Method for producing plant suspension culture

Abstract

The present invention relates to a method for producing a suspension culture of plant tissue particles developing plant calli. The present invention further relates to a method for producing a suspension culture of plant cells as well as to a method for producing biomass from plant cells. Also, the present invention relates to a suspension culture of plant tissue particles developing plant calli as well as to a suspension culture of plant cells. Finally, the present invention relates to biomass from plant cells.

Description

[0001] Method for producing plant suspension culture

[0002] The present invention relates to a method for producing a suspension culture of plant tissue particles developing plant calli. The present invention further relates to a method for producing a suspension culture of plant cells as well as to a method for producing biomass from plant cells. Also, the present invention relates to a suspension culture of plant tissue particles developing plant calli as well as to a suspension culture of plant cells. Finally, the present invention relates to biomass from plant cells.

[0003] Cacao and coffee beans are economically highly relevant soft commodity trading goods and are used in several food applications including, e.g., beverages, butter, ice cream, chocolate and other candies, baked goods, and also dietary supplements. The world production of coffee exceeded 10 million metric tons in 2020 and the production of cacao exceeded 5.5 million metric tons. Most production and harvesting takes place in southern countries, primarily countries in Africa, South America and Oceania.

[0004] There are several in vitro methods known in the art to cultivate cacao and coffee plants. For example, US 4204366 describes a method for the production of cotyledons by non-agricultural means in cacao. However, this method requires propagating or culturing asexual embryos. Regeneration of plants by somatic embryogenesis has also been described in US 5312801.

[0005] US 9167840 describes the use of Theobroma cacao cell lines producing procyanidins which are antioxidants useful as dietary supplement. WO 2008 / 081275 describes the production of biomass from differentiated tissue of cacao seeds and culture medium for biomass production from differentiated plant seeds with specific ingredients at specific concentration ranges. However, these methods still require long periods of time of manual work to grow primary calli as well as time to produce a suspension culture.

[0006] These and further disadvantages need to be overcome. The present invention therefore addresses these needs and technical objectives and provides a solution as described herein and as defined in the claims.

[0007] Accordingly, the present invention relates to a method for producing a suspension culture of plant tissue particles developing plant calli, comprising (a) wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles in liquid medium;

[0008] (b) directly culturing said plant tissue particles obtained in step (a) in plant cell medium, preferably liquid plant cell medium, under conditions and for a time sufficient for said plant tissue particles to develop plant calli.

[0009] Preferably, said plant tissue particles are cultured for 1 to 15 days, preferably, 7 to 15 days, e.g. 7, 8, 9, 10, 11 , 12, 13, 14 or 15 days, with 15 days being preferred.

[0010] The method of the present invention may sometimes also called “RCC” herein. “RCC” stands for rapid cell culture. In fact, as is described herein and shown in the Examples, the method of the present invention is advantageous insofar as it results in a shorter time period from producing a suspension culture of plant cells using plant tissue as starting material.

[0011] In accordance with the present invention, a suitable liquid plant cell medium may comprise, e.g., MS (Murashige & Skoog) basal salts, MS vitamins with an enriched concentration of saccharose in a concentration between 15 and 40 g / L, kinetin in a concentration between 0.2 and 4 mg / L, zeatin in a concentration between 0.2 and 5 mg / L, ascorbic acid in a concentration between 20 and 150 mg / L, and plant agar in a concentration between 7 and 12 g / L, wherein the culture medium has the pH adjusted between 5.5 and 6.0. In a more specific example, in accordance with the present invention, a suitable liquid plant cell medium may comprise, e.g., at least agar, MS basal salts, MS vitamins with an enriched concentration of saccharose in a concentration of 20 - 40 g / L, kinetin in a concentration of 0.1 and 5 mg / L, Indol Butyric Acid (I BA) in a concentration of 0.1 - 5 mg / L, zeatin in a concentration of 0.1 mg / L and 5 mg / L, Indol Acetic Acid (IAA) in a concentration of 0.1 mg / L and 5 mg / L, ascorbic acid in a concentration of 20 mg / L and 120 mg / L, and plant agar in a concentration of 9 g / L, wherein the culture medium has the pH adjusted to 5.8, and wherein the MS basal salt and vitamin concentrations per liter are prepared according to the standards known in the art.

[0012] In one embodiment of the present invention, the liquid medium of step (a) of the method described and provided herein confers anti-oxidative conditions. For example, in accordance with the present invention, said liquid medium may contain an anti-oxidative agent (e.g., ascorbic acid).

[0013] A “callus” as used herein is a growing mass or clumps of usually more or less non-organized and / or undifferentiated or partially differentiated plant cells . Therefore, the term callus includes cells with various degrees of differentiation. A callus is usually loosely organized. A “plant tissue particle” or “particle” as used herein refers to disaggregated plant tissue and includes fragments, pieces or slices of plant material. Such particles have preferably a size of about 0.1 mm to about 10 mm, more preferably from about 0.1 mm to about 4 mm, e.g., after blending. Such particles have preferably a thickness of about 1 to 5 mm, e.g. 1 mm, 2 mm, 3 mm, 4 mm or 5 mm. Accordingly, such particles may preferably have a size of about 0.1 mm to about 10 mm, more preferably from about 0.1 mm to about 4 mm and / or a thickness of about 1 to 5 mm, e.g. 1 mm, 2 mm, 3 mm, 4 mm or 5 mm.

[0014] Disaggregation is achieved as described herein. Preferably, disaggregation is done mechanically, acoustically or by light, e.g. laser, such as laser microdissection. Preferably, disaggregation may result in slices having preferably a thickness as described before and herein. Particles - due to disaggregation - have preferably multiple injuries on their surface, such as more than about 2, 3, 4, 5, 6, 7, 8, 9, 10 injuries. Preferably, these injuries are scratches and / or bumps. Disaggregation of plant tissue is preferably done by mechanical blending or mechanical cutting, preferably by a cell aggregation device, e.g. as described herein. Accordingly, mechanical blending is preferably done with rotating blades, e.g., plastic blades. However, rotating blades are not limited to plastic blades, because - without being bound by theory - the potential oxidative effect of, e.g. rotating metal blades can preferably be reduced or even avoided by disaggregating plant tissue under essentially non-oxidative conditions. For example, rotating blades such as plastic blades may be used in parallel with one or more antioxidative agent (s). In accordance with the present invention, mechanical cutting is preferably done by a metallic cutting tool, e.g., razor blades, knives, scissors, or the like. In one embodiment of the present invention, mechanical blending results in particles having a size of about 0.1 mm to about 1 mm, preferably about 0.1 mm to about 4 mm.

[0015] As for mechanical blending, without being bound by theory, for mechanical cutting it is preferred to reduce or even avoid disaggregating plant tissue under oxidative conditions, i.e. to apply essentially non-oxidative conditions. In one embodiment of the present invention, mechanical cutting results in slices having a thickness of about 1 to 5 mm, e.g. 1 mm, 2 mm, 3 mm, 4 mm or 5 mm.

[0016] In accordance with the present invention, disaggregation, in particular mechanical blending or cutting is preferably done under non-oxidative conditions. For example, either non-oxidative conditions may be applied, or anti-oxidative agents (e.g., ascorbic acid) used, or non-oxidative means employed, e.g., plastic or other non-oxidating (or, as also used herein, non-oxidizing) cutting tools. However, under non-oxidative conditions which are preferred, as described herein, also a metallic cutting tool may be employed for disaggregation. As has surprisingly turned out in context with the present invention, wounding plant tissue by disaggregating said plant tissue into plant tissue particles allows much quicker development of plant calli compared to methods known in the art. In the examples, the method of the present invention was compared to a “traditional” method for producing plant calli and a suspension culture derived therefrom and it is shown that the time to suspension culture is much shorter (see Table 1). It is also shown that by making use of the method of the present invention, more biomass dry weight is obtained (see Table 2).

[0017] Without being bound by theory, the advantageous effect of the method of the present invention may be to disaggregate plant tissue into plant tissue particles in liquid medium and / or to disaggregate plant tissue into plant tissue particles under essentially non-oxidative conditions, preferably under non-oxidative conditions, e.g. as described herein. For example, the liquid medium may preferably have non-oxidative conditions. These non-oxidative conditions can be achieved as described herein.

[0018] Indeed, prior art methods for wounding plant tissue disaggregate such plant tissue by dry disaggregation, e.g. dry cutting, i.e., no liquid medium is used when wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles. Hence, when wounding, such plant tissue and / or plant tissue particles is / are not kept submerged, e.g., in liquid medium. Also, prior art methods do not apply a liquid medium which confers advantageously anti-oxidative conditions.

[0019] In fact, Table 1 and Table 2 show that wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles in liquid medium, preferably conferring non-oxidative conditions, and directly culturing said plant tissue particles in plant cell liquid medium under conditions and for a time sufficient for said plant tissue particles to develop plant calli is, in comparison to prior art methods, advantageous as regards the time from plant tissue to suspension culture and / or biomass dry weight.

[0020] That said, mechanical disaggregation (preferably automated mechanical disaggregation, e.g., using a cell disaggregator such as gentleMACS™ Dissociator from Miltenyi Biotec B.V. & Co KG, Germany) of plant tissue allows such quick callus formation, particularly where the resulting plant tissue particles are directly transferred to the liquid culture medium. That is, in accordance with the present invention, preferably there is no intermediate step of culturing plant tissue in solid state (e.g., explants on agar plate to grow a callus followed by transfer into liquid medium). By the method described and provided in context with the present invention, more cells can be taken in comparison to state-of-the-art methods since more explants are generated at a time.

[0021] Also, without being bound by theory, disaggregation of plant tissue into plant tissue particles may cause multiple injuries on the surface of said particles, wherein these injuries are - due to the disaggregation - preferably scratches and / or bumps. It is assumed that due to disaggregation plant tissue particles obtained from plant tissue are not damaged to a degree which would be disadvantageous for the induction of plant calli. Indeed, as evidence, the present inventors compared callus induction when processing plant tissue in accordance with the teaching of the present invention and when processing plant tissue pursuant to the traditional way of cutting plant tissue into pieces and subjecting such wound plant tissue to callus induction. It was found that plant tissue processed in accordance with the teaching of the present invention provides for calli which give rise to suspension cultures having more biomass dry weight in comparison to suspension cultures derived from calli which are the result of the traditional way of dry cutting plant tissue and growing wound plant tissue on solid media; see also examples herein.

[0022] Accordingly, the method of the present invention provides for a significant gain of time in the step from plant tissue into suspension culture. Also, due to the option to apply automated disaggregation (e.g., using gentleMACS™ Dissociator from Miltenyi Biotec B.V. & Co KG, Germany), there is significantly less manual work required. The method of the present invention also provides for a higher amount of biomass dry weight in comparison to the traditional way known in the prior art (see Table 2).

[0023] In accordance with the teaching of the present invention, plant calli are induced in a shorter time period compared to the traditional way and / or are induced in a higher number per plant tissue particle obtained by wounding plant tissue by disaggregation. It was also observed by the present inventors that suspension cultures obtained from plant calli developed by plant tissue particles obtained by wounding plant tissue by disaggregation are more homogenous and / or produce a higher amount of biomass dry weight in comparison to plant tissue obtained by the traditional way of dry cutting plant tissue and growing wound plant tissue on solid media; see also examples herein.

[0024] In context with the present invention, unless specified herein otherwise, the terms “disaggregation”, “disaggregator”, “disaggregate” and the like are used synonymously with “dissociation”, “dissociator”, “dissociate” and the like, respectively, particularly in context with cell disaggregation / cell dissociation, etc. Preferably, the plant tissue which is first wounded according to step (a) of the present invention, is in its surrounding area surface disinfected, e.g., using a fungicide and further solutions (e.g., NaCIO, preferably in a concentration below 5%). Accordingly, the plant tissue which is then used in accordance with the methods of the present invention for being disaggregated is preferably not in direct contact with a disinfectant, e.g. a fungicide or a bleaching compound. For example, pods which contain seeds inside are disinfected, while the seeds inside are merely extracted.

[0025] In accordance with the present invention, in one embodiment, the embryo may be removed and / or discarded before disaggregating said plant tissue into plant tissue particles. This way, it is possible to obtain endosperm tissue and / or cotyledon tissue, which may then be dissociated (e.g., manually or automatically) to obtain particles according to step (a) of the present invention, which are then cultured in (preferably liquid) plant cell medium according to step (b) of the present invention. Optionally, in accordance with the present invention, the particles which have been obtained by disaggregating said plant tissue according to step (a) of the present invention, may be separated by size using a filter or, preferably, a sorting device, or flow cytometry before further cultivation in liquid medium according to step (b) of the present invention. Additionally or alternatively, said particles may also - after separation via a filter or flow cytometry, if applied - further be separated by selecting only living particles (e.g., by applying the particles on plates, allowing them to grow under suitable conditions, and then only selecting live cells) before proceeding with cultivation of said particles in (preferably liquid) cell medium.

[0026] The present invention further relates to a method for producing a suspension culture of plant cells, comprising

[0027] (c) culturing separated plant cells obtained from the plant calli developed by said plant tissue particles of the method described and provided herein in plant cell medium (preferably liquid plant cell medium).

[0028] Preferably, said separated plant cells are cultured for 1 to 15 days, preferably, 7 to 15 days, e.g. 7 to 14 days, 7 to 13 days, 7 to 12 days, 7 to 11 days, 7 to 10 days.

[0029] In other words, in accordance with the present invention, the plant calli obtained in step (b) of the method described and provided herein are treated to obtain separated plant cells from said calli (e.g., using a knife, or dissociator, or the like), which are then cultured according to said step (c) to obtain a suspension of culture of plant cells. The present invention further relates to a method for producing biomass from plant cells, comprising

[0030] (d) culturing plant cells obtained in step (c) of the method described and provided herein in plant cell medium under conditions and for a time sufficient to produce biomass from plant cells;

[0031] (e) harvesting said biomass.

[0032] Preferably, said plant cells are cultured for 14 days or more, e.g. 15, 20, 25, 30, 35, 40, 45, 50 or more days.

[0033] In other words, in accordance with the present invention, the plant cells obtained in optional step (c) of the method described and provided herein, are further cultured in (preferably liquid) plant cell medium (e.g. in suspension) under conditions and for a time sufficient to produce biomass from plant cells, followed by a step of harvesting said biomass. Such conditions to produce biomass from plant cells in suspension are generally known in the art and described in, e.g., Eibl (2018), Appl. Microbiol. Biotechnol. 102, 8661-8675.

[0034] Preferably, the present invention relates to a method for producing a suspension culture of plant cells, comprising

[0035] (a) wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles in liquid medium;

[0036] (b) directly culturing said plant tissue particles obtained in step (a) in plant cell medium under conditions and for a time sufficient for said plant tissue particles to develop plant calli; and

[0037] (c) culturing separated plant cells obtained from the plant calli developed by said plant tissue particles obtained in steps (a) and / or (b) in plant cell medium.

[0038] Preferably, the present invention relates to a method for producing biomass from plant cells, comprising

[0039] (a) wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles in liquid medium;

[0040] (b) directly culturing said plant tissue particles obtained in step (a) in plant cell medium under conditions and for a time sufficient for said plant tissue particles to develop plant calli;

[0041] (c) culturing separated plant cells obtained from the plant calli developed by said plant tissue particles obtained in steps (a) and / or (b) in plant cell medium; (d) culturing plant cells obtained in step (c) in plant cell medium under conditions and for a time sufficient to produce biomass from plant cells; and

[0042] (e) harvesting said biomass.

[0043] In accordance with the present invention, in one embodiment the part of a plant from which the plant tissue is obtained which is then wounded by disaggregation as described in step (a) of the method of the present invention may be or derived from root, stem, leaf, flower, fruit or seed. In a specific embodiment of the present invention, where the part of a plant from which the plant tissue is obtained which is then wounded by disaggregation as described in step (a) of the method of the present invention is fruit, said fruit may contain seed and / or pulp. In another specific embodiment of the present invention, where the part of a plant from which the plant tissue is obtained which is then wounded by disaggregation as described in step (a) of the method of the present invention is seed, said seed may contain endosperm and / or cotyledon tissue.

[0044] In one embodiment of the present invention, the part of a plant from which the plant tissue is obtained which is then wounded by disaggregation as described in step (a) of the method of the present invention may be extracted under sterile conditions. That is, e.g., the tool for extracting said plant part may be sterilized before extraction of said part, the plant part may be disinfected before and / or after extraction, and / or other measures for sterilization may be applied. For example, in accordance with the present invention, particularly where the part of a plant from which the plant tissue is obtained which is then wounded by disaggregation as described in step (a) of the method of the present invention is seed, said plant part (e.g., seed) may be extracted under sterile conditions from the fruit of a plant when extracting endosperm tissue and / or cotyledon tissue. In this context, in one embodiment of the present invention, embryonic tissue of said plant part (e.g., seed) may be discarded when extracting said endosperm tissue and / or cotyledon tissue. In a more specific embodiment of the present invention, particularly where the part of a plant from which the plant tissue is obtained which is then wounded by disaggregation as described in step (a) of the method of the present invention is seed, and where said plant part (e.g., seed) is extracted under sterile conditions from the fruit of a plant when extracting endosperm tissue and / or cotyledon tissue, said endosperm and / or cotyledon tissue may be essentially free from embryonic tissue of said plant part (e.g., seed).

[0045] In one embodiment of the present invention, said particles obtained in step (a) of the method described and provided herein by wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue may be separated by size. This can be done using methods and devices known in the art, e.g., using a filter or cell cytometry, preferably cell cytometry. In a particular embodiment of the present invention, said particles obtained in step (a) and further cultured in step (b) of the method described and provided herein have a size of about 0.1 mm to about 10 mm, preferably about 0.1 mm to about 4 mm.

[0046] In accordance with the present invention, disaggregating the plant tissue into plant tissue particles in liquid medium as described in step (a) of the method described and provided herein may be done mechanically, acoustically, or by light, preferably mechanically (e.g., by mechanical blending or cutting). Such mechanical disaggregation can be done manually using suitable devices, e.g., a mortar with pestle, hammer, blades (e.g., razor blades, rotating blades, plastic blades, rotating plastic blades), knives, scissors, or the like. Alternatively, or additionally, such mechanical disaggregation can be done automatically using a dissociation device, e.g., by shaking, bumping, scratching, or cutting (preferably bumping or scratching) the plant tissue. Examples for such devices include, e.g., gentleMACS™ Dissociator from Miltenyi Biotec B.V. & Co KG, Germany. In one embodiment of the present invention, the disaggregation of the plant tissue into plant tissue particles in liquid medium as described herein is done by keeping said plant tissue and plant tissue particles submerged, e.g., in liquid medium. In one embodiment of the present invention, the liquid medium confers anti-oxidative conditions. For example, in accordance with the present invention, said liquid medium may contain an anti- oxidative agent (e.g., ascorbic acid).

[0047] In one embodiment of the present invention, said particles obtained in step (a) of the method described and provided herein by wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue may be characterized by having multiple injuries on their surface due to said disaggregation. For example, in accordance with the present invention, such injuries may comprise inter alia scratches and / or bumps.

[0048] As mentioned, in accordance with the present invention, disaggregating the plant tissue into plant tissue particles in liquid medium as described in step (a) of the method described and provided herein may be done mechanically, acoustically, or by light, preferably mechanically. In a more particular embodiment of the present invention, such mechanical disaggregation may be done by mechanical blending or cutting. Preferably in accordance with the present invention, such mechanical blending or cutting may be done under conditions to wound said plant tissue, e.g., to apply scratches and / or bumps to said plant tissue. In one embodiment of the present invention, the disaggregation of the plant tissue into plant tissue particles in liquid medium as described herein is done by keeping said plant tissue and plant tissue particles submerged, e.g., in liquid medium. In one embodiment of the present invention, the liquid medium confers anti-oxidative conditions. For example, in accordance with the present invention, said liquid medium may contain an anti-oxidative agent (e.g., ascorbic acid).

[0049] Generally, in accordance with the present invention, mechanical disaggregation, e.g., mechanical blending or cutting, may be done manually or automatically. In one embodiment of the present invention, mechanical blending may be done by a cell disaggregation device. Examples for such a device include, e.g., gentleMACS™ Dissociator from Miltenyi Biotec B.V. & Co KG, Germany. In one embodiment of the present invention, disaggregating plant tissue into plant tissue particles in liquid medium according to step (a) of the method described and provided herein may be done mechanically, e.g., by mechanical blending or grinding, e.g., using blades, rotating blades, plastic blades, or rotating plastic blades. That is, in one embodiment of the present invention, disaggregating plant tissue into plant tissue particles in liquid medium according to step (a) of the method described and provided herein may be done mechanically, e.g., by mechanical cutting, e.g., using razor blades, knives, scissors, or the like. In one embodiment of the present invention, the disaggregation of the plant tissue into plant tissue particles (e.g., by mechanical blending or cutting) in liquid medium as described herein is done by keeping said plant tissue and plant tissue particles submerged, e.g., in liquid medium. Put differently, “dry cutting” as is traditionally done in the prior art, is preferably avoided, i.e., no dry cutting is done.

[0050] In one embodiment of the present invention, the liquid medium confers anti-oxidative conditions. For example, in accordance with the present invention, said liquid medium may contain an anti-oxidative agent (e.g., ascorbic acid).

[0051] Generally, in accordance with the present invention, disaggregating plant tissue into plant tissue particles in liquid medium according to step (a) of the method described and provided herein may be done by any suitable process and under suitable conditions. In one embodiment of the present invention, disaggregating plant tissue into plant tissue particles in liquid medium according to step (a) of the method described and provided herein may be done under essentially non-oxidative conditions, or completely non-oxidative conditions (preferably essentially non-oxidative conditions). Such non-oxidative conditions may be achieved, e.g., by adding one or more anti-oxidative agents (e.g., ascorbic acid) to the liquid medium of step (a). In a more specific embodiment of the present invention, said non-oxidative conditions may be achieved, e.g., by adding one or more anti-oxidative agents (e.g., ascorbic acid) to the liquid medium of step (a) and, optionally, by a washing step with an anti-oxidative agent (e.g., ascorbic acid). As described herein, non-oxidative conditions may also be given, if non- oxidative means may be employed, e.g., plastic or other non-oxidating (or, as also used herein, non-oxidizing) cutting tools. In accordance with the present invention, disaggregating plant tissue into plant tissue particles in liquid medium according to step (a) of the method described and provided herein may be done by applying bumps, scratches, or similar wounds or injuries to the plant tissue and / or particles as described herein. In another embodiment of the present invention, disaggregating plant tissue into plant tissue particles in liquid medium according to step (a) of the method described and provided herein may be done by cutting the particles into thin slices as described herein, e.g., by mechanical cutting (using, e.g., razor blades, knives, scissors, or the like) resulting in slices having a thickness of about 1 to 5 mm. In accordance with the present invention, disaggregating plant tissue into plant tissue particles is preferably done under (essentially) non-oxidative conditions. Such non-oxidative conditions may be achieved, e.g., by adding one or more anti-oxidative agents (e.g., ascorbic acid) to the liquid medium of step (a). In a more specific embodiment of the present invention, said non-oxidative conditions may be achieved, e.g., by adding one or more anti-oxidative agents (e.g., ascorbic acid) to the liquid medium of step (a) and, optionally, by a washing step with an anti-oxidative agent (e.g., ascorbic acid).

[0052] In one embodiment of the present invention, the liquid medium in which said plant tissue is disaggregated into plant tissue particles is a defined plant cell medium containing MS basal salt mixture and vitamins, sucrose, and phytoregulators to induce calli. In a further embodiment of the present invention, a cell wall degrading enzyme may be added to the liquid medium such as cellulase, pectinase, amyl glucosidase, hemicellulose. In one embodiment of the present invention, the liquid medium of step (a) of the method described and provided herein confers anti-oxidative conditions. For example, in accordance with the present invention, said liquid medium may contain an anti-oxidative agent (e.g., ascorbic acid).

[0053] In one embodiment of the present invention, said disaggregated plant tissue obtained in step (a) of the method described and provided herein may be washed in a step (a’) prior to directly culturing said disaggregated plant tissue in plant cell medium in step (b). In a specific example of the present invention, said washing step may include washing with an anti-oxidative agent (e.g., ascorbic acid). For example, said washing may be done by a wash solution comprising an anti-oxidative agent (e.g., ascorbic acid).

[0054] In another embodiment of the present invention, where separated plant cells obtained from the plant calli developed by said plant tissue particles of the method described and provided herein are cultured in plant cell medium, such separated plant cells are particularly obtained from a filtrate and / or retentate of the suspension culture of plant calli of the method described and provided herein.

[0055] In another embodiment of the present invention, where biomass is to be produced as described and provided herein, i.e. where plant cells are cultured in plant cell medium under conditions and for a time sufficient to produce biomass from plant cells according to step (d) and then biomass is harvested according to step (e) as described and provided herein, said step (d) and / or (e) may be carried out in a bioreactor selected from a stirred tank bioreactor, an orbitally shaken bioreactor, a bubble column bioreactor, a wave reactor, a circular reactor, or an air-lift bioreactor.

[0056] In accordance with the present invention, there is no general limitation as to the origin of the plant from which the plant tissue is obtained which is then wounded by disaggregation as described and provided herein. In one embodiment of the present invention, the plant or the plant cells to be cultured according to step (c) according to the method of the present invention are from monocotyledonous or dicotyledonous plants. In a specific example in context with the present invention, the monocotyledonous plants may be wheat, barley, rye, oat or palm tree. In another specific example in context with the present invention, the dicotyledonous plants may be coffee, cacao, nuts, fruits, avocado, berries (e.g. blueberry), acai, grape or citrus fruits, herbs, and spices like, e.g., vanilla, saffron, guarana, turmeric / curcuma, sage, pepper, or wasabi.

[0057] As mentioned, in accordance with the present invention, culturing of plant particles and / or plant cells is preferably done in liquid plant cell medium. Likewise, preferably also the disaggregation of plant tissue to obtain plant particles and the development of plant calli is done in liquid medium. Accordingly, in a preferred embodiment of the present invention, there is no intermediate step of cultivation on solid medium (maybe except for selecting living plant tissue particles which may be done using plates with solid medium as described herein above). In a particular embodiment of the present invention, the development of plant calli is not done on solid medium.

[0058] The present invention further relates to a suspension culture of plant tissue particles developing plant calli obtained by the method as described and provided herein.

[0059] The present invention further relates to a suspension culture of plant cells obtained by the method as described and provided herein, including step (c) where separated plant cells obtained from the plant calli developed by said plant tissue particles of the method described and provided herein are cultured in plant cell medium (preferably liquid plant cell medium). In this context, in a specific embodiment of the present invention, said plant cells may be subjected to sieves of about 100 - 2000 .m, preferably about 100 - 1000 .m, particularly about 300 .m to filter the cells and to induce cell suspension. In another specific embodiment of the present invention, such suspension culture (where plant cells were subjected to sieves of about 100 - 2000 .m, preferably about 100 - 1000 .m, particularly about 300 .m to filter the cells and to induce cell suspension) may contain about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more homogenous plant cells.

[0060] Preferably, a “suspension culture” as referred to herein encompasses

[0061] (i) plant tissue particles developing plant calli obtained from disaggregated plant tissue, i.e. , plant tissue particles obtained in step (a) and cultured in step (b) of the method described and provided herein

[0062] (ii) separated plant cells obtained from plant calli developed by plant tissue particles; i.e., separated cells obtained in step (a), cultured in step (b) and cultured in step (c) of the method described and provided herein

[0063] (iii) plant cells obtained from said separated plant cells, i.e., plant cells obtained in step (a), cultured in step (b), cultured in step (c) and cultured in step (d) of the method described and provided herein.

[0064] Methods to process plant cells in suspension are generally known in the art and also described in, e.g., Gubser (2021), Eng. Life Sci. 21 , 87-98. Methods to adapt plant cells to laboratory conditions are generally known in the art and also described in, e.g., Gallego (2017), Bioprocess Biosyst. Eng. 40, 1479-1492.

[0065] The present invention further relates to biomass from plant cells obtained by the method as described and provided herein, including step (c) of culturing separated plant cells obtained from the plant calli developed by said plant tissue particles in step (b) of the method described and provided herein in (preferably liquid) plant cell medium, as well as steps (d) of culturing plant cells obtained in step (c) in plant cell medium under conditions and for a time sufficient to produce biomass from plant cells and (e) harvesting said biomass.

[0066] In a preferred embodiment of the suspension culture of the present invention as described herein, said suspension culture yields within 32 days of culturing at least 2-fold the amount of biomass dry weight in comparison to plant tissue particles obtained by dry cutting plant tissue and culturing said pieces, preferably said pieces are cultured on solid medium. Said suspension culture preferably comprises

[0067] (i) plant tissue particles developing plant calli obtained from disaggregated plant tissue, i.e., plant tissue particles obtained in step (a) and cultured in step (b) of the method described and provided herein, and / or

[0068] (ii) separated plant cells obtained from plant calli developed by plant tissue particles; i.e., separated cells obtained in step (a), cultured in step (b) and cultured in step (c) of the method described and provided herein.

[0069] In a preferred embodiment of the suspension culture of the present invention as described herein, said suspension culture yields within the same period of time of culturing a higher amount of biomass dry weight in comparison to plant tissue obtained in accordance with the method as described and provided herein, except for omitting in step (a) of the method of the present invention the liquid medium. That is, plant tissue processed for the comparison is disaggregated by dry-cutting, i.e., wound plant tissue is not disaggregated in liquid medium. Said suspension culture preferably comprises

[0070] (ii) separated plant cells obtained from plant calli developed by plant tissue particles; i.e., separated cells obtained in step (a), cultured in step (b) and cultured in step (c) of the method described and provided herein; and / or

[0071] (iii) plant cells obtained from said separated plant cells, i.e., plant cells obtained in step (a), cultured in step (b), cultured in step (c) and cultured in step (d) of the method described and provided herein.

[0072] In another embodiment of the present invention, said suspension culture yields within a period of time of culturing a higher amount of biomass dry weight in comparison to plant tissue obtained in accordance with the method as described and provided herein, except for not having in the liquid medium in step (a) of the method of the present invention anti-oxidative conditions.

[0073] Said suspension culture preferably comprises

[0074] (ii) separated plant cells obtained from plant calli developed by plant tissue particles; i.e., separated cells obtained in step (a), cultured in step (b) and cultured in step (c) of the method described and provided herein; and / or

[0075] (iii) plant cells obtained from said separated plant cells, i.e., plant cells obtained in step (a), cultured in step (b), cultured in step (c) and cultured in step (d) of the method described and provided herein

[0076] Preferably, the “time of culturing” as referred to above is between 28 and 35 days, more preferably 32 days. “Biomass dry weight” is the biomass obtained in accordance with the methods of the present invention after drying. “Biomass” as used herein encompasses plant tissue particles developing plant calli obtained from disaggregated plant tissue; separated plant cells obtained from plant calli developed by plant tissue particles; or plant cells obtained from said separated plant cells. Usually, such biomass is separated from, e.g. culture medium and dried to obtain biomass dry weight.

[0077] For example, when disaggregated plant tissue particles are cultured, they develop plant calli, from which separated cells are obtained as described herein, e.g. separated plant cells are obtained from a filtrate and / or retentate of the suspension culture of plant tissue particles developing plant calli as described herein. Such separated cells then give rise to biomass, in particular biomass dry weight.

[0078] The embodiments which characterize the present invention are described herein, shown in the Figures, illustrated in the Examples, and reflected in the claims.

[0079] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0080] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0081] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all" or any other combination of the elements connected by said term.

[0082] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% or 2% of a given value or range, and also comprises the respective exact numeric value. Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

[0083] When used herein “consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0084] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0085] When used herein, the term “essentially”, e.g., “essentially free of” or “essentially non-[...]” or the like, the term also includes the absolute condition, i.e. “free of”, “non-[...]” or the like, respectively.

[0086] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0087] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The Figures show:

[0088] Figure 1 : shows in the top panel a schematic and mere illustrative overview of the method of the present invention (“RCC method”) for obtaining a suspension culture from plant tissue and in the bottom panel a schematic overview of a typical traditional method for obtaining a suspension culture from plant tissue.

[0089] Examples

[0090] Example 1 - comparison of the RCC method and prior art methods with respect to the time from plant tissue to suspension culture

[0091] Results of the data are summarized in Table 1 .

[0092] Standard method protocol:

[0093] Cacao beans were selected for the induction of cell cultures. Pods of CCN51 were initially disinfected with 2 g / L of carbendazim (378674-Sigma Aldrich) over 12-18 h, followed by an immersion in commercial NaCIO (2 %) over 1 h and ethanol 70 % for 15 minutes. Then the pods were rinsed three times with distil lated water. Seeds were extracted from the pods and the mucilage was manually removed. For the state-of-the-art standard method, the beans were cut into smaller explants (randomized piece sizes) and one bean per agar plate was placed into the solid medium (MS basal medium supplemented with sugar, vitamins and phytohormones). The agar plates were incubated at a 29°C static incubator in darkness. Afterwards the plates were observed regularly to determine the callus growth.

[0094] Rapid cell culture (RCC) method protocol:

[0095] Cacao beans were selected for the induction of cell cultures. Pods of CCN51 were initially disinfected with 2 g / L of carbendazim (378674-Sigma Aldrich) over 12-18 h, followed by an immersion in commercial NaCIO (2 %) over 1 h and ethanol 70 % for 15 minutes. Then the pods were rinsed three times with distil lated water. Seeds were extracted from the pods and the mucilage was manually removed. For RCC, cacao seeds were first chopped in an antioxidant solution (300 ppm ascorbic acid solution) and after blended for 30 seconds using a disaggregator device (e.g. gentleMACS from Miltenyi Biotec). The blended beans were later passed through a sieve and washed twice using media supplemented with 120 mg / L ascorbic acid. Only the retained seed pieces in the sieve were further used in the experiment. The biomass of one blended bean was used per flask and placed in a 125 ml flask containing 30 ml of liquid medium. The shake flasks were incubated in a shaken incubator at 29°C without light. Fifteen days after introduction, shake flasks were sieved. The retentate and the filtrate were both used to induce more cell suspensions and they were allowed to grow for another 7- 10 days. Afterwards the shake flasks were observed regularly to determine the callus growth.

[0096] Table 1

[0097] Example 2 - comparison of the RCC method and a prior art method with respect to biomass

[0098] Cacao beans were selected for the induction of cell cultures. Pods of CCN51 were initially disinfected with 2 g / L of carbendazim (378674-Sigma Aldrich) over 12-18 h, followed by an immersion in commercial NaCIO (2 %) over 1 h and ethanol 70 % for 15 minutes. Then the pods were rinsed three times with distilled water. Seeds were extracted from the pods and the mucilage was manually removed. Cell cultures were established to compare RCC with standard methods and produce primary calli and further cell suspensions. For RCC, cacao seeds were first chopped in an antioxidant solution (ascorbic acid 300 mg / L) and after blended for 30 seconds using a disaggregator device. The blended beans were later passed through a sieve and washed twice using liquid media containing an antioxidant solution (ascorbic acid 120 mg / L). Only the retained seed pieces in the sieve were further used in the experiment. An amount of 1.1 g fresh weight (FW) blended beans were used per flask and placed in a 125mL flask containing 30 mL of liquid medium. For the standard method, cacao beans were cut using scalpel and then 1.1 g FW were weighted and placed on solid medium. A total of 10 flasks and 10 petri dishes were raised for the RCC and standard method respectively.

[0099] Fifteen days after introduction, shake flasks in the RCC method were sieved and the retentateand the filtrate were both used to induce more cell suspensions and were allowed to grow for another 17 days. The length of the experiment for both the RCC and the standard methods was 32 days. After this, fresh weight in g / L was calculated. The biomass from both methods were dried using an oven at 45°C for two days and the dry weight was calculated in g / L. A schematic outline of the experimental design is shown in Figure 1 .

[0100] Results of the data are summarized in Table 2.

[0101] Table 2

[0102] Biomass dry weight after 32 is higher using RCC compared to the standard method.

[0103] The present invention may also be characterized by the following items:

[0104] 1. A method for producing a suspension culture of plant tissue particles developing plant calli, comprising

[0105] (a) wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles in liquid medium;

[0106] (b) directly culturing said plant tissue particles obtained in step (a) in plant cell medium under conditions and for a time sufficient for said plant tissue particles to develop plant calli.

[0107] 2. A method for producing a suspension culture of plant cells, comprising

[0108] (c) culturing separated plant cells obtained from the plant calli developed by said plant tissue particles of the method of item 1 in plant cell medium.

[0109] 3. A method for producing biomass from plant cells, comprising

[0110] (d) culturing plant cells obtained from the method of item 2 in plant cell medium under conditions and for a time sufficient to produce biomass from plant cells;

[0111] (e) harvesting said biomass.

[0112] 4. The method of any one of the preceding items, wherein said part of a plant is root, stem, leaf, flower, fruit or seed.

[0113] 5. The method of item 4, wherein said fruit contains seed and / or pulp.

[0114] 6. The method of item 5, wherein said seed contains endosperm and / or cotyledon tissue.

[0115] 7. The method of any one of the preceding items, wherein said part of a plant of step (a) is extracted under sterile conditions.

[0116] 8. The method of item 6 or 7, wherein said seed is extracted under sterile conditions from the fruit of a plant when extracting endosperm tissue and / or cotyledon tissue.

[0117] 9. The method of item 8, wherein embryonic tissue of said seed is discarded when extracting said endosperm tissue and / or cotyledon tissue.

[0118] 10. The method of item 8 or 9, wherein said endosperm and / or cotyledon tissue is essentially free from embryonic tissue of said seed. 11. The method of any one of the preceding items, wherein in step (a) disaggregating said plant tissue is done mechanically, acoustically, or by light.

[0119] 12. The method of any one of the preceding items, wherein in step (a) said particles are characterized by having multiple injuries on their surface due to said disaggregation.

[0120] 13. The method of item 12, wherein said injuries are scratches and / or bumps.

[0121] 14. The method of item 11, wherein mechanically disaggregating said plant tissue is done by mechanical blending or by mechanical cutting.

[0122] 15. The method of item 14, wherein mechanical blending or mechanical cutting is done under conditions to wound said plant tissue.

[0123] 16. The method of item 14 or 15, wherein mechanical blending is done by a cell disaggregation device.

[0124] 17. The method of any one of items 14 to 16, wherein mechanical blending is done with rotating blades.

[0125] 18. The method of any one of items 14 to 17, wherein mechanical blending results in particles having a size of about 0.1 mm to about 1 mm, preferably about 0.1 mm to about 4 mm.

[0126] 19. The method of item 14 or 15, wherein mechanical cutting is done by a metallic cutting tool.

[0127] 20. The method of item 14, 15 or 19, wherein mechanical cutting results in slices having a thickness of about 1 to 5 mm.

[0128] 21. The method of any one of the preceding items, wherein in step (a) disaggregating plant tissue is done under essentially non-oxidative conditions.

[0129] 22. The method of item 21, wherein said non-oxidative conditions are achieved by adding an anti-oxidative agent to the liquid medium of step (a) and, optionally a washing step (a’) with an anti-oxidative agent. 23. The method of any one of the preceding items, wherein said liquid medium of step (a) confers anti-oxidative conditions.

[0130] 24. The method of item 23, wherein said liquid medium of step (a) contains an anti-oxidative agent.

[0131] 25. The method of any one of the preceding items, wherein disaggregating said plant tissue into plant tissue particles in liquid medium is done by keeping said plant tissue and plant tissue particles submerged.

[0132] 26. The method of any one of the preceding items, wherein the liquid medium in step (a) is defined plant cell medium.

[0133] 27. The method of any one of the preceding items, wherein in step (a) a cell wall degrading enzyme is added.

[0134] 28. The method of any one of items 1 to 27, further comprising step (a’) directly washing said disaggregated plant tissue obtained in step (a) prior to directly culturing said disaggregated plant tissue in plant cell medium in step (b).

[0135] 29. The method of item 28, wherein said washing is done by a wash solution comprising an anti-oxidative agent.

[0136] 30. The method of any one of items 2 to 29, wherein separated plant cells are obtained from a filtrate and / or retentate of the suspension culture of plant call i of the method of item 1.

[0137] 31. The method of any one of the preceding items, wherein step (d) and / or step (e) is carried out in a bioreactor selected from a stirred tank bioreactor, an orbitally shaken bioreactor, a bubble column bioreactor, a wave reactor or an air-lift bioreactor.

[0138] 32. The method of any one of the preceding items, wherein said plant cells are from a monocotyledonous or dicotyledonous plant.

[0139] 33. The method of item 32, wherein said monocotyledonous plant is wheat, barley, rye, oat or a palm tree. The method of item 32, wherein said dicotyledonous plant is coffee, cacao, nuts, fruits, avocado, berries acai, blueberry, grape or citrus fruits, herbs, spices, e.g. vanilla, saffron, guarana, turmeric / curcuma, sage, pepper or wasabi. The method of any one of the preceding items, wherein development of plant calli is not done on solid medium. A suspension culture of plant tissue particles developing plant calli obtained by the method of any one of items 1 and 4 to 35. A suspension culture of plant cells obtained by the method of any one of items 2 and 4 to 35. The suspension culture of plant cells of item 37, wherein said plant cells are subjected to sieves of about 300 .m to filter the cells and to induce cell suspension. The suspension culture of plant cells of item 37 or 38, which contains 50% or more homogenous plant cells. The suspension culture of any one of items 36 to 39, wherein said suspension culture yields within 32 days of culturing at least 2-fold the amount of biomass dry weight in comparison to plant tissue particles obtained by dry cutting plant tissue and culturing said pieces, preferably said pieces are cultured on solid medium. The suspension culture of any one of items 36 to 39, wherein said suspension culture yields within the same period of time of culturing a higher amount of biomass dry weight in comparison to plant tissue obtained in accordance with the method of any one of items 1 to 35 except for omitting in step (a) the liquid medium. The suspension culture of any one of items 36 to 39, said suspension culture yields within a period of time of culturing a higher amount of biomass dry weight in comparison to plant tissue obtained in accordance with the method of any one of items 1 to 35 except for not having in the liquid medium in step (a) anti-oxidative conditions. Biomass from plant cells obtained by the method of any one of items 3 to 35.

Claims

Claims1. A method for producing a suspension culture of plant tissue particles developing plant calli, comprising(a) wounding plant tissue obtained from a part of a plant by disaggregating said plant tissue into plant tissue particles in liquid medium;(b) directly culturing said plant tissue particles obtained in step (a) in plant cell medium under conditions and for a time sufficient for said plant tissue particles to develop plant calli.

2. A method for producing a suspension culture of plant cells, comprising(c) culturing separated plant cells obtained from the plant calli developed by said plant tissue particles of the method of claim 1 in plant cell medium.

3. A method for producing biomass from plant cells, comprising(d) culturing plant cells obtained from the method of claim 2 in plant cell medium under conditions and for a time sufficient to produce biomass from plant cells;(e) harvesting said biomass.

4. The method of any one of the preceding claims, wherein said part of a plant is root, stem, leaf, flower, fruit or seed.

5. The method of any one of the preceding claims, wherein in step (a) disaggregating said plant tissue is done mechanically, acoustically, or by light.

6. The method of claim 5, wherein mechanically disaggregating said plant tissue is done by mechanical blending or by mechanical cutting.

7. The method of any one of the preceding claims, wherein in step (a) disaggregating plant tissue is done under essentially non-oxidative conditions.

8. The method of claim 7, wherein said non-oxidative conditions are achieved by adding an anti-oxidative agent to the liquid medium of step (a) and, optionally a washing step (a’) with an anti-oxidative agent.

9. The method of any one of the preceding claims, wherein disaggregating said plant tissue into plant tissue particles in liquid medium is done by keeping said plant tissue and plant tissue particles submerged.

10. A suspension culture of plant tissue particles developing plant calli obtained by the method of any one of claims 1 and 4 to 9.

11. A suspension culture of plant cells obtained by the method of any one of claims 2 and 4 to 9.

12. The suspension culture of plant cells of claim 11 , wherein said plant cells are subjected to sieves of about 100 - 2000 .m to filter the cells and to induce cell suspension.

13. The suspension culture of any one of claims 10 to 12, wherein said suspension culture yields within 32 days of culturing at least 2-fold the amount of biomass dry weight in comparison to plant tissue particles obtained by dry cutting plant tissue and culturing said pieces, preferably said pieces are cultured on solid medium.

14. The suspension culture of any one of claims 10 to 12, wherein said suspension culture yields within the same period of time of culturing a higher amount of biomass dry weight in comparison to plant tissue obtained in accordance with the method of any one of claims 1 to 9 except for omitting in step (a) the liquid medium.

15. The suspension culture of any one of claims 10 to 12, said suspension culture yields within a period of time of culturing a higher amount of biomass dry weight in comparison to plant tissue obtained in accordance with the method of any one of claims 1 to 9 except for not having in the liquid medium in step (a) anti-oxidative conditions.

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