACT-3' and for exon 4 5'- TTTTTCGAGCCCAATCCTAT-3' and 5'- ATGAAAGGAGCAGCTGGAAT-3'.

[101] The resulting PCR products were sequenced to identify shoots carrying indel mutations at the CENH3 target site.

[102] Calli that had been sorted by the COPAS were genotyped directly using the direct PCR kit (Phire Plant Direct PCR kit, Thermo Scientific) using the primers described above and the resulting PCR products were sequenced to identify calli that had an indel at the CENH3 target sites. These calli were then transferred to medium that promotes shoot regeneration. The resulting shoots were then sampled for DNA isolation and the PCR was once again performed on the CENH3 target site(s) to confirm that the indel mutation present in the callus was also present in the regenerated shoot.

Results

[103] The number of shoots or calli analyzed using either the manual picking method or the COPAS sorting and the number of mutants detected is shown in table 1. Table 1 : Genotyping shoots / calli for mutations at the CENH3 locus

[104] We were unable to detect any indel mutations in CENH3 in shoots derived from calli that had been picked manually. However, indel mutants were readily identified in the calli that had been sorted using the COPAS. The types of indel mutations found are shown in figure 3. Shoots were then regenerated from these mutant calli and were genotyped for the presence of the indel found in the original callus.

[105] In each case we were able to detect the correct indel in the shoot demonstrating that the COPAS sorted calli were not chimeric. The majority of these mutations resulted in frame shifts in the CENH3 coding sequence and a null mutation that eliminates CENH3 function.

[106] This experiment demonstrates that the ability to obtain mutant plants from protoplasts is dependent on the method that is used for sampling, even when the technology itself is able to create mutations at a high efficiency.

Claims

1) Method for obtaining a population of plant calli that is enriched for plant calli that comprise a genome-editing event and/or a genetic-modification event, wherein the method comprises a) providing a multitude of plant protoplasts;

b) subjecting said multitude of plant protoplasts to a treatment to introduce a genome- editing event and/or a genetic-modification event;

c) allowing the multitude of plant protoplasts to grow into plant calli until 0.5 -5% of the plant calli is larger than 700 micrometer;

d) selecting at least part of the plant calli that have a size between 80 micrometer and 500 micrometer. 2) The method of claim 1 , wherein the plant calli selected in step d) are plant calli that can grow in the absence of other plant calli.

3) The method of any one of the previous claims, wherein the plant calli selected in step d) are smaller than 400 micrometer, or smaller than 300 micrometer, or smaller than 200 micrometer.

4) The method of any of the previous claims, wherein the plant calli selected in step d) are larger than 100 micrometer. 5) The method of any of the previous claims wherein the plant calli selected in step d) are within the size range of about 100 - 400 micrometer.

6) The method of any one of the previous claims, wherein the multitude of plant protoplasts provided in step a) is a multitude of plant protoplasts that have been subjected to a treatment to introduce a genome-editing event and/or a genetic-modification event and wherein, optionally step b) is omitted.

7) The method of any one of the previous claims, further comprising

e) (e1) screening at least part of the selected plant calli for the genome-editing event and/or a genetic-modification event and regeneration of at least one plant cell from a callus comprising the genome-editing event and/or a genetic-modification event; or (e2) regenerating of at least one plant cell from at least part of the selected plant calli and screening of at least part of the regenerated plant cells for the genome-editing event and/or a genetic-modification event.

8) The method of claim 7, wherein the regenerated at least one plant cell is an aggregate of at least two plant cells, a plant shoot, a plant tissue or a plant.

9) The method of any one of the previous claims, wherein the genome-editing event is by a) introducing or expressing at least one site-specific nuclease in the multitude of plant protoplasts, preferably wherein said nuclease is selected from the group consisting of (engineered) nucleases, Cas/RNA CRISPR nuclease, zinc-finger nuclease, meganuclease and TAL-effector nuclease, preferably wherein said nuclease is Cas/RNA CRISPR nuclease, preferably wherein said Cas/RNA CRISPR nuclease comprises sgRNA and Cas9 protein and/or expression vectors therefor; and/or b) oligonucleotide-directed mutagenesis using a oligonucleotide, preferably wherein the oligonucleotide is a single-stranded oligonucleotide.

10) The method of any one of the previous claims wherein the screening is performed by a genotyping assay, preferably by PCR followed by genotyping.

1 1) The method of any of the previous claims wherein the plant protoplasts are tomato plant protoplasts.

12) The method of any of the previous claims, wherein selection in step d) is performed using an automated device, preferably an automated sorter, even more preferably a flow cytometer.

13) Method of obtaining at least one plant cell, wherein the method comprises

a) providing a multitude of plant protoplasts;

c) allowing the multitude of plant protoplasts to grow into plant calli until 0.5 -5% of the plant calli is larger than 700 micrometer;;

d) selecting at least part of the plant calli that have a size between 80 micrometer and 500 micrometer; e) (e1) screening at least part of the selected plant calli for the genome-editing event and/or a genetic-modification event and regeneration of at least one plant cell from a callus comprising the genome-editing event and/or a genetic-modification event; or (e2) regenerating of at least one plant cell from at least part of the selected plant calli and screening of at least part of the regenerated plant cells for the genome-editing event and/or a genetic-modification event.

14) The method of claim 13, wherein the plant calli selected in step d) are plant calli that can grow in the absence of other plant calli.

15) The method of any one of the previous claims 13 - 14, wherein the plant calli selected in step d) are smaller than 400 micrometer, or smaller than 300 micrometer, or smaller than 200 micrometer. 16) The method of any of the previous claims 13 - 15, wherein the plant calli selected in step d) are larger than n 100 micrometer.

17) The method of any of the previous claims 13 - 16 wherein the plant calli selected in step d) are within the size range of about 100 - 400 micrometer.

18) The method of any one of the previous claims 13 - 17, wherein the at least one plant cell is an aggregate of at least two plant cells, a plant shoot, a plant tissue or a plant.

19) The method of any one of the previous claims 13 - 18, wherein the multitude of plant protoplasts provided in step a) is a multitude of plant protoplasts that have been subjected to a treatment to introduce a genome-editing event and/or a genetic-modification event and wherein, optionally step b) is omitted.

20) The method of any one of the previous claims 13 - 19, wherein the genome-editing event is by a) introducing or expressing at least one site-specific nuclease in the multitude of plant protoplasts, preferably wherein said nuclease is selected from the group consisting of (engineered) nuclease, Cas/RNA CRISPR nuclease, zinc-finger nuclease, meganuclease and TAL-effector nuclease, preferably wherein said nuclease is

Cas/RNA CRISPR nuclease, preferably wherein said Cas/RNA CRISPR nuclease comprises sgRNA and Cas9 protein and/or expression vectors therefor; and/or b) oligonucleotide-directed mutagenesis using a oligonucleotide, preferably wherein the oligonucleotide is a single-stranded oligonucleotide.

21) The method of any one of the previous claims 13 - 20 wherein the screening is performed by a genotyping assay, preferably by PCR followed by genotyping.

22) The method of any of the previous claims 13- 21 wherein the plant protoplasts are tomato plant protoplasts. 23) The method of any of the previous claims 13 - 22, wherein selection in step d) is performed using an automated device, preferably an automated sorter, even more preferably a flow cytometer.