Gelling agents in protoplast regeneration

Abstract

This invention generally pertains to means and methods for cultivation of plant protoplasts. Specifically, the invention pertains to a method for cultivating plant protoplasts, wherein the method at least comprises (a) providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin, (b) causing the pectin to form a hydrogel comprising the plant protoplasts and (c) contacting the hydrogel comprising the plant protoplast with a first culture medium. The invention further pertains to a pectin hydrogel comprising plant protoplasts and a pectin hydrogel comprising clonal microcalli.

Description

[0001] Title: GELLING AGENTS IN PROTOPLAST REGENERATION

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention generally pertains to means and methods for cultivation of plant protoplasts. Specifically, the invention pertains to a method for cultivating plant protoplasts, wherein the method at least comprises (a) providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin, (b) causing the pectin to form a hydrogel comprising the plant protoplasts and (c) contacting the hydrogel comprising the plant protoplast with a first culture medium. The invention further pertains to a pectin hydrogel comprising plant protoplasts and a pectin hydrogel comprising clonal microcalli.

[0004] BACKGROUND OF THE INVENTION

[0005]

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006]

[0003] Plants have a remarkable reprogramming potential, which enables plant regeneration from organs, tissues, or even a single cell. Protoplasts, i.e., cells excluding a cell wall, have the ability to dedifferentiate, and cultured protoplasts have the ability to form cell walls and undergo cell division, allowing whole plant regeneration. However, using a single cell such as a protoplast as a starting point for whole plant regeneration poses many challenges. The single cell needs to be grown under controlled conditions such that eventually a multi-cellular organism is obtained, having a multitude of varying cell types and functions. For example, the single cell has to differentiate into cells part the root system, stem system, leaves, flowers and so forth. Key processes involved in protoplast generation and regeneration, including cell wall removal, handling of the protoplast, cell wall recovery, cell cycle re-entry, callus formation (for example, and in particular clonal microcalli formation), pluripotency acquisition, and de novo tissue regeneration, are essential in order to obtain a fully grown plant. Thus, means for growing a single cell (e.g., protoplast) to a fully grown plant poses many challenges and can only be successful if at every stage of growth, or regeneration, the right conditions are provided especially at an early stage of growth, i.e., from the stage of a single cell or protoplast to a microcallus. For example, at this stage, often there will be no initial cell division of the single cells, or growth arrest because the right triggers or nutrients are not provided or there is too little oxygen (often at the edges of standard alginate gels one observes bigger call i) or the calli in an alginate gel toxify itself toxic compounds it produces and secretes (for example because of stress triggers) stay trapped around the cell in alginate gels.

[0007]

[0004] Protoplast regeneration methods have been developed in several plant species. Conventional methods of protoplast regeneration involve liquid culture. Liquid culture of protoplasts is a simple and easy technique used to induce cell division and callus formation, but it has a low efficiency of tissue regeneration, owing to cell aggregation-induced cell death, low cell proliferation activity or the cells do not stay adhered to one another, failing to form a microcallus. A further downside of liquid cultures is that chimeras occur frequently, i.e., non-monoclonal microcalli, due to the plurality of protoplasts free ‘floating’ in the liquid culture which results in two or more protoplasts engaging with one another and forming a chimeric microcallus. Other methods of protoplast regeneration involve embedding in gels comprising gelling agents such as alginate or low-melting agaroses but do not always lead to satisfactory or reproducible regeneration of protoplasts and may have the same drawbacks as mentioned for liquid cultures, i.e., cell aggregation-induced cell death and low cell proliferation activity. Thus, protoplast regeneration remains challenging, especially for many important cultivars of crop and model species.

[0008]

[0005] Accordingly, there is a need for improved cultivating of plant protoplasts and microcalli proliferation. Accordingly, there is a need for a fast, cheap, and effective method to achieve this goal. Advantageously, the inventors herein provide such means and methods of regenerating isolated plant protoplasts such that a fully grown plant can be grown from a single cell.

[0009]

[0006] The inventors have surprisingly found that by embedding and immobilizing protoplasts in a hydrogel comprising a hydrogel-forming pectin, the protoplasts efficiently and reproducibly proliferate and form microcalli, which in turn can be further cultivated and grown into a mature, fully grown plant. The inventors have also found that with the method according to the invention it has now become possible to more easily obtain clonal microcalli, i.e., microcalli obtained from a single cell. The inventors have found that this is even possible when using reduced volumes of the hydrogel and / or when using reduced number of protoplast per volume unit of the hydrogel. Without being bound by any particular theory, the inventors believe proliferation of protoplasts according to the invention is improved due to a better exchange, i.e., ‘communication’ of e.g., nutrients, molecules, (phyto)hormones, chemical messengers and the like between the protoplasts when using the hydrogel according to the invention.

[0010]

[0007] The means and methods according to the invention highly advantageously provide for the ability to proliferate a large diversity and plurality of protoplasts to microcalli. Such microcalli may be further proliferated into microcolonies, and ultimately into fully grown plants. Indeed, the invention provides for improved methods to obtain such plants. The protoplasts, or plant cells from which the protoplasts are derived, can further be genetically modified, e.g., using known genetic engineering methods such as the CRISPR gene editing system. This allows the cultivation of genetically modified microcalli, and thus ultimately genetically modified plants that have highly advantageous traits. Genetic engineering strategies such as the CRISPR gene editing may cause cellular stress for example, but not limited due to induced double stranded breaks which in turn elicit a DNA damage response, transformation of the CRISPR gene editing system into a cell and / or elevated temperatures used for such transformations. By providing improved means and methods for proliferation of protoplasts according to the current invention, allows such genetically engineered protoplasts to handle cellular stresses better compared to the same cell in a different condition, for example in a liquid culture. Hence, the inventors advantageously provide for the means and methods for improved proliferation of genetically edited cells, such as protoplasts genetically engineered using the CRISPR gene editing system.

[0011] SUMMARY OF THE INVENTION

[0012]

[0008] As embodied and broadly described herein, the present invention is directed to a method of cultivating plant protoplasts in a pectin gel, a pectin gel comprising plant protoplasts and a pectin gel comprising clonal microcalli grown from the plant protoplast. Surprisingly, the inventors have found that when a hydrogel-forming pectin is used, preferably at defined concentrations of said pectin as disclosed herein and / or defined density of protoplasts in the hydrogel as defined herein, protoplast regeneration and hence formation of clonal microcalli is improved, for example, compared to standard methods in the prior art.

[0009] In a first aspect the invention pertains to a method for cultivating plant protoplasts, wherein the method at least comprises (a) providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin, (b) causing the pectin (in the aqueous solution of step (a)) to form a hydrogel comprising the plant protoplasts, preferably by contacting the aqueous solution of step (a) with (a source of) calcium, and (c) contacting the hydrogel comprising the plant protoplast with a first culture medium.

[0013]

[0010] In a second aspect, the invention further pertains to a pectin hydrogel comprising plant protoplasts and a pectin hydrogel comprising microcalli, preferably clonal microcalli.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

[0011] Figure 1: Schematic representation of the pectin gel structure. The polysaccharide’s pectin backbone is mostly made by a-(1,4)-linked D-galacturonic acid units (zigzag black lines in the figure). The gelation mechanism includes divalent cations, such as for example calcium ions (grey dots), interacting with the carboxyl groups in the galacturonic acid residues. This results in the formation of “egg-box" structures in which calcium ions bridge adjacent galacturonic acid chains, resulting in a 3D gel network. Protoplasts (big circles with “P”) are trapped and stabilized in such 3D structure. Pectin shows various levels of methyl esterification at the level of its carboxylic group which are changing the ability of pectins to interact with calcium to form gels (small open circles in the figure). An optimal balance of free carboxylic and methyl-esterified groups is required to achieve the proper gel strength upon calcium supply.

[0016]

[0012] Figure 2: Schematic representation of the procedure to make pectin gels on plates. (A) The hydrogel-forming pectin suspension and the protoplasts suspension are combined to obtain the final mixture with the expected pectin and protoplast concentration. (B) The pectin-protoplasts mix is put in contact with a source of calcium (C), e.g., pipetted on the surface of a petri dish containing a semi-solid substrate rich in calcium) until hydrogel formation. Hydrogels are transferred into sterile vessels of the appropriate size (e.g., wells of a multi-well plate; (D)) containing culture medium. Once microcalli are formed, pectin hydrogels are transferred to a semi-solid culture medium (E), removed from hydrogels, and further cultivated until a microcolony is formed.

[0013] Figure 3: Schematic representation of the protocol to prepare pectin suspension. The pectin powder is dissolved in the mannitol solution at 80°C under stirring for at least three hours. The homogeneous pectin suspension is cooled down to room temperature under stirring and then filter-sterilized.

[0017]

[0014] Figure 4: microscopy images of microcallus formation for growing Tomato Marglobe.

[0018]

[0015] Figure 5: Microscopy images of microcallus formation for growing strawberry (everbearing variety).

[0019]

[0016] Figure 6: Representative data of monoclonality analysis of single-cell regenerated ErCas12a edited Nbhexol mutant lines using the method as disclosed herein. Three plants of Nicotiana benthamiana individually mutated in the gene Hexol (|3-hexosaminidase) named Nbhexo1-1, Nbhexo1-5 & Nbhexo1-2, were sampled in duplo at three distinct locations (leaves, flowers, and roots) and genotyped by targeted ONT-NGS (Oxford Nanopore Technology; next-generation sequencing). For a non¬ chimeric tetrapioid N. benthamiana plant four copies of the gene are present and each copy can have a different editing profile; here indicated by the size of the deletion. Each of the three plants tested has an unique set of edits over the four copies and it is clear from this data that this pattern is identical in each part of the plant. This demonstrates that the regenerated plants are monoclonal and non-chimeric.

[0020]

[0017] Figure 7: Microscopy images of microcallus formation for growing romaine lettuce at a density of 5000 cells / ml in pectin (0.2 wt.%) or alginate (1 wt.% or 0.5 wt.%).

[0021]

[0018] Figure 8: Microscopy images of microcallus formation for growing six different tomato varieties (variety

[0001] -[6]) at a density of 100.000 ceils / ml or 200.000 cells / ml in pectin (0.2 wt.%).

[0022]

[0019] Figure 9: Microscopy images of microcallus formation for growing four different June-bearing strawberry varieties (variety [1]-[4]) at a density of 100.000 cells / ml or 200.000 cells / ml in pectin (0.2 wt.%)

[0023]

[0020] Figure 10: Microscopy images of microcallus formation for growing recalcitrant white cabbage variety (Fig. 10; left) and non-recalcitrant cauliflower variety (Fig 10; right) at a density of 100.000 cells / ml in pectin (0.2 wt.%).

[0021] Figure 11: Microscopy images of microcallus formation for growing two different sugar beet varieties (variety

[0001] -[2]) at a density of 75.000 cells / ml or 100.000 cells / ml in pectin (0.2 wt.%).

[0024]

[0022] Figure 12: Microscopy images of microcallus formation for growing acorn squash cotelydons (Fig. 12; top left), pumpking cotelyodons (Fig. 12; top right), acorn squash leaf (Fig. 12; bottom left) and pumpkin leaf (Fig. 12; bottom right) at a density of 50.000 cells / ml (for cotelydon protoplasts) or 300.000 cells / ml (for leaf protoplasts) in pectin (0.2 wt.%)

[0025]

[0023] Figure 13: Microscopy images of microcallus formation after four days from tomato (variety marglobe) at a density of 50.000 cells / ml in pectin (0.5 wt.%) or alginate (1.0%). Cells in pectin are developing after said four days into cell colonies and the cells in alginate have not yet divided

[0026]

[0024] Figure 14: Microscopy images of microcallus formation from white cabbage variety at a density of 250.000 cells / ml in pectin (0.1 wt.%, 0.2 wt.% 0.5 wt.% and 1 wt.%). Regeneration of the protoplasts at the indicated pectin concentrations were performed in triplicate (left, middle and right columns).

[0027]

[0025] Figure 15: Microscopy images of microcallus formation from N. benthamiana protoplasts when grown in alginate (1 wt.%) at a density of 10.000 cells / ml with- and without the presence of nurse cells. Without nurse cells no microcalli development was observed at this cell density (10.000 cells / ml) in alginate. Scale bars indicate size.

[0028] DESCRIPTION

[0029] Definitions

[0030]

[0026] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0031]

[0027] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0032]

[0028] For purposes of the present invention, the following terms are defined below.

[0033]

[0029] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0034]

[0030] The terms “about” and “approximately”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±10% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0035]

[0031] When used in these specification and claims, the terms “comprises" and "comprising" and variations thereof mean that the specified features, steps, or integers are included. The terms are not to be interpreted to exclude the presence of other features, steps, or components.

[0036]

[0032] As used herein, the term “and / or” indicates that 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.

[0037]

[0033] When used in these specification and claims, the term “at least” followed by a particular value means that 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, 11, 12, 13, 14, 15, etc.

[0038]

[0034] As used herein, the term “protoplasts” or “plant protoplast” refers to its generally recognized meaning in the field, i.e., refers to an entire, single, plant cell, excluding the cell wall. Protoplasts may be obtained by methods known in the art and the skilled person is capable of obtaining protoplasts, for example as described in Chen et al., J. Mol. Sc / . 2023, 24(23), 16892 (doi: 10.3390 / ijms242316892). Generally, protoplasts are obtained by obtaining one or more plant cells, explant material, preferably leaves from a plant, preferably sterile, from which the cell wall is removed, for example by enzymatic digestion using cellulase and / or pectinase. Further processing of the protoplasts, i.e., isolation or removal of enzymes, can be done by any known means in the art, e.g., by filtering, phase separation or centrifugation. Obtained protoplasts are then ready for various applications, for example in plant tissue culture and / or transient expression studies. A protoplast may be obtained from a plant cell that is genetically engineered, e.g., by modifications to the DNA using for example a CRISPR system. Protoplasts as such may be genetically engineered, e.g., by modifications to the DNA using for example a CRISPR system.

[0039]

[0035] As used herein, the term “pectin”, i.e., pectic polysaccharides, refers to a composition of complex polysaccharides that are present in the primary cell walls of a plant. Within the context of the current invention the term “pectin” also refers to a single component comprised in pectin e.g., polygalacturonic acid or salt thereof. Depending on the plant source, a pectin structure may comprise of (polymers from) homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) domains. The structure and chemical composition of pectin is different among plants, within a plant over time, and in various parts of a plant. The skilled person is able to isolate (i.e., extract) pectin from a suitable pectin source, for example citrus or apple by using common methods such as, but not limited to, conventional heating (CE), microwave heating (MAE), ultrasonic (UAE), and enzymatic extraction (EAE) methods. Alternatively, the skilled person may obtain a suitable pectin through e.g., a commercial vendor for example polygalacturonic acid sodium salt obtained from Merck (product id: P3850). Pectin is made up primarily of galacturonic acid units linked together in a chain, with varying degrees of esterification. Pectin is classified based on its degree of esterification and is classified as high methoxyl (HM) pectin or low methoxyl (LM) pectin. Within the context of the current invention, preferably LM pectin is used. The pectin, preferably LM pectin according to the invention can form a hydrogel, for example in the presence of calcium ions. In other words, the pectin is a “hydrogel-forming pectin”.

[0040]

[0036] As used herein, the term "hydrogel-forming pectin” refers to a pectin that can be formed into a hydrogel. It refers to the ability of pectin to undergo gelation, turning a liquid solution, i.e., a liquid solution that can be for example poured or pipetted, into a semi-solid state. Any pectin that can form a hydrogel is considered to be a hydrogelforming pectin according to the invention. For example, an aqueous solution comprising a hydrogel-forming pectin, protoplasts and water can form a hydrogel comprising the protoplasts in the presence of (or when contacted with) divalent cations, such as calcium ions (Ca2+). Thus, the aqueous solution comprising the hydrogel-forming pectin forms into a hydrogel. The skilled person understands how to form a hydrogel from a hydrogel-forming pectin in the context of the current invention. Hydrogels according to the invention refer to polymeric materials with a 3D network capable of absorbing and / or retaining water, making them highly hydrophilic. Advantageously, the hydrophilic nature of pectin allows the hydrogel to retain a significant amount of water which allows efficient transfer of, for example, nutrients from a culture medium to said protoplasts and of molecules, for example signaling molecules, between individual protoplasts.

[0041]

[0037] As used herein, the term “microcalli” or “microcallus” refers to a clusters of undifferentiated plant cells formed during tissue culture, e.g., from a single isolated protoplast. Microcalli may serve as an intermediate stage in (whole) plant regeneration. Microcalli in accordance with the invention are about 0.2 - 1.5 mm, 0.3 - 1.2 mm, or 0.5 - 1.0 mm in diameter defined by the longest axis of said microcalli from any particular angle, e.g., the longest axis based on a picture taken of said microcalli from above, when the microcalli are embedded in the hydrogel. As a microcallus grows, it can form a microcolony which in turn can form a plant organ such as a shoot, which in turn, or ultimately, can form into a complete plant. Under specific hormonal and / or nutrient conditions in the culture medium, the microcolony can be induced to differentiate into plant organs (such as shoots and roots) or to form somatic embryos, which can then develop into complete plants.

[0042]

[0038] As used herein, the term “microcolonies” or “microcolony” refers to a cluster of cells at the earliest stage of dedifferentiation and proliferation. Microcolonies as used herein are formed from microcalli, i.e., microcalli form into microcolonies. Microcolonies in accordance with the invention are about 1.5 - 10.0 mm, 1.5 - 7.5 mm, or 1.5 - 5.0 mm in diameter in diameter defined by the longest axis of said microcolonies from any particular angle, e.g. the longest axis based on a picture taken of said microcolonies from above, when the microcolonies from are embedded in the hydrogel. The stage of dedifferentiation with respect to microcolony precedes the development of a more organized callus, from which (ultimately) regeneration into complete plants can occur. Microcolony formation is influenced by factors like the type of explant, media composition, and hormonal balance. It is understood that, between the stage of microcolony and stage of a fully grown plant, a plurality of other stages may be defined.

[0043]

[0039] As used herein, the term “clonal” or “clonal microcalli” “monoclonal microcalli” refers a group of genetically identical cells, i.e., the cells of a single microcallus derived, or derivable from, a single ‘parent’ protoplast. In the context of genetic engineering, e.g., using the CRISPR gene editing system, a plurality of protoplasts can comprise the same modification to their genetic code and proliferate into a plurality of microcalli. However, each genetically modified protoplast that proliferates into a single microcalli thus forms a clonal microcalli, regardless of whether other protoplasts contain the same modification. A clonal microcallus can split into two or more microcalli, e.g., due to mechanical disruption of the microcallus. The two or more microcalli are considered to be derived from, or derivable from, the same protoplast and are thus clonal microcalli.

[0044]

[0040] As used herein, the term “plant” as used throughout the present disclosure is in general refers to any type of plant such as, but not limited to, trees, bushes, herbs, ferns, crops, mosses, and the like, preferably the term “plant” refers to vascular plants. As meant herein, the term “plant” preferably refers to plants cultivated in agriculture, horticulture, or aquaculture, such as, but not limited to, food crops, feed crops, fiber crops, oil crops, ornamental crops, industrial crops, and the like. The term “plant” also refers to any part of a plant, such as the shoots, leaves, fruits, roots, or any other plant part, either growing or harvested.

[0045] Detailed description

[0046]

[0041] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0047]

[0042] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment envisaged herein. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are also envisaged herein, and form different embodiments, as would be understood by those in the art.

[0043] It is contemplated that embodiments described herein in relationship to any method, use, or composition can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0048]

[0044] The present invention is detailed below.

[0049]

[0045] Method for proliferation of ptent protoplasts

[0050]

[0046] In a first aspect, the invention pertains to a method for cultivating plant protoplasts, wherein the method at least comprises:

[0051] (a) providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin,

[0052] (b) causing the pectin to form a hydrogel comprising the plant protoplasts, preferably by contacting the aqueous composition of step (a) with calcium,

[0053] (c) contacting the hydrogel comprising the plant protoplast with a first culture medium and allowing the plant protoplast to proliferate and to form one or more microcalli

[0054]

[0047] As will be elucidated in further detail below, the method according to the invention, as well as embodiments pertaining to the method or otherwise, is highly advantageous for, for example, improving plant protoplast proliferation, generating at least one microcallus, preferably at least one clonal microcallus, generating at least one plant cell line, preferably at least one clonal cell line, regenerating at least one plant cell, improving microcallus formation, improving plant cell regeneration efficiency, and / or regenerating of at least one plant, preferably at least one clonal plant.

[0055]

[0048] As will be understood by the skilled person, the method of the invention may further comprise additional steps before, during or after any of steps (a)-(c). For example, in some embodiments, the method may include an additional step between step (a) and step (b), and / or between step (b) and (c), or the method may comprise additional steps before step (a), and / or after step (c), and any combination thereof. The steps according to the method, as well as additional steps will be further elucidated below.

[0056]

[0049] Step (a) - Providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin.

[0057]

[0050] In step (a) according to the invention an aqueous solution is provided comprising plant protoplasts and a hydrogel-forming pectin. The aqueous solution is understood to comprise water, preferably sterilized, deionized or milli-Q water, and may further comprise other substances such as macro- and / or micronutrients such as nitrogen, phosphorus, potassium, iron, zinc, and the like. The aqueous solution may further comprise a carbon source such as sucrose, glucose, fructose, or the like. The aqueous solution may further comprise (plant growth) hormones such as auxins, cytokines, brassinosteroids or a combination thereof. The aqueous solution may further comprise vitamins such as thiamine, pyridoxine, niacin. The aqueous solution may further comprise pH adjusters such as hydrochloric acid or sodium hydroxide to obtain a suitable pH, for example a pH of between 5-7, preferably 5-6, more preferably 5.5-6.

[0058]

[0051] In an embodiment, the aqueous solution comprises osmotic stabilizers to keep protoplasts from bursting or shriveling, thus the osmotic stabilizers provide for the desired osmolarity in e.g., the aqueous solution of step (a), the hydrogel of step (b) or the first, second of a further culture medium. In a preferred embodiment the osmotic stabilizers are selected from mannitol, sorbitol, sucrose, or glucose or a combination thereof. It was found that preferably, the osmotic stabilizer is mannitol. In a further preferred embodiment, the aqueous solution of step (a) comprises an osmotic stabilizer selected from mannitol, glucose, sorbitol, or a combination thereof, preferably mannitol and glucose, more preferably mannitol. During the formation of the hydrogel in step (b) the osmotic stabilizer, e.g., mannitol, which is added in step (a) will be present in step (b). Thus, in an embodiment, the hydrogel of step (b) also comprises an osmotic stabilizer selected from mannitol or sorbitol, preferably mannitol. Preferably, the medium osmolarity of the aqueous solution of step (a), the hydrogel of step (b) or the first, second of a further culture medium is between 100 - 1000 mOsmol / Kg, preferably between 200 and 800 mOsmol / Kg, more preferably between 400 - 600 mOsmol / Kg. It is understood that the osmolarity may change between steps of over time e.g., the osmolarity may change from step (a) to step (b). The osmolarity can be measured using common means known by the skilled person and the skilled person can adjust the osmolarity to the desired range, e.g., to between 100 - 1000 mOsmol / Kg, preferably between 200 and 800 mOsmol / Kg, more preferably between 400 - 600 mOsmol / Kg.

[0059]

[0052] The plant protoplasts provided in step (a) may be obtained by methods known in the art and the skilled person is capable of obtaining protoplasts. Generally, protoplasts are obtained by obtaining one or more plant cells, from which the cell wall is removed, for example by enzymatic digestion using cellulase and / or pectinase.

[0060]

[0053] Further processing of the protoplasts, i.e., isolation or removal of enzymes, can be done by any known means in the art, e.g., by filtering or centrifugation. Obtained protoplasts are then ready for various applications, for example in plant tissue culture or genetic engineering.

[0061]

[0054] The provided protoplasts in step (a) are provided in such a way that they can, preferably, be distributed evenly, i.e., distributed homogeneously, (e.g., by mixing) in the aqueous solution of step (a) and therefore are homogeneously distributed in the formed hydrogel of step (b). Advantageously, by providing a homogeneous distribution of protoplasts, said protoplast can efficiently form microcalli and are less prone to fuse together creating hybrids of two or more plant protoplasts. The aqueous solution of step a) has a viscosity such that when the protoplasts are mixed therein, they preferably do not settle, but remain, spatially, in the same position during and after the hydrogel is formed in step b). It is understood that the protoplasts in the aqueous solution will, given enough time (e.g., hours / days), settle to the bottom of the aqueous solution. However, as the time for forming a hydrogel in step b) is e.g., 20-25 minutes, the protoplasts will not settle or become non-homogeneously distributed before the hydrogel is formed in step b).

[0062]

[0055] The protoplasts provided in step (a) are viable and can carry out normal metabolic functions, such as photosynthesis, respiration, uptake or secretion of substances and / or protein synthesis to ensure that a microcallus may be formed. Under certain circumstances, e.g., when cells are genetically engineered using for example the CRISPR gene editing system, cells are viable but may be stressed due to the presence of a double stranded DNA break. Advantageously, the means and methods according to the present invention provide for improved proliferation conditions and thus provide optimal conditions for even stressed cells.

[0063]

[0056] In an embodiment, the provided protoplasts in step (a) are all derived from the same plant species or strain, preferably the same single plant, more preferably the same part of the plant. In an embodiment, the protoplasts are derived from a plant selected from corn, wheat, barley, sunflower, grapes, apples, bananas, oranges, mandarins, avocados, blackberries, blueberries, raspberries, kiwi fruit, lemons, oil palm fruit, rapeseed, sugar cane, cotton, coffee, sweet potatoes, potatoes, cucumber, peppers, lettuce, cauliflower, tomato, soybean, strawberry, zucchini, rice, sugar beet, brassica, squash, pumpkin, N. benthamiana, rose. In a preferred embodiment the protoplasts are derived from a plant selected from tomato, soybean, strawberry, zucchini, rice, sugar beet, brassica, squash, pumpkin, N. benthamiana, rose. In an embodiment protoplasts are derived from a part of the plant selected from the root, stem, leaf, flower, fruit seed, cotyledon, preferably wherein the part is the leaf of the cotyledon.

[0064]

[0057] In an embodiment, the provided protoplasts of step (a) are derived from a plant callus culture, preferably wherein said plant callus culture is from a plant selected from corn, wheat, barley, sunflower, grapes, apples, bananas, oranges, mandarins, avocados, blackberries, blueberries, raspberries, kiwi fruit, lemons, oil palm fruit, rapeseed, sugar cane, cotton, coffee, sweet potatoes, potatoes, cucumber, peppers, lettuce, cauliflower, tomato, soybean, strawberry, zucchini, rice, sugar beet, brassica, squash, pumpkin, N. benthamiana, rose. In a preferred embodiment the protoplasts are derived from a plant selected from tomato, soybean, strawberry, zucchini, rice, sugar beet, brassica, squash, pumpkin, N. benthamiana, rose. Advantageously, obtaining the protoplasts derived from the same plant results in protoplasts that are highly identical, barring any naturally occurring variations in the genetic code.

[0065]

[0058] In an embodiment, the plant protoplasts provided in step (a) are cells that have been contacted or are contacted with a gene editing system, preferably wherein the gene editing system is selected from the group consisting of CRISPR systems, TALENs, meganucleases, transposases, recombinases, and zinc finger nucleases.

[0066]

[0059] In an embodiment, the gene editing system is transiently, i.e., temporarily, active or expressed in a plant cell, protoplast, or progeny thereof. For example, by delivery of CRISPR-Cas9 as a ribonucleoprotein (RNP) complex or as one or more nucleic acid sequences encoding for a functional CRISPR gene editing system. Hence, such a gene editing system is transient because the gene editing system is not integrated into the plant cell’s genome. For example, one or more nucleic acid sequences encoding for a functional CRISPR gene editing system are introduced into a protoplasts of step (a) and said CRISPR gene editing system is no longer active after formation of a microcallus in step (c) due to the decline in the amount of, or degradation of, one or more of the nucleic acid sequences. Advantageously, a transient gene editing system may be beneficial to e.g., alter the gene expression of a particular gene to improve microcallus formation. However, the alteration to the gene expression is no longer needed, or disadvantageous when the microcallus further proliferates into e.g., a microcolony a fully grown plant. Hence, the transient nature of the gene editing system may be highly advantageous.

[0067]

[0060] In an embodiment, the invention provides a method for introducing an molecular payload into a protoplast, the molecular payload comprising a nucleic acid, a protein, or a nucleic-acid-protein complex, non-limiting examples of such molecular payloads are circular plasmids, double or single stranded DNA or RNA, ribonucleoproteins (RNP), one or more parts of a CRISPR system, TALENS, meganucleases and zinc finger nucleases. The molecular payload is delivered using a transformation modality selected from: polyethylene glycol (PEG)-mediated transfection, electroporation, chemical permeabilization agents, lipid- or polymer-based delivery complexes, mechanical or microfluidic membrane-deformation systems, biolistic particle delivery, peptide-based delivery vectors, Agrobacterium-mediated transfer, and extracellular-vesicle-based transfer. Introducing a molecular payload using e.g. PEG can transiently disrupt membrane integrity and impose osmotic stress on protoplasts, which may reduce cell viability. As a result, protoplast regeneration efficiency may be adversely affected if the treatment compromises the cells’ ability to resume normal growth and developmental programs. Advantageously protoplast regeneration in a pectin hydrogel according to the invention can mitigate effects that arise due to introduction of a payload e.g. using PEG. Without being bound by any particular theory, the inventors contemplate that the hydrogel provides a mechanically supportive and osmotically stable microenvironment that helps protoplasts recover from membrane stress and re¬ establish cell wall formation. This stabilized milieu promotes sustained viability and division, enabling efficient regeneration even after introduction of a payload.

[0061] In an embodiment, intact plant cells may have been edited using e.g., the CRISPR gene editing system prior to step (a) according to the invention and the gene editing of said plant cells has been finalized, i.e., the gene editing system is no longer present or is inactive, thus providing gene edited plant cells. From these edited, intact, plant cells, protoplast are provided and used in step (a) according to the invention. The genetic modifications, i.e., edits, to the protoplasts is preferably also present in the progeny of the protoplasts.

[0068]

[0062] In an embodiment protoplast may have been edited using e.g., the CRISPR gene editing system prior to step (a) and the gene editing of said protoplasts has been finalized, i.e., the gene editing system is no longer present or is inactive, thus providing gene edited protoplasts. The genetic modifications, i.e., gene edits, to the protoplasts is preferably also present in the progeny of the protoplasts.

[0069]

[0063] In an embodiment the gene editing system is brought into contact with the protoplasts before step (a) and continues to be active during step (a), (b) and / or (c). For example, the gene editing system is brought into contact with the protoplasts before step (a), e.g., when preparing the protoplasts and is transiently expressed during steps (a), (b) and / or (c).

[0070]

[0064] In another embodiment, the gene editing system is brought into contact with the protoplasts during step (a), e.g., by adding the gene editing system to the aqueous solution. In yet another embodiment, the gene editing system is brought into contact with the protoplasts during step (b) or (c), e.g., by adding the gene editing system to the first culture medium.

[0071]

[0065] Any means known in the art may be used to contact the protoplast with one or more gene editing system, for example by treating said protoplasts with a gene editing system, or, by providing one or more polynucleotides encoding for the one or more gene editing system, e.g., a polynucleotide encoding for a zinc finger nuclease or a CRISPR gene editing system. It is understood that, by contacting the protoplasts with a gene editing system, said gene editing system is capable of editing nucleic acids in a cell such as a protoplasts, thus said gene editing system is capable of entering the cell such as protoplasts. Hence, in an embodiment, the protoplasts have been contacted with, or are contacted with, a nucleic acid sequence encoding a gene editing system thereby expressing said gene editing system in the protoplasts and allowing for genetic modification of the protoplasts, wherein the expression of the gene editing system in the protoplasts is permanent or transient, preferably transient.

[0072]

[0066] In an embodiment, where the protoplasts are contacted with a gene editing system, the protoplasts provided in step (a) all contain the same gene editing (i.e., a single gene modification or a plurality thereof to the same or multiple genes). For example, all protoplasts have been edited to delete genes X and Y and are provided in step (a) according to the method of the example. This may be achieved for example by genetically modifying a protoplast, proliferation said protoplast such that a microcallus is formed, confirming that the cells of the microcallus are obtained from a single (genetically modified) protoplast, i.e. are monoclonal, and obtaining or preparing protoplasts from the (monoclonal) microcallus to use in step (a) according to the invention. It is understood that other means for obtaining a plurality of plant cells or plant protoplasts comprising the same genetic modification may be used. Advantageously, having a plurality of protoplasts with the same edited genes can be used quickly and efficiently provide a plurality of microcalli comprising the desired edited genes. In another embodiment, where the protoplasts are contacted with a gene editing system, the protoplasts provided in step (a) may contain or suspected to contain varying edits to their genes. For example, one protoplast has an edited gene X, whereas another protoplast has an edited gene Y, whereas yet another protoplast has an edited gene Z. Another example is wherein a first protoplast has an edited gene X at a first position, whereas another protoplast has an edited gene X at a second position. Advantageously, having protoplasts with varying edits to their genes allows the screening of protoplasts that form microcalli, and / or microcalli comprising the desired characteristics.

[0073]

[0067] In an embodiment, where the protoplasts are contacted with a gene editing system, culture temperature is set to between 20 - 37 °C, preferably 25 - 37 °C, preferably 25 - 30 °C preferably 28 - 30 °C to allow for efficient use of the gene editing system.

[0074]

[0068] In some embodiments, the aqueous solution of step (a) comprises 1000 - 500.000 protoplasts per milliliter, 2500 - 400.000 protoplasts per milliliter, or, preferably between 5000 and 150.000 protoplasts per milliliter. In an embodiment, the aqueous solution of step (a) comprises 1000 - 2500, protoplasts per milliliter or 1000 - 5000 protoplasts per milliliter 150.000 - 500.000 protoplasts per milliliter, 400.000 - 500.000 protoplasts per milliliter. Depending on the type of plant from which the protoplasts are derived, the protoplasts per milliliter may differ. In embodiments, the protoplasts per milliliter are selected such that at least 1, 2, 3, 4, 5 10, 20, 50, 100 or more plant or plant shoot are regenerated from the plurality or protoplasts that are initially embedded in the hydrogel-forming pectin. The protoplasts per milliliter required to obtain e.g. 1, 2, 3, 4, 5 10, 20, 50, 100 or more plant or plant shoot that are regenerated from the plurality or protoplasts can be determined by performing a comparative test. For example comparing e.g. two conditions of different concentrations of protoplasts, or, different volumes of the hydrogel, and, based on the outcome of the comparison, selecting an appropriate condition, as described elsewhere herein.

[0075]

[0069] In a preferred embodiment, 5000 - 10.000 protoplasts per milliliter are used when proliferating protoplasts from N. benthamiana. 50.000 - 75.000 protoplasts per milliliter are used when proliferating protoplasts from rice. 25.000 - 200.000 protoplasts per milliliter are used when proliferating protoplasts from tomato. 100.000 - 200.000 protoplasts per milliliter are used when proliferating protoplasts from strawberry. 75.000 - 100.000 protoplasts per milliliter are used when proliferating protoplasts from sugar beet. 50.000 - 100.000 protoplasts per milliliter are used when proliferating cotyledon protoplasts from pumpkin and squash. In an embodiment 50.000 - 200.000 protoplasts per milliliter are used when proliferating protoplasts from cabbage. 5.000 - 50.000 protoplasts per millilitre are used when proliferating protoplasts from lettuce.

[0076]

[0070] The plant protoplasts provided in step (a) according to the method are preferably protoplast from a plant selected from the list of tomato, soybean, strawberry, zucchini, rice, sugar beet, brassica genus, squash, pumpkin, N. benthamiana, rose, potato.

[0077]

[0071] The hydrogel-forming pectin provided in step a) may be derived (i.e., extracted, or isolated) from any suitable source, such as citrus fruits (e.g., oranges and lemons), apples, pears, quince, plums, and some types of berries like currants and blackberries, and may, for example be obtained from commercial sources.

[0078]

[0072] In an embodiment, the hydrogel-forming pectin, i.e., pectin, is a High Methoxyl (HM) or a Low Methoxyl (LM) pectin. Advantageously and regardless of whether a HM or LM pectin is used, the inventors realized that a proliferation of protoplasts in a pectin hydrogel, as disclosed herein, results in good formation of one or more microcalli.

[0079]

[0073] In an embodiment, the hydrogel-forming pectin provided in step a) is a HM pectin. In HM pectin, more than 50% of the galacturonic acid units in the pectin chain are esterified with methanol (methyl groups). HM pectin can gel in the presence of a sugar, for example at least 55-75 wt.% sugar. The sugar interacts with the pectin molecules, drawing water out and allowing the pectin chains to form a network, trapping the water, and creating a gel. HM pectin can gel at low pH levels, preferably at a pH below 3.5. Advantageously, the combination of relatively high amounts of sugar and low pH is preferred for proliferation of certain protoplasts. Hence, in an embodiment, although not preferred, the hydrogel-forming pectin is a HM pectin, wherein the hydrogel is formed by providing a pH of below 3.5, preferably a pH of between 2.0 and 3.5 and further providing 55-75 wt.% of a sugar, preferably wherein the sugar is glucose, fructose, sucrose, lactose mannitol, sorbitol maltose or a combination thereof.

[0080]

[0074] In another embodiment, the hydrogel-forming pectin provided in step a) is a LM pectin. In LM pectin, less than 50% of the galacturonic acid units in the pectin chain are esterified with methanol (methyl groups). LM pectin can gel in the presence of divalent cations, such as a divalent cation selected from calcium ions, magnesium ions, iron ions or copper ions or a combination thereof, preferably wherein the divalent cations are calcium ions. LM pectin can gel in the presence of divalent cations without the need for high sugar concentration. LM pectin forms gels through ionic cross-linking facilitated by, for example, calcium ions. The calcium ions form ionic bonds with the carboxyl groups on the pectin molecules, creating cross-links that form a gel. LM pectin can gel at pH levels between 2.0 and 9.0, preferably 2.0 and 6.0, more preferably 4.0 and 6.0. Advantageously, the inventors have found that, when an LM pectin is used as the hydrogel-forming pectin, protoplasts efficiently proliferate to form one or more microcalli. Hence, in an embodiment, the hydrogel-forming pectin is a LM pectin, wherein the hydrogel is formed by providing a pH between 2.0 and 9.0, preferably 2.0 and 6.0, more preferably 4.0 and 6.0 and further providing between 0.1 - 10 g / L, between 1 - 10 g / L, preferably between 5 - 10 g / L, more preferably between 7.5 - 10 g / L divalent cations, preferably calcium ions. In an embodiment, the hydrogel-forming pectin is a LM pectin, wherein the hydrogel is formed by providing a pH between 2.0 and 9.0, preferably 2.0 and 6.0, more preferably 4.0 and 6.0 and further providing preferably between 1 and 250 mM, between 5 and 250, between 50 and 250 mM, between 100 and 250 mM, between 150 and 250 mM divalent cations, preferably calcium ions.

[0081]

[0075] In embodiments, the hydrogel-forming pectin is selected based on the desired gelation mechanism and concentration. In an embodiment a High Methoxyl (HM) pectin is used when gelation is to be induced via Ca2+ -mediated cross-linking (chelation) and low to high sugar content (depending on protoplast type), suitable for low- to moderate-concentration formulations, for example between 0.05 - 1.5 wt.% hydrogel-forming pectin. In an embodiment a Low Methoxyl (LM) pectin is used when higher ionic crosslinking with divalent cations, such as calcium, is preferred, allowing more stable gel formation at similar pectin concentrations. Furthermore, homogeneous hydrogels can be obtained from LM pectin at higher concentrations in comparison to HM pectin, for example 1.5 and 3 wt.% hydrogel-forming pectin, due to a higher solubility. The choice of pectin type can therefore be tailored to optimize hydrogel strength, cell viability, and regeneration efficiency in plant cell culture. In an embodiment, the divalent cations for forming the hydrogel according to the invention is selected from calcium, magnesium, iron, or copper, preferably calcium.

[0082]

[0076] In an embodiment, the hydrogel-forming pectin provided in step (a) is a polygalacturonic acid. It is understood that polygalacturonic acid is a polymer of many galacturonic acid units. Polygalacturonic acid may be derived from any suitable source such as, but not limited to, citrus fruit. In a further preferred embodiment, the hydrogelforming pectin provided in step a) may be a polygalacturonic acid salt, wherein the salt is selected from calcium, potassium, magnesium, aluminum, or sodium. Preferably the polygalacturonic acid salt is a sodium or potassium salt, i.e., polygalacturonic acid sodium salt or polygalacturonic acid potassium salt. The choice of salt depends on the intended application and desired properties, such as solubility, stability, and gelling ability. The skilled person is able to obtain polygalacturonic acid, a polygalacturonic acid salt, or polygalacturonic acid sodium salt e.g., by isolation from for example citrus fruit using methods known in the art, or, by acquiring polygalacturonic acid from a commercial vendor (e.g., Merck P3889 or Merck P3850).

[0083]

[0077] In embodiment, the aqueous solution of step (a) according to the method of the invention comprises at least 0.05 wt.% hydrogel-forming pectin, and preferably at most 3.0 wt.% hydrogel-forming pectin. The weight of the hydrogel-forming pectin in the aqueous solution is measured relative to the aqueous solution without protoplasts. For example, to 99.5 grams of an aqueous solution without the protoplast 0.5 grams of hydrogel-forming pectin is added to obtain an aqueous solution comprising 0.5 wt.% hydrogel-forming pectin.

[0084]

[0078] In an embodiment, the aqueous solution comprises 0.05 wt.%, 0.1 wt.%, 0.2 wt.%, 0.5 wt.%, 1.0 wt.%, 1.5 wt.%, 2.0 wt.%, 2.5 wt.% or 3.0 wt.% hydrogel-forming pectin. In an embodiment, the aqueous solution comprises about 0.05 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.5 wt.%, about 1.0 wt.%, about 1.5 wt.%, about 2.0 wt.%, about 2.5 wt.% or about 3.0 wt.% hydrogel-forming pectin.

[0085]

[0079] For example, an aqueous solution comprising 1 wt.% hydrogel-forming pectin is obtained by adding 1 gram of hydrogel-forming pectin to 99 grams of an aqueous solution, e.g., water. It is understood that as disclosed herein, the aqueous solution may comprise other substances as long as 99 grams of such an aqueous solution is mixed with 1 gram, or about 1 gram, or hydrogel-forming pectin.

[0086]

[0080] In embodiment, hydrogel of step (b) according to the method of the invention comprises at least 0.05 wt.% hydrogel-forming pectin, and preferably at most 3.0 wt.% hydrogel-forming pectin. In an embodiment, the aqueous solution comprises 0.05 wt.%, 0.1 wt.%, 0.2 wt.%, 0.5 wt.%, 1.0 wt.%, 1.5 wt.%, 2.0 wt.%, 2.5 wt.% or 3.0 wt.% hydrogel-forming pectin. In an embodiment, the aqueous solution comprises about 0.05 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.5 wt.%, about 1.0 wt.%, about 1.5 wt.%, about 2.0 wt.%, about 2.5 wt.% or about 3.0 wt.% hydrogel-forming pectin. Hence, when for example an aqueous solution comprising 1 wt.% hydrogel-forming pectin is obtained by adding 1 gram of hydrogel-forming pectin to 99 grams of an aqueous solution, a hydrogel is obtained having 1 wt.% of pectin. It is understood that, when adding further substances, for example 1 gram of calcium to 1 gram hydrogelforming pectin to 99 grams of an aqueous solution reduces the weight percentage of pectin in the formed hydrogel. The skilled person can readily calculate the weight percentage of pectin in the aqueous solution of step a) such that the hydrogel of step b) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin.

[0087]

[0081] In a preferred embodiment, the hydrogel of step (b) according to the invention comprises 0.2 wt.% pectin and plant protoplasts are derived from N. benthamiana, rice, strawberry, tomato, sugar beet, soybean, rose, zucchini, acorn squash, pumpkin, white cabbage.

[0088]

[0082] In embodiment, the aqueous solution of step a) comprises between 0.05 and 3.0 wt.% pectin, between 0.1 and 2.5 wt.% pectin, between 0.1 and 2.0 wt.% pectin, between 0.1 and 1.0 wt.% pectin, between 0.1 and 0.5 wt.% pectin, or between 0.1 and 0.3 wt.% pectin. In embodiment, the aqueous solution comprises of step (a) comprises between about 0.05 and about 3.0 wt.% pectin, between about 0.1 and about 2.5 wt.% pectin, between about 0.1 and about 2.0 wt.% pectin, between about 0.1 and about 1.0 wt.% pectin, between about 0.1 and about 0.5 wt.% pectin, or between about 0.1 and about 0.3 wt.% pectin. The term “about” with respect to the amount of hydrogel-forming pectin refers to a range of plus / minus 10%, e.g., between about 0.1 and about 2.0 wt.% pectin refers to a range of between 0.09 - 2.2 wt.%, or 0.11 to 1.9 wt.%, or 0.09 to 2.2 wt.% or 0.11 to 2.2 wt.%.

[0089]

[0083] In embodiment, the hydrogel of step (b) according to the method of the invention comprises between 0.05 and 3.0 wt.% pectin, between 0.1 and 2.5 wt.% pectin, between 0.1 and 2.0 wt.% pectin, between 0.1 and 1.0 wt.% pectin, between 0.1 and 0.5 wt.% pectin, or between 0.1 and 0.3 wt.% pectin. In embodiment, the aqueous solution comprises of step a) comprises between about 0.05 and about 3.0 wt.% pectin, between about 0.1 and about 2.5 wt.% pectin, between about 0.1 and about 2.0 wt.% pectin, between about 0.1 and about 1.0 wt.% pectin, between about 0.1 and about 0.5 wt.% pectin, or between about 0.1 and about 0.3 wt.% pectin. The skilled person can readily calculate the weight percentage of pectin in the aqueous solution of step a) such that the hydrogel of step b) comprises between 0.05 and 3.0 wt.% pectin.

[0090]

[0084] In an embodiment, the aqueous solution comprising plant protoplasts and a hydrogel-forming pectin does not comprise any nurse cells of the same or a different species of the plant protoplasts that are comprised in said hydrogel-forming pectin. Adding nursing cells to e.g. the aqueous solution comprising plant protoplasts and a hydrogel-forming pectin or to the first culture medium adds complexity to the method, the more rapid depletion of nutrients from the regeneration medium and may increase the chance of microbial contamination. Adding nursing cells further requires the time¬ consuming step of creation of a cell culture that can be used as nursing cells. Advantageously, the method in accordance with the invention allows for the generation of microcalli from protoplasts without nursing cells, thereby avoiding unnecessary complexity or potential contaminations.

[0091]

[0085] Step (b) - formation of a hydrogel comprising plant protoplasts.

[0092]

[0086] In step (b) according to the invention, the pectin is allowed to form a hydrogel comprising the plant protoplasts, preferably by contacting the aqueous solution of step (a) with calcium. Hence, in step (b) according to the invention, protoplasts are embedded in the hydrogel. Preferably the embedded protoplasts are distributed evenly, i.e., homogenously, in the hydrogel. Having an even distribution allows for optimal growth of microcalli, exchange of substances such as nutrients or cellular signaling molecules. It further prevents fusion of protoplasts or microcalli and improves isolation of individual protoplasts or microcalli if desired.

[0093]

[0087] The skilled person understand how to allow the hydrogel-forming gel to form the hydrogel in the presence of, for example, calcium (ions). The amount of calcium required for gelation is determined by the degree of esterification. The amount of calcium further depends on the size, and distribution of non-methyl esterified galacturonic acid, as well as the process parameters. In an embodiment, the amount of calcium ions to contact the aqueous solution is between 1 - 10 g / L, preferably between 5 - 10 g / L, more preferably between 7.5 - 10 g / L. In an embodiment, the calcium to contact the aqueous solution such that a gel is formed is between 10 - 100 mM, preferably between 20 - 80 mM, more preferably 30 - 60 mM.

[0094]

[0088] In an embodiment, the aqueous solution comprising the hydrogel forming pectin is contacted with calcium by adding a solution comprising a suitable concentration of calcium, e.g., by mixing the aqueous solution of step (a) with a solution comprising calcium in a suitable container.

[0095]

[0089] In an embodiment, the formation of the hydrogel in step (b) occurs at a temperature of between 20 - 37 °C, preferably 25 - 37 °C, preferably 25 - 30 °C preferably 28 - 30 °C. In an embodiment the hydrogels is contacted with the first culture medium, and the culture temperature is at least about 25 °C, such as about 26 °C, about 27 °C about 28 °C about 29 °C, about 30 °C. In an embodiment, after formation of the hydrogel, the temperature is kept at a temperature of between 20 - 37 °C, preferably 25 - 37 °C, preferably 25 - 30 °C preferably 28 - 30 °C. In an embodiment the hydrogels is contacted with the first culture medium, and the culture temperature is at least about 25 °C, such as about 26 °C, about 27 °C about 28 °C about 29 °C, about 30 °C

[0096]

[0090] In a preferred embodiment, the aqueous solution comprising the hydrogel forming pectin is contacted with calcium by transfer of calcium comprised in a solid medium or hydrogel to the aqueous solution. For example, the aqueous solution of step (a) is pipetted in ‘droplets’ of, for example, 250 microliter on a solid medium comprising calcium (as exemplified in Figure 2c). The calcium ions will transfer from the solid medium to the aqueous solution thereby forming a hydrogel. In an embodiment, the solid medium comprising the calcium is an agar medium, for example a purified agar such as micro agar. The inventors advantageously found that by allowing the pectin hydrogel to form in this way, i.e. on a solid medium comprising calcium, it is particular suitable for use in the invention because it allows for obtaining a hydrogel of defined volume and size, e.g. 250 mL and because it was found that culturing, i.e. proliferating protoplasts, in a gel formed that way provide good, improved, results.

[0097]

[0091] In an embodiment, the hydrogel of step (b) has a volume of 1 - 1000 microliter, 1 - 500 microliter, 10 - 1000 microliter, 10 - 500 microliter, 10 - 250 microliter, 50 -250 microliter, or 150 - 250 microliter. It is understood that the volume of the aqueous solution of step (a) is substantially the same as the volume of the formed hydrogel of step (b). For example, when pipetting 250 microliter of the aqueous suspension of step (a), a gel is formed having a volume of substantially 250 microliter. Over time, the volume of the formed gel may change, e.g., increase due to swelling when water is absorbed, or decrease due to evaporation of water.

[0098]

[0092] In an embodiment, the hydrogel has a thickness of at most 10 millimeters or at most 5 millimeters. The formed hydrogel can have varying shapes; thus, the thickness of the hydrogel is dependent on the shape thereof and / or determined by the surface or vesicle it is formed in or on. For example, when the aqueous solution of step (a) is pipetted in a well of a e.g., a 24-well plate, the shape of the formed hydrogel will be ‘disc’ shaped. In a more preferred embodiment, the aqueous solution of step (a) is pipetted on a surface as a droplet (e.g., as shown in Figure 2c). Thus, the shape of the formed hydrogel is that of a ‘droplet’ on a surface. The thickness of the hydrogel is measured as the distance from the surface the formed hydrogel is formed or placed on to the tallest point of the hydrogel, i.e., ‘droplet’.

[0093] In an embodiment the method according to the invention comprises the following features, i.e., wherein:

[0099] - the aqueous solution of step (a) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin and / or wherein the hydrogel of step (b) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin,

[0100] - the aqueous solution of step (a) comprises 1000 - 500.000 protoplasts per milliliter, 2500 - 400.000 protoplasts per milliliter, or, preferably between 5000 and 150.000 protoplasts per milliliter.

[0101] - the hydrogel of step (b) has a volume of 1 - 1000 microliter, 1 - 500 microliter, 10 - 1000 microliter, 10 - 500 microliter, 10 - 250 microliter, 50 - 250 microliter, or 150 - 250 microliter, and

[0102] - a ratio (vol / vol) between the hydrogel and the first culture medium of step (c) is about 1:1 - 1:25, preferably about 1:2.5 - 1:20, more preferably about 1:2.5 - 1:10.

[0103]

[0094] For example, in one embodiment, the aqueous solution of step (a) comprises 0.05 - 1 wt.% pectin and between 5000 - 50.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 10 - 100 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is about 1: 1.

[0104]

[0095] For example, in one embodiment, the aqueous solution of step (a) comprises 0.05 - 0.5 wt.% pectin and between 5000 - 50.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 10 - 100 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is about 1: 1

[0105]

[0096] For example, in one embodiment, the aqueous solution of step (a) comprises 0.5 - 1 wt.% pectin and between 5000 - 50.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 10 - 100 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is about 1: 1

[0106]

[0097] For example, in one embodiment, the aqueous solution of step (a) comprises 1 - 2 wt.% pectin and between 50.000 - 150.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 100 - 250 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is about 1: 1.

[0107]

[0098] For example, in one embodiment, the aqueous solution of step (a) comprises 2 - 3 wt.% pectin and between 50.000 - 150.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 100 - 250 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is about 1: 1.

[0108]

[0099] For example, in one embodiment, the aqueous solution of step (a) comprises 0.5 - 1.5 wt.% pectin and between 5000 - 150.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 100 - 250 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is about 1: 1.

[0109]

[0100] For example, in one embodiment, the aqueous solution of step (a) comprises 0.2 wt.% pectin and between 5000 - 10000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 200 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is 1: 3.

[0110]

[0101] For example, in one embodiment, the aqueous solution of step (a) comprises 0.2 wt.% pectin and between 50000 - 100.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 200 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is 1: 5.

[0111]

[0102] For example, in one embodiment, the aqueous solution of step (a) comprises 0.2 wt.% pectin and between 50000 - 100.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 200 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is 1: 2.5.

[0112]

[0103] For example, in one embodiment, the aqueous solution of step (a) comprises 0.2 wt.% pectin and between 50000 - 300.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 200 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is 1: 2.5.

[0113]

[0104] For example, in one embodiment, the aqueous solution of step (a) comprises 0.2 wt.% pectin and between 50000 - 300.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 200 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is 1: 5.

[0114]

[0105] For example, in one embodiment, the aqueous solution of step (a) comprises 0.2 wt.% pectin and between 50000 - 300.000 protoplasts per milliliter, the hydrogel of step (b) has a volume of 100 microliter, and wherein the ratio (vol / vol) between hydrogel and the first culture medium of step (c) is 1: 5.

[0115]

[0106] Step (c) - proliferation of plant protoplasts into microcalli

[0107] In step (c) according to the invention, the hydrogel, i.e., pectin hydrogel comprising the plant protoplasts is contacted with a first culture medium and allowing the plant protoplasts to proliferate and to form one or more microcalli. By contacting the hydrogel comprising the protoplasts with a first culture medium, components, and substances of said first culture medium can enter, i.e., exchange with, the hydrogel and the protoplasts, allowing the proliferation of the protoplasts. Vice versa, factors or substances secreted by the protoplasts can be exit the hydrogel and are diluted in the culture medium either to re-enter the hydrogel or be removed e.g., when the first culture medium is removed and / or replenished. Factors or substances secreted by one protoplast may be absorbed, i.e., taken up, by another protoplast, which can improve proliferation of protoplasts to form microcalli in the hydrogel.

[0116]

[0108] In an embodiment, the microcalli formed in step (c) according to the invention are preferably monoclonal microcalli. Monoclonal microcalli refer to a population of cells that originate from a single protoplast and are thus all genetically identical. Non-monoclonal microcalli are for example microcalli that are obtained e.g., due to fusion of protoplasts or clumping of two or more microcalli. Monoclonal microcalli can be obtained by using an appropriate density of protoplasts per milliliter and having an even distribution of protoplasts in the hydrogel, thereby preventing protoplasts from fusing and / or two or more microcalli from clumping together. Without being bound by any particular theory the inventors contemplate that obtaining clonal microcalli using the method according is improved in view of other means and methods for obtaining microcalli. That is despite a relatively low concentrations of protoplasts, said protoplasts can efficiently proliferated into microcalli and thus are prevented from fusing with another microcallus.

[0117]

[0109] In an embodiment, the hydrogel is contacted by the first culture medium, by submerging the hydrogel in the first culture medium. For example, the hydrogel is placed in a vesicle and the appropriate volume of first culture medium is added, such that the hydrogel is below the liquid surface of the first culture medium. Advantageously, this allows the largest surface area of the hydrogel to be in contact with the first culture medium and therefore an optimal exchange of e.g. nutrients from the first culture medium into the hydrogel This is especially advantageous in view of preferred volumes of the hydrogel according to the invention, preferably wherein said hydrogels have a volume of 1 - 1000 microliter, 1 - 500 microliter, 10 - 1000 microliter, 10 - 500 microliter, 10 - 250 microliter, 50 - 250 microliter, or 150 - 250 microliter, and. In another embodiment, the hydrogel is contacted by the first culture medium, such that the hydrogel is not fully submerged. For example, the hydrogel is placed in a vesicle and the appropriate volume of first culture medium is added, such that the hydrogel is above the liquid surface of the first culture medium. Advantageously, this allows the transfer of e.g., nutrients from the first culture medium to enter the hydrogel, whilst also allowing efficient transfer of gasses directly between the hydrogel and the part of the hydrogel that is above the surface of the hydrogel.

[0118]

[0110] In step (c) the (in step (b)) formed hydrogel comprising protoplasts is preferably transferred to a suitable vesicle such that it can be contacted with the first culture medium. In an embodiment, the formed hydrogel is placed in a well of e.g., a 24-well plate and the first culture medium is added to the well (e.g., as exemplified in Figure 2d). In an embodiment, two or more of the formed hydrogels are placed in a vesicle e.g., well of a 24, 48, or 96 well plate.

[0119]

[0111] The first culture medium is a medium comprising a mixture of substances that allow the proliferation of a protoplast into a microcalli. In a preferred embodiment, the first regeneration medium comprises a base salt mix with one or more vitamins, one or more carbon source, one or more osmotic stabilizer, and one or more (phyto)hormone.

[0120]

[0112] In an embodiment, the base salt mixture is a known mixture such as Kao & Michayluk basal salt mixture, Murashige, and Skoog medium (i.e., MSO), Gamborg basal medium. Such base salt mixtures are known in the art and result in efficient proliferation of protoplasts.

[0121]

[0113] In an embodiment, the one or more carbon source in the culture medium is selected from glucose, sucrose, fructose, galactose, or a combination thereof. Preferably the carbon source is glucose and / or sucrose. Generally, in the context of protoplast proliferation and microcalli growth in accordance with the invention, the carbon source is able to provide the necessary carbon atoms that plants use to build organic molecules such as carbohydrates, proteins, and lipids.

[0122]

[0114] In an embodiment, the one or more osmotic stabilizer in the culture medium is selected from mannitol, sorbitol, sucrose, glycerol, polyethylene glycol (PEG) or a combination thereof, preferably wherein the osmotic stabilizer is mannitol. Preferably, the medium osmolarity of the first culture medium is such that final medium osmolarity is between 100 - 1000 mOsmol / Kg, preferably between 200 - 800 mOsmol / Kg, more preferably between 400 - 600 mOsmol / Kg.

[0123]

[0115] In an embodiment, the one or more (phyto)hormone in the culture medium is selected as auxins, cytokines, gibberellins, brassinosteroids or a combination thereof. Preferably wherein the phytohormones are selected from benzyl aminopurine (BAP) or Naphthaleneacetic acid (NAA). Preferably BAP is provided in the first culture medium at a concentration of between 0.1 - 10 mg / L, more preferably between 0.1 and 5 mg / L, even more preferably between 0.1 and 2.5 mg / L, most preferably between 0.1 and 1.5 mg / L. Preferably NAA is provided in the first culture medium at a concentration of between 0.01 - 2 mg / L, more preferably between 0.05 and 2 mg / L, even more preferably between 0.05 and 1 mg / L, most preferably between 0.05 and 0.5 mg / L. Preferably, BAP and NAA are provided both in the first culture medium.

[0124]

[0116] In an embodiment, the first culture medium comprises a mixture of glucose, mannitol, to a final medium osmolarity of between 400 - 600 mOsmol / Kg, further comprising 1 mg / L NAA, and 0.2 mg / L BAP and a suitable base salt medium. The inventors have found that, this first culture medium resulted in efficient proliferation of plant protoplasts.

[0125]

[0117] In an embodiment, the pH of the first culture medium is between 4 - 7, preferably between 5 - 6, more preferably between 5.5 - 6. In an embodiment, the pH of the first culture medium is between about 4 - 7, preferably between about 5 - 6, more preferably between about 5.5 - 6.

[0126]

[0118] In an embodiment, the ratio (vol / vol) between the hydrogel and the first culture medium of step (c) is 1:1 - 1:25, preferably 1:2.5 - 1:20. In an embodiment, the ratio (vol / vol) between the hydrogel and the first culture medium of step (c) is about 1:1 - 1:25, preferably about 1:2.5 - 1:20. For example, when the hydrogel has a volume of 250 ml, the amount of first culture medium that is added to said hydrogel is also 250 mL, i.e. the ratio (vol / vol) of hydrogel to first culture medium is 1:1. In an embodiment, where two or more of the formed hydrogels are placed in a well (e.g. a well of a 24-well plate), the volume of culture medium is equal to the total volume of hydrogels in said well. For example, when a well contains three hydrogels each of 250 mL, 750 mL of the first culture medium is added to said well such that the ratio (vol / vol) of each hydrogel to first culture medium is 1:1. Without being bound by any particular theory, the inventors contemplate that these volumes work well in the context of the invention and that by providing such ratios (vol / vol) improved exchange of substances such as nutrients or cellular signaling molecules

[0127]

[0119] In an embodiment, the hydrogel comprising the protoplasts is contacted with the first culture medium and the protoplast are cultivated. After a (predetermined) time, said first culture medium is exchanged with a different (i.e., new, or fresh) first culture medium. For example, the first culture medium is removed (e.g., by vacuum suction) and new first culture medium is added to the hydrogel. Preferably the volume of the culture medium before and after the exchange is the same and / or with the ratio’s as disclosed herein. In an embodiment, the exchange of the first culture medium, with a different first culture medium has an interval of 4, 5, 6, 7, 8, 9 or 10 days, preferably the interval is 7 days.

[0128]

[0120] In an embodiment, the first culture medium is exchanged with a different first culture medium, i.e., wherein the different first culture medium has a different composition compared to the first culture medium. For example, the first culture medium is optimized to proliferate protoplasts during the initial stages of microcalli growth, and the different first culture medium is optimized later stages of microcalli growth.

[0129]

[0121] In an embodiment, the contacting of the hydrogel with the first culture medium is for a period of at least 3 days. In an embodiment, the contacting of the hydrogel with the first culture medium is for a period of no more than 100 days, 75 days, 50 days, or 30 days. In an embodiment, the contacting of the hydrogel with the first culture medium is for a period of between 3 - 100 days, 3 - 75 days, 3 - 50 days, or 3 - 30 days. In an embodiment, the contacting of the hydrogel with the first culture medium is for a period of between 3 - 100 days, 3 - 75 days, 3 - 50 days, or 3 - 30 days and the first culture medium is exchanged with an interval of 7 days.

[0130]

[0122] In an embodiment, the contacting of the hydrogel with the first culture medium is in the dark. In a preferred embodiment, the contacting of the hydrogel with the first culture medium is in the dark for a period of at least 3 days, and / or, for a period of no more than 100 days, 75 days, 50 days, or 30 days. In an embodiment, the contacting of the hydrogel with the first culture medium is in the dark for a period of between 3 -100 days, 3 - 75 days, 3 - 50 days, or 3 - 30 days. In an embodiment, the contacting of the hydrogel with the first culture medium is for a period of between 3 - 100 days, 3 - 75 days, 3 - 50 days, or 3 - 30 days and the first culture medium is exchanged with an interval of 7 days. Contacting the hydrogel with the first culture medium in the dark is beneficial as light may cause stress in the protoplasts or microcalli, causing differentiation instead of the desired proliferation. It is further preferred to use dark conditions as this causes the protoplasts to put most of their energy into proliferation.

[0131]

[0123] In an embodiment, the hydrogel is contacted with the first culture medium wherein the hydrogel is then cultured at temperature is set to between 20 - 37 °C, preferably 25 - 37 °C, preferably 25 - 30 °C preferably 28 - 30 °C. In an embodiment the hydrogels is contacted with the first culture medium, and the culture temperature is at least about 25 °C, such as about 26 °C, about 27 °C about 28 °C about 29 °C, about 30 °C. In an embodiment, the hydrogel and first culture medium have the same temperature at the moment the first culture medium is brought into contact with the hydrogel.

[0132]

[0124] In an embodiment, the one or more microcalli of formed at the end of step (c) are 0.2 - 1.5 mm, 0.3 - 1.2 mm, or 0.5 - 1.0 mm in diameter. In an embodiment, the one or more microcalli formed at the end of step (c) are about 0.2 - 1.5 mm, 0.3 - 1.2 mm, or 0.5 - 1.0 mm in diameter. Hence, in an embodiment, the formed microcalli are 0.2 - 1.5 mm, 0.3 - 1.2 mm, or 0.5 - 1.0 mm in diameter, or about 0.2 - 1.5 mm, 0.3 - 1.2 mm, or 0.5 - 1.0 mm in diameter. Microcalli are not perfectly spherical objects and typically have an irregular shape, hence, not all cross sections of a microcalli are of the same length. The size of a microcalli is defined as the longest distance between any two points along the boundary of the shape.

[0133]

[0125] ] Following the steps (a) - (c) as described above is highly advantageous as it allows useful for improving plant protoplast proliferation, generating at least one microcallus, preferably at least one clonal microcallus, generating at least one plant cell line, preferably at least one clonal cell line, regenerating at least one plant cell, improving plant cell regeneration efficiency, and / or regenerating of at least one plant, preferably at least one clonal plant.

[0134]

[0126] Other steps of the method according to the invention

[0135]

[0127] In an embodiment, hydrogel comprising the formed microcalli in step (c) are transferred to a culture plate comprising solidified culture medium to allow the microcalli obtained in step c) to further proliferate, e.g., to form microcolonies (as exemplified in Figure 2e).

[0128] In an embodiment, the transfer of the microcalli in step (c) to the culture plate occurs when the microcalli are between 0.2 - 1.5 mm, 0.3 - 1.2 mm, preferably between 0.5 - 1.0 mm in diameter.

[0136]

[0129] In an embodiment, the hydrogel or part of the hydrogel comprising a plurality of microcalli is transferred to the culture plate. It is preferred that clonal microcalli are grown. Hence, in an embodiment, an individual and isolated microcallus from the hydrogel is transferred to the culture plate. For example, the hydrogel according to the invention may comprise a plurality of clonal microcalli each grown from an individual protoplast. To avoid microcalli from fusing thereby forming a fused, non-monoclonal, microcallus, the hydrogel is split such that individual microcalli can be grown on the culture plate such that they may grow into e.g., monoclonal microcolonies. Microcalli may be split by manually cutting / isolating each microcalli from a hydrogel comprising a plurality of said microcalli. Another way of isolating microcalli, thereby preventing one or more microcalli from fusing, is to smear, i.e., spread, a dissolved hydrogel comprising the microcalli over a sufficiently large surface such that the microcalli can proliferate further. Microcalli may also be split by means of microfluidics or (automated) cell sorting, e.g., by dissolving the hydrogel comprising the microcalli, separate / isolate the microcalli using the e.g., microfluidics or cell sorting means and then allowing for further proliferation of the isolated microcalli. Preferably, transfer to the culture plate occurs when the microcalli have a size of 0.2 - 1.5 mm, 0.3 - 1.2 mm, preferably between 0.5 - 1.0 mm in diameter was observed to result in effective proliferation of microcalli and formation of microcolonies.

[0137]

[0130] In an embodiment, the culture plate comprising solidified culture medium comprises the same components as the first culture medium described herein i.e., comprises base salt mix with one or more vitamins, one or more carbon source, one or more osmotic stabilizer, and one or more (phyto)hormone. The solidified culture medium further comprises a substance that enables the culture medium to solidify, e.g., agar, pectin phytoagar, microagar, gelrite (e.g., as obtained from Duchefa Biochemie; G1101), or low melting temperature agarose. It is understood that the solidified culture medium is such that it allows transfer, i.e., exchange, of for example nutrients from the solidified culture medium to the hydrogel comprising the protoplasts and / or microcalli.

[0131] In another embodiment, the culture plate comprising solidified culture medium comprises the different components to the first culture medium. For example, different or additional hormones could be present in the solidified culture medium as microcalli might require different stimuli for further growth on the solid plate. Also, osmotic stabilizers may be omitted from the solidified culture medium, and the carbon source may further be different, e.g., replacing glucose with sucrose is. The skilled person understands that protoplasts have different nutrient requirements than microcalli and subsequent development stages and can adapt the nutrient requirements accordingly.

[0138]

[0132] In an embodiment, after transfer of the hydrogel, part of the hydrogel, or an individual microcallus in the hydrogel to the culture plate comprising solidified culture medium, the hydrogel comprising protoplasts, microcalli and / or microcolonies are incubated for a period of at least 3 days and preferably for a period of no more than 100 days, 75 days, 50 days, or 30 days. For example, for a period of between 3 - 100 days, 3 - 75 days, 3 - 50 days, or 3 - 30 days.

[0139]

[0133] In a further embodiment, after transfer of the hydrogel to the culture plate comprising solidified culture medium, the hydrogel comprising protoplasts, microcalli and / or microcolonies are incubated at a temperature set to between 20 - 37 °C, preferably 25 - 37 °C, preferably 25 - 30 °C preferably 28 - 30 °C. In an embodiment the hydrogels is contacted with the first culture medium, and the culture temperature is at least about 25 °C, such as about 26 °C, about 27 °C about 28 °C about 29 °C, about 30 °C.

[0140]

[0134] In an embodiment, the one or more microcalli are further cultivated, preferably wherein the one or more microcalli are isolated from the gel and further cultivated. For example, the microcalli can be further cultivated to microcolonies and subsequently regenerated into full plants.

[0141]

[0135] To allow for further cultivation and regeneration one or more different culture media may be used and wherein one or more of the different culture media are in a solidified form. For example, for proliferation of protoplasts to microcalli a first culture medium is used, then to form microcolonies from microcalli a second culture medium is used comprising substances optimized for e.g., differentiations of cells and / or organogenesis, and a third, fourth, fifth or further culture medium is used during even later stages of cultivation.

[0136] In an embodiment, at least part of a microcallus is analyzed, for example for the presence of a genetic modification or for establishing the microcallus is a clonal microcallus. The presence of a genetic modification is determined using known methods in the art, for example using sanger sequencing, whole genome sequencing, site-directed PCR amplification, RNA sequencing or the like. Determining if a microcallus is a clonal microcallus is done by determining if all cells in said microcallus are genetically identical, i.e., originating from a single protoplast. This can be done by analyzing two or more cells from a microcallus and determining whether they originate from a single protoplast or not.

[0142]

[0137] In an embodiment the analysis of the microcalli comprises analysis of ploidy of the one or more cells of the microcalli, e.g., determining if the cells are haploid, diploid, or polypoid. In an embodiment, the analysis of the microcalli comprises analysis of shoot potential, i.e., determining the ability of the microcalli tissue to form (new) shoots. Determining shoot potential is performed e.g., by genetic analysis, analysis for the presence and / or absence of certain hormones or a combination thereof. In an embodiment, a proteomics analysis is performed, e.g., by western blotting or mass spectrometry. In an embodiment, fluorescent microscopy is used as an analysis, e.g., to determine the presence or absence of specific proteins. In an embodiment, flow cytometry is used as an analysis, e.g., to separate microcalli, or categorize them according to shape or size.

[0143]

[0138] In an embodiment, the method according to the invention further comprises:

[0144] Comparing at least two conditions with different concentrations of pectin. This allows for determining an optimal concentration of pectin, e.g., for growth of a protoplast obtained from a specific plant variety or species. Protoplasts from different plant varieties or species, or protoplasts from the same plant variety or species but prepared somewhat differently may grow more efficiently on hydrogels with different pectin concentrations. For example, three hydrogels comprising 0.5 wt.% 0.75 wt.% and 1.0 wt.% pectin are prepared each comprising the same number of protoplasts and other substances. Protoplasts are then cultivated in accordance with the method of the invention and microcalli growth is analyzed after e.g., 30 days. Comparing at least two conditions with different concentrations of protoplasts. Protoplasts may grow better when in close physical proximity to other protoplasts e.g., due to exchange of factors between protoplasts. Hence, the concentration of protoplasts may affect the effective cultivation of said protoplasts to microcalli. For example. Three identical hydrogels but each comprising 10.000, 25.000 or 50.000 cells per milliliter are prepared. Protoplasts are then cultivated in accordance with the method of the invention and microcalli growth is analyzed after e.g., 30 days. Comparing at least two conditions with different volumes of the hydrogel. Depending on the volume, i.e., the size, of the hydrogel, protoplasts grow more or less efficiently. For example, in a relatively small volume protoplasts in the center of the hydrogel can more efficiently exchange substances with the surrounding environment, such as with the first culture medium. Further, a small volume has a relatively large surface area compared to the surface area of a larger volume of the same shape. Thus, exchange of compounds and or other substances with the surrounding environment, such as the first culture medium are more efficiently exchanged with the hydrogel and / or protoplasts. For example, the comparison comprises three identical hydrogels which are prepared having a volume of 100, 250 or 500 microliter. Protoplasts are then cultivated in accordance with the method of the invention and microcalli growth is analyzed after e.g., 30 days.

[0145] Comparing at least two conditions with different ratio (vol / vol) between the hydrogel and the first culture medium of step (c). Proliferation of protoplasts may depend on the availability of nutrients in the surrounding environment, or secretion of compounds from the cell into the surrounding environment. By comparing different ratios (vol / vol) between hydrogel and the first culture medium, optimal conditions can be determined for protoplast proliferation. For example, a ratio of 1:1, 1:1.5 and 1:2 hydrogel to first culture medium is used. Protoplasts are then cultivated in accordance with the method of the invention and microcalli growth is analyzed after e.g., 30 days. based on the outcome of one or more of the comparisons, a condition is selected that allows for generating a clonal plant microcalli, and optionally repeating the method according to any one of the previous claims with the selected condition.

[0146]

[0139] In an embodiment, one comparison is performed and subsequently one or more different comparisons are performed to determine optimal conditions.

[0147]

[0140] Products according to the invention

[0148]

[0141] In a further aspect of the invention provided is for a pectin hydrogel as defined herein comprising one or more plant protoplast, preferably wherein the gel comprises one or more clonal plant protoplasts.

[0149]

[0142] In a further aspect of the invention provided is for a pectin hydrogel as defined herein comprising one or more microcalli, preferably wherein the gel comprises one or more clonal microcalli.

[0150] EXAMPLES

[0151]

[0143] Materials and methods

[0152]

[0144] Protoplast isolation

[0153]

[0145] Protoplasts were obtained from different plant materials (e.g., sterile plant tissues, callus, cell suspensions) by enzymatic digestion of tissues within a buffer with specific osmolarity and a certain configuration of cell wall degrading enzymes. Protoplasts used for the regeneration method described were obtained following the CPW-based method described in Frearson et al. (1973) with the following modifications.

[0154]

[0146] The CPW buffer was supplemented with mannitol (Duchefa, NL; M0803) in the range from 80 to 130 g / L. The amount of mannitol and the combination and concentration of cell wall degrading enzymes to use was determined empirically as it varies among distinct species and cell types. Cell wall degrading enzymes include, but are not limited to, Cellulase Onozuka R10 (Duchefa, NL; C8001), Cellulase Onozuka RS (Duchefa, NL; C8003), hemicellulase (Sigma-Aldrich, NL; H2125), Macerozyme Onozuka R10 (Duchefa, NL; M8002), and Viscozyme (Sigma-Aldrich, NL; V2010). After 16 hours, protoplasts were isolated and washed to eliminate residual cell debris. Isolated protoplasts were resuspended in Ma-buffer (80-130 g / L mannitol, 1 g / L MES (Duchefa, NL; M1503) dissolved in H2O, set pH to 5.8), and were assessed on viability before continuing with the embedding.

[0155]

[0147] Preparation of the pectin suspension

[0156]

[0148] The gellable pectin, i.e., hydrogel-forming pectin, solution was prepared by dissolving 0.05 - 3 wt. % of pectin powder (polygalacturonic acid sodium salt, Merck; P3850) in a H2O solution containing 72.88 g / L D-Mannitol (Duchefa, NL; M0803). To prepare 1 wt.% gellable pectin solution, 7.29 gram of mannitol was added slowly to 90 ml of milliq H2O under constant and vigorous stirring until completely solubilized. The pH of the mannitol solution was adjusted to 5.6 and the total volume of the solution was brought to 100 mL. The mannitol solution was transferred to a 2-neck round bottom flask and placed in a thermostatic bath with a vapor condenser and on a magnetic stirring plate set to 80 °C. 1 gram of pectin was added (slowly) to the heated mannitol solution under sustained stirring until homogeneous. The pectin suspension (i.e., mannitol solution comprising pectin) was cooled down under stirring to about 40 °C and transferred to a graduate cylinder. The volume was adjusted to 100 mL cooled down to room temperature and filtered under sterile conditions with a 0.2 um bottle top filter. The pectin suspension was diluted to any desired concentration (e.g., 0.2 wt.%) using sterile 0.4 M mannitol solution. The pectin suspension may be stored at room temperature.

[0157]

[0149] Protoplast embedding

[0158]

[0150] The embedding mixture comprises of protoplasts resuspended in the pectin solution containing the desired amount of pectin polymer (e.g., 0.2 wt. % of pectin, see preparation of the pectin solution). Resuspension buffer present in the solution comprising protoplasts was removed by centrifugation for 5 minutes at 85 xg at room temperature. For most regeneration applications, protoplasts were resuspended in the pectin solution to a final cell density between 1x103and 5x105cell-mL’1. Pectin discs were cast by pipetting 50-200 uL of the embedding mixture on solid medium rich in calcium (72.5 g / L D-Mannitol (Duchefa, NL; M0803), 7.35 g / L CaCl2'2H2O (Carl Roth, USA; HN04.1), 8 g / L Microagar (Duchefa, NL; M1002.1000)). The formation of a stable (pectin) gel is due to the transfer of the calcium ions from the solid medium to the embedding mixture and was achieved after 30 minutes of incubation. Pectin disks containing the immobilized protoplasts were transferred to suitable microtiter plates containing regeneration medium. The format of the microtiter plate (i.e., 24, 48, or 96) and the amount of regeneration medium depends on the volume of disks. For example, 200 pl discs are transferred to 24-well plates containing 500pl - 1000 pl regeneration medium, 100 pl discs to 48-well plates containing 500 pl of medium, whereas 96-well plates and 100 ul medium are used to regenerate 50 pl discs.

[0159]

[0151] Protoplast regeneration

[0160]

[0152] Protoplasts were centrifuged, and resuspended in 2% alginate (72.88 g / L D-Mannitol (Duchefa, NL; M0803), 20 g / L Alginic acid sodium salt (Sigma-Aldrich, USA; A0682)), or 0.2 wt. % of pectin (polygalacturonic acid sodium salt, Merck; P3850) and cast onto CA-agar (72.5 g / L D-Mannitol (Duchefa, NL; M0803), 7.35 g / L CaCl2'2H2O (Carl Roth, USA; HN04.1), 8 g / L Microagar (Duchefa, NL; M1002.1000)) to polymerize. The alginate disks were transferred to K8P without nursing cells or auxin and grown under light 24°C / 20°C (16 / 8 h photoperiod). After 14 more days, microcalli were isolated by excising individual microcalli from the hydrogel and cultured on solid fullstrength Murashige and Skoog including vitamins medium (Duchefa, NL; M0222), supplemented with 30 g / L sucrose (Duchefa, NL; S0809.1000), 8 g / L microagar (Duchefa, NL; M1002.1000) and 1 mg / L TDZ (Duchefa, NL; T0916) at pH 5.8 and under light at 24°C / 20°C (16 / 8 h photoperiod). The culture medium was refreshed every 14 days until shoot primordia began developing on the callus material, at which point the microcalli were transferred to solid full-strength Murashige and Skoog including vitamins medium (Duchefa, NL; M0222), supplemented with 30 g / L sucrose (Duchefa, NL; S0809.1000), 8 g / L microagar (Duchefa, NL; M1002.1000) and 1 mg / L 2-iP (Duchefa, NL; D0906) at pH 5.8. Once the shoots were elongated, the calli were transferred to solid full-strength Murashige and Skoog, including vitamins medium (Duchefa, NL; M0222), supplemented with 30 g / L sucrose (Duchefa, NL; S0809.1000), 8 g / L microagar (Duchefa, NL; M1002.1000) free of plant growth regulators. After one subculture, shoots were isolated. Isolated shoots were transferred to solid full-strength Murashige and Skoog including vitamins medium (Duchefa, NL; M0222), supplemented with 30 g / L sucrose (Duchefa, NL; S0809.1000), 8 g / L microagar (Duchefa, NL; M1002.1000) and 0.1 mg / L IBA (Duchefa, NL; I0902) at pH 5.8. Rooted shoots were deflasked and transferred to a climate-controlled greenhouse where the plants were grown at 22°C / 21°C for seed production.

[0161]

[0153] Regeneration medium and regeneration conditions

[0154] Regeneration medium comprises a base salt mix with vitamins, a carbon source (usually glucose or sucrose), an additional substance to adjust osmolarity (usually mannitol), and (phyto)hormones. Different base media were successfully used for the regeneration of protoplasts embedded with this method from distinct species and tissues, including Kao & Michayluk 8P (K8P), Murashige and Skoog (Duchefa, NL; M0222), Gambourg B5. Among sugars and osmotic substances, representative results were obtained with glucose in combination with mannitol to a final medium osmolarity falling in the 400 to 600 mOsmol / Kg range. Type and concentrations of hormones varied across species but representative results in most species were be obtained using 1 mg / L NAA and 0.2 mg / L BAP. The pH of regeneration media was adjusted to 5.8.

[0162]

[0155] Embedded protoplasts were incubated at a temperature of about 25-40 °C in the dark with 7-day medium refreshing intervals and were observed to generate microcalli of about 0.5-1 mm within one month. Size of microcalli was determined using a bright field microscope. It is noted that the minimum size for clonal microcalli cultures varies by species. After microcalli were obtained, whole pectin discs were transferred to tissue culture plates containing solidified culture media to develop microcolonies. Alternatively, pectin discs were disrupted by pipetting before transferring the microcalli to solid media plates to develop microcolonies. Microcolonies were further grown and sampled for downstream analyses (e.g., editing profile, ploidy and monoclonality) and / or sub cultured to shoot induction medium to regenerate plant shoots and finally rooted plants. In Figure 6 the editing profile of three rooted mutant plants is shown for different organs of the plant (flower, leaf & root) to demonstrate the genetic uniformity of the plant.

[0163]

[0156] Example 1 - improved microcalli development for protoplasts grown in pectin hydrogel

[0164]

[0157] This example compares the industry standard alginate hydrogel for cultivating protoplasts to a pectin hydrogel according to the invention for cultivating protoplasts.

[0165]

[0158] Protoplasts were obtained, embedded, and regenerated as described in the Methods section above. Protoplasts were embedded in either an alginate gel (1 wt.%) or a pectin gel (0.2 wt.%) according to the invention. Protoplasts were embedded in alginate and / or pectin hydrogel disks at a cell density indicated in the table below. Protoplast were regenerated according to the methods described above.

[0159] Table i:

[0166] Sample Protoplasts from Improved Cell density More Crop / variety microcallus (cells / mL) microcalli development? developed per 0.2 ml pectin vs alginate disk (%), average numbers per disk #1 N. benthamiana Yes 5.000 cells / mL 151.3 / 11.3

[0167] + 1235.1 % #2 Rice / Oc Yes 100.000 cells / mL 222.5 / 154.5

[0168] +44% #3 Tomato / MicroTom Yes 30.000 cells / mL 125.8 / 17.2

[0169] +633.2% #4 Tomato / Marglobe Yes 50.000 cells / mL - #5 Tomato / Yes 50.000 cells / mL 47.3 / 2.7

[0170] Moneymaker + 1651.8% #6 Strawberry Yes 150.000 cells / mL - (everbearing variety)

[0171]

[0172]

[0160] The results of example 1 show that the number of microcalli per disk was higher in pectin hydrogels compared to alginate hydrogels for the indicated cell densities.

[0173]

[0161] Example 2 - concentration of pectin

[0174]

[0162] This example compares the effect the weight percentage (wt.%) pectin in the gel on the protoplast regeneration. N. benthamiana protoplasts were grown for a 2- week regeneration period embedded at 50.000 cells / ml in pectin disks comprising a weight percentage (wt.%) of pectin according to the table below. Conditions for regeneration are described above. Protoplast cell density at the start of the regeneration period is indicated in the table below. The number of microcalli was determined after the 2-week regeneration period.

[0175]

[0163] Example 2 indicates that the concentration of pectin is a parameter to keep in mind when optimizing regeneration conditions. Although different concentrations can lead to microcallus formation, the use of too high concentration is not preferred as the discs do not form proper lens-like structures with high concentrations of pectin. Finally, Example 2 shows that a concentration of 0.2 wt.% pectin can be used to obtain hydrogel discs that are solid enough and provide good regeneration, i.e., proliferation of protoplasts therein.

[0176]

[0164] Table 2: indication of protoplasts formation from no microcalli formation (-) to high microcalli formation (+++++)

[0177] | Sample Pectin Microcalli formation

[0178] (wt.%)

[0179] #1 0.2 +++++

[0180] #2 0.5 +++++

[0181] #3 1.0 + +

[0182] #4 1.5 +

[0183] #5 2.0 ++++

[0184] #6 3.0 -

[0185]

[0186]

[0165] Example 3a - Comparison of Alginate to Pectin for growing N. benthamiana

[0187]

[0166] Data was collected 18 days after the embedding.

[0188]

[0167] Table 3 results comparison alginate to pectin for growing N. benthamiana Cell Number of Regenerati SD Regenera SD density samples on in tion in

[0189] (x1000 alginate 1% pectin

[0190] cells / mL) (N 0.2% (N microcolon microcol

[0191] ies per mL) onies per

[0192] mL)

[0193] 5 3 11.3 5.9 151.3 21.5

[0194]

[0195]

[0168] Example 3b - Comparison of Alginate to Pectin for growing Rice

[0169] Table 4 - results comparison alginate to pectin for growing rice

[0196] Regenera Cell Number Regenera SD Regene SD tion density of tion in ration medium (x1000 samples alginate in

[0197] cells / mL) 1% (N pectin microcol 0.2% (N

[0198] microc onies per olonies

[0199] mL) per mL)

[0200] 1 100 4 154.5 27.8 222.5 39.7 2 100 4 85.0 5.5 104.5 21.3

[0201]

[0202]

[0170] Example 3c - Comparison of Alginate to Pectin for growing Tomato

[0171] Table 5 - results comparison alginate to pectin for growing tomato Variety Cell Number Regenera SD Regene SD density of tion in ration

[0203] (x1000 samples alginate in cells 1% (N pectin

[0204] / mL) microcol 0.2% (N

[0205] onies per microc

[0206] mL) olonies

[0207] per mL) MicroTom 30 6 17.2 13.8 125.8 26.1 MoneyMaker 50 6 2.7 2.4 47.3 40

[0208]

[0209]

[0172] Example 3d - Comparison of Alginate to Pectin for growing Tomato Marglobe

[0210]

[0173] Microscopy pictures (Fig. 4; full well scans) depicting the microcallus formation in discs of alginate 1.0 wt.% (left) or pectin 0.5 wt.% (right) four weeks after the embedding and cultivation in regeneration medium. Protoplasts of Tomato Marglobe variety were embedded at a density of 50.000 cells / ml. Only protoplasts embedded in pectin discs proliferated into microcalli.

[0211]

[0174] Example 3g - Comparison of Alginate to Pectin for growing Strawberry (everbearing variety)

[0212]

[0175] Microscopy pictures (Fig. 5; four panels each condition) depicting the microcallus formation in discs of alginate 1 wt.% (left) or pectin 0.2 wt.% (right) four weeks after the embedding and cultivation in regeneration medium. Protoplasts of the everbearing strawberry variety were embedded at a density of 100.000 cells / ml. Only protoplasts embedded in pectin discs formed microcolonies / microcalli.

[0213]

[0176] Example 4 - improved microcallus growth in pectin versus alginate

[0177] Protoplasts derived from romaine lettuce were embedded at a density of 5000 cells / ml in pectin (0.2 wt.%) or alginate (1 wt.% or 0.5 wt.%). Microcalli formation after four weeks was markedly higher in pectin when compared to alginate (Figure 7). When cultivated in 0.2 wt.% pectin 75.8+ / -4.0 (n=4) microcalli were found. When cultivated in 1 wt.% alginate 32.8+ / -33.6 (n=4) microcalli were found and when cultivated in 0.5 wt.% alginate 20.3+ / -5.0 microcalli were found (table 6).

[0214]

[0178] Table 6

[0215] Well Pectin 0.2 wt.% Alginate 1 wt.% Alginate 0.5 wt.% 1 78 5 27

[0216] 2 79 79 19

[0217] 3 76 11 20

[0218] 4 70 36 15

[0219] Mean 75.8 32.8 20.3

[0220]

[0221] SD 4.0 33.6 5.0

[0222]

[0179] Example 5 - tomato protoplasts regeneration to microcalli in 0.2 wt.% pectin

[0223]

[0180] Protoplasts derived from six different tomato varieties (variety [1]-[6]) were embedded at a density of 100.000 cells / ml or 200.000 cells / ml in pectin (0.2 wt.%). Microcalli formation was observed after two weeks for each of the varieties (Figure 8). The results highly advantageous show that embedding in pectin results in microcalli formation regardless of the variety.

[0224]

[0181] Example 6 - strawberry protoplasts regeneration to microcalli in 0.2 wt.% pectin

[0225]

[0182] Protoplasts derived from four different June-bearing strawberry varieties (variety

[0001] -[4]) were embedded at a density of 100.000 cells / ml or 200.000 cells / ml in pectin (0.2 wt.%). Microcalli formation was observed after four weeks for each of the varieties (Figure 9). The results highly advantageous show that embedding in pectin results in microcalli formation regardless of the variety as outlined within this example but further also in view of an other strawberry varieties such as the everbearing strawberry variety of example 1 disclosed elsewhere herein.

[0226]

[0183] Example 7 - white cabbage protoplasts regeneration to microcalli in 0.2 wt.% pectin

[0227]

[0184] Protoplasts derived from a regeneration recalcitrant white cabbage variety (Fig.

[0228] 10; left) and non-recalcitrant cauliflower variety (Fig 10; right) were embedded at a density of 100.000 cells / ml in pectin (0.2 wt.%). The failure to form tissue, such as microcalli or shoots, roots, flowers leafs etc., post-transformation is defined as regeneration recalcitrance. The white cabbage of this example is a regeneration recalcitrant variety. Microcalli formation was observed after four weeks for each of the varieties (Figure 10). The results highly advantageous show that embedding in pectin results in microcalli formation regardless of the variety and despite the regeneration recalcitrance.

[0229]

[0185] Example 8 - sugar beet protoplasts regeneration to microcalli in 0.2 wt.% pectin

[0230]

[0186] Protoplasts derived from two different sugar beet varieties (variety [1]-[2]) were embedded at a density of 75.000 cells / ml or 100.000 cells / ml in pectin (0.2 wt.%). Microcalli formation was observed after four weeks for each of the cultivars (Figure 11). The results highly advantageous show that embedding in pectin results in microcalli formation regardless of the variety.

[0231]

[0187] Example 9 - acorn squash and pumpkin protoplasts regeneration to microcalli in 0.2 wt.% pectin

[0232]

[0188] Protoplasts derived from acorn squash cotelydons (Fig. 12; top left), pumpking cotelyodons (Fig. 12; top right), acorn squash leaf (Fig. 12; bottom left) and pumpkin leaf (Fig. 12; bottom right) were embedded at a density of 50.000 cells / ml (for cotelydon protoplasts) or 300.000 cells / ml (for leaf protoplasts) in pectin (0.2 wt.%). Microcalli formation was observed after two weeks for each of the cultivars (Figure 12). The results highly advantageous show that embedding in pectin results in microcalli formation regardless of the variety.

[0233]

[0189] Example 10 - tomato protoplasts regeneration to microcalli in 0.2 wt.% pectin

[0234]

[0190] Protoplasts derived from tomato (variety marglobe) were embedded at a density of 50.000 cells / ml in pectin (0.5 wt.%) or alginate (1.0%). Surprisingly, cell division and cell colony formation was observed already after 4 days in pectin (Fig. 13; top). Conversely, when cultivated in alginate, cells were found to be spherical and no cell division or colony formation was observed after 4 days (Fig. 13; bottom).

[0235]

[0191] Example 11 -microcallus growth under various pectin concentrations

[0192] Protoplasts derived from a white cabbage variety were embedded at a density of 250.000 cells / ml in pectin (0.1 wt.%, 0.2 wt.% 0.5 wt.% and 1 wt.%). Regeneration of the protoplasts at the indicated pectin concentrations were performed in triplicate (left, middle and right columns)

[0236]

[0193] Microcalli formation was observed four weeks after protoplasts were embedded in 0.2 wt.%, 0.5 wt.% or 1 wt.% pectin (Figure 14). An optimal regeneration of protoplasts for this cabbage variety was observed at 1 wt.% pectin (table 7).

[0237]

[0194] Table 7

[0238] Well Pectin 0.1 wt.% Pectin 0.2 wt.% | Pectin 0.5 wt.% Alginate 1 wt.% 1 0 69 50 372

[0239] 2 0 64 127 390

[0240] 3 0 106 164 622

[0241] Mean 0.0 79.7 123.7 461.3

[0242]

[0243] SD 0.0 22.9 72.1 139.4

[0244]

[0195] Example 12 - comparative: microcallus growth in alginate with and without nursing cells

[0245]

[0196] This comparative example shows the lack of regeneration of N. benthamiana protoplasts when grown in alginate (1 wt.%) with- and without the presence of nurse cells Nurse cells are free (not embedded) N. tabacum BY-2 cells. Protoplasts derived from N. benthamiana were embedded at a density of 10.000 cells / ml in alginate (1 wt.%) and microcalli formation was tracked at 0 days, 7 days and 2 weeks (Fig. 15).

[0246]

[0197] In this comparative example, it is shown that without nursing cells (-Nurse cells) the protoplasts embedded in alginate do not regenerate to microcalli at this density at 0 days, 7 days or 2 weeks (Fig. 15). In examples disclosed elsewhere herein (e.g. example 1), it is shown that when embedded in pectin N. benthamiana does regenerate even at 5.000 cells / ml and without nursing cells. Hence, highly advantageously, pectin enables protoplasts regeneration even at low cell densities and without nursing cells.

[0247]

[0198] Example 13 - monoclonality of plants from regenerated from single protoplast

[0248]

[0199] Monoclonality was analyzed of 52 tomatoes plants from four different diploid tomato varieties. The plants were regenerated from a single cell (protoplast) in pectin (0.2 wt.%) at a density of 200.000 cells / ml as described elsewhere herein. All protoplasts were edited via RNP transfection with 2 guides simultaneous targeting the same gene in each of the plants. Genotyping of the plants comprised PCR amplification of the edited region and sequencing of the amplified edited regions using the Oxford Nanopore sequencing platform in accordance with standard practice.

[0200] Sequencing data revealed that 44 out of 52 plants had at least one allele edited (85%), 25 out of 52 plants had all alleles edited (48%), and 8 out of 52 plants had no edits (15%). In 42 plants two alleles could be distinguished in the correct ratio (1:1) (81%). It could be concluded that these 42 plants are monoclonal and non-chimeric. For 7 plants four alleles were observed (13%), either these 7 plants have doubled ploidy before editing (tetrapioid) or are not monoclonal. Hence, the method in accordance with the invention highly advantageously allows for the regeneration of monoclonal plants regenerated from a single (gene-edited) protoplast.

Claims

CLAIMS1. A method for cultivating plant protoplasts, wherein the method at least comprises:(a) providing an aqueous solution comprising plant protoplasts and a hydrogel-forming pectin,(b) causing the pectin to form a hydrogel comprising the plant protoplasts, preferably by contacting the aqueous solution of step (a) with calcium, (c) contacting the hydrogel comprising the plant protoplast with a first culture medium and allowing the plant protoplast to proliferate and to form one or more microcalli2. The method according to any one of the previous claims, wherein the aqueous solution of step (a) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin and / or wherein the hydrogel of step (b) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin.

3. The method according to any one of the previous claims, wherein the aqueous solution of step (a) comprises between 0.05 and 3.0 wt.% pectin, between 0.1 and 2.5 wt.% pectin, between 0.1 and 2.0 wt.% pectin, between 0.1 and 1.0 wt.% pectin, between 0.1 and 0.5 wt.% pectin, or between 0.1 and 0.3 wt.% pectin and / or wherein the hydrogel of step (b) comprises between 0.05 and 3.0 wt.% pectin, between 0.1 and 2.5 wt.% pectin, between 0.1 and 2.0 wt.% pectin, between 0.1 and 1.0 wt.% pectin, between 0.1 and 0.5 wt.% pectin, or between 0.1 and 0.3 wt.% pectin.

4. The method according to any one of the previous claims, wherein the hydrogelforming pectin is a polygalacturonic acid, preferably a polygalacturonic acid salt, preferably a polygalacturonic acid sodium salt.

5. The method according to any one of the previous claims, wherein the hydrogel of step (b) has a volume of 1 - 1000 microliter, 1 - 500 microliter, 10 - 1000 microliter, 10 - 500 microliter, 10 - 250 microliter, 50 - 250 microliter, or 150 - 250 microliter.

6. The method according to any one of the previous claims wherein the ratio (vol / vol) between the hydrogel and the first culture medium of step (c) is about 1:1 - 1:25, preferably about 1:2.5 - 1:20.

7. The method according to any one of the previous claims, wherein the hydrogel has a thickness of at most 10 millimeter or at most 5 millimeter.

8. The method according to any one of the previous claims, wherein the aqueous solution of step (a) comprises 1000 - 500.000 protoplasts per milliliter, 2500 - 400.000 protoplasts per milliliter, or, preferably between 5000 and 150.000 protoplasts per milliliter.

9. The method according to any one of the previous claims, wherein the plant protoplasts are plant protoplasts derived from tomato, soybean, strawberry, zucchini, rice, sugar beet, brassica genus, squash, pumpkin, N. benthamiana, rose, potato.

10. The method according to any one of the previous claims wherein:- the aqueous solution of step (a) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin and / or wherein the hydrogel of step (b) comprises at least 0.05 wt.% pectin, and preferably at most 3.0 wt.% pectin,- the aqueous solution of step (a) comprises 1000 - 500.000 protoplasts per milliliter, 2500 - 400.000 protoplasts per milliliter, or, preferably between 5000 and 150.000 protoplasts per milliliter,- the hydrogel of step (b) has a volume of 1 - 1000 microliter, 1 - 500 microliter, 10 - 1000 microliter, 10 - 500 microliter, 10 - 250 microliter, 50 - 250 microliter, or 150 - 250 microliter, and- the ratio (vol / vol) between the hydrogel and the first culture medium of step (c) is about 1:1 - 1:25, preferably about 1:2.5 - 1:20.

11. The method according to any one of the previous claims wherein the aqueous solution of step (a) comprises mannitol and / or wherein the hydrogel of step (b) comprises mannitol.

12. The method according to any one of the previous claims, wherein the contacting of the hydrogel with the first culture medium is for a period of at least 3 days.

13. The method according to any one of the previous claims, wherein the contacting of the hydrogel with the first culture medium is for a period of no more than 100 days, 75 days, 50 days, or 30 days.

14. The method according to any one of the previous claims, wherein the one or more microcalli of step (c) are between 0.2 - 1.5 mm, 0.3 - 1.2 mm, or 0.5 - 1.0 mm in diameter.

15. The method according to any one of the previous claims wherein the plant protoplasts in step a) have been contacted with a gene editing system, preferably wherein the gene editing system is selected from the group consisting of CRISPR systems, TALENs, meganucleases and zinc finger nucleases.

16. The method according to any one of the previous claims, wherein the one or more microcalli are further cultivated, preferably wherein the one or more microcalli are isolated from the gel and further cultivated.

17. The method according to any one of the previous claims, wherein at least part of a microcallus is analyzed, for example for the presence of a genetic modification or for establishing the microcallus is a clonal microcallus.

18. The method according to any one of the previous claims, wherein the method is for:improving plant protoplast proliferation,generating at least one microcallus, preferably at least one clonal microcallus,- generating at least one plant cell line, preferably at least one clonal cell line,regenerating at least one plant cell,improving microcallus formation,improving plant cell regeneration efficiency, and / orregenerating of at least one plant, preferably at least one clonal plant.

19. The method according to any one of the previous claims wherein the method comprises:- comparing protoplast proliferation and / or microcalli formation using at least two conditions with different concentrations of pectin,- comparing protoplast proliferation and / or microcalli formation using at least two conditions with different concentrations of protoplasts, comparing protoplast proliferation and / or microcalli formation using at least two conditions with different volumes of the hydrogel, and / or - comparing protoplast proliferation and / or microcalli formation using at least two conditions with different ratio (vol / vol) between the hydrogel and the first culture medium of step (c)and, based on the outcome of the comparison, selecting a condition that allows for generating a clonal plant microcalli, and optionally repeating the method according to any one of the previous claims with the selected condition.

20. A pectin hydrogel as defined in any of claims 1 - 15 comprising one or more plant protoplast, preferably wherein the gel comprises one or more clonal plant protoplasts.

21. A pectin hydrogel as defined in any of claims 1 - 15 comprising one or more microcalli, preferably wherein the gel comprises one or more clonal microcalli.

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