Intergrafting tool and method of generating an intergraft using the same

Abstract

The invention relates an intergrafting tool (1) for supporting a plant during an intergrafting process and to a method of generating a shoot of a plant using the same. The intergrafting tool (1) comprising a body (12) extending in a first direction (X); a retaining portion (14) projecting frontally from the body (12) and including at least two opposing retaining elements (15a, 15b) formed by two parallel bars extending in the first direction (X), and positioned next to each other in a second direction (Y) substantially perpendicular to the first direction (X), the at least two opposing retaining elements (15a, 15b) being configured to at least partially surrounding a stem of the plant and apply a gripping force thereto to secure the plant to the body (12); and an intergraft seat (16), located at a central region of the retaining portion (14) and providing frontal and lateral access to an interior of the retaining portion (14), thereby exposing a stem segment when the plant is secured to the body (12). The invention further relates to a method of generating an intergraft using the intergrafting tool and to an intergrafting tool array comprising said intergrafting tool.

Description

[0001] Intergrafting tool and method of generating an intergraft using the same

[0002] Field of the invention

[0003] The invention relates an intergrafting tool for supporting a plant during an intergrafting process and to a method of generating an intergraft using the same. The invention further relates to an intergrafting tool array comprising the intergrafting tool.

[0004] Background

[0005] In vitro micrografting is a crucial step in emerging tissue technology processes for various crop improvement technologies, such as for the production of graft hybrids (e.g., periclinal chimeras) with improved biotic stress resistance and / or capable of producing seed with improved germination properties, and for regeneration of recalcitrant plants (WO2018 / 1 15395; WO2018 / 115396; WO2022 / 219181 ; WO2024 / 133851). Many of these applications rely on or benefit from a process called intergrafting, which often involves inserting callus tissue (such as internode segments or calli or internode segments) into the stem, preferably between a scion and rootstock under aseptic conditions. In such protocols, the scion and rootstock are preferably of young plants, preferably seedlings of between 1-4 weeks after sowing.

[0006] Hence, a significant limitation is the small size of the materials involved, typically around 1 mm, which makes the grafting and handling processes challenging. The commercial advancement of grafting technologies would greatly benefit from new, scalable methods for in vitro intergrafting that are both practical and easy to implement.

[0007] Grafting tools for holding a plant are known, they are generally designed for traditional stock-scion grafts (i.e., root-shoot grafts). For example, NL9300625A discloses a tool for grafting seedlings, comprising a hollow cylindrical component with a vertical gap and an attachment mechanism for securing the tool to a stalk.

[0008] It is an object of the present invention to overcome or mitigate one or more of the disadvantages from the prior art.

[0009] Summary

[0010] In a first aspect of the invention, there is provided an intergrafting tool for supporting a plant during an intergrafting process, the intergrafting tool comprising a body extending in a first direction; a retaining portion projecting frontally from the body and including at least two opposing retaining elements formed by two parallel bars extending in the first direction, and positioned next to each other in a second direction substantially perpendicular to the first direction, the at least two opposing retaining elements being configured to at least partially surrounding a stem of the plant and apply a gripping force thereto to secure the plant to the body; and an intergraft seat, located at a central region of the retaining portion and providing frontal and lateral access to an interior of the retaining portion, thereby exposing a stem segment when the plant is secured to the body.

[0011] The intergrafting tool is specifically configured for intergrafting plant material. Examples of intergrafted plant material are callus or internode segments. The intergraft seat provides frontal and lateral access to an interior of the retaining portion to facilitate the intergrafting process while providing a secure retention of the stem. The intergraft seat also facilitates the separation of a section of the stem for subsequent insertion of the intergrafted plant material and without exerting pressure on the intergrafted plant material, while the remainder of the plant is securely held. Advantageously, the intergrafting tool allows for the precise insertion of the intergrafted plant material and later removal of the plant stem after the intergrafting process is finalised. By virtue of the retaining portion, particularly the two opposing retaining elements formed by parallel bars, the tool applies a distributed gripping force across the stem surface, reducing localized pressure, an important advantage for small and / or fragile plants. This configuration is especially suited for intergrafting because the parallel bars stabilize the stem sections adjacent to the intergraft seat, keeping them in a fixed and aligned position throughout the process. This stability is critical when removing a stem segment and inserting intergrafted plant material between the remaining sections, as it prevents wobbling or misalignment during cutting and placement.

[0012] Furthermore, the retaining portion of the intergrafting tool secures the plant in such a manner that it can be positioned on a growth medium either vertically or horizontally for subsequent growth. After inserting the intergrafted plant material, a graft union forms between the stem sections and the intergrafted plant material. Positioning the plant vertically offers the advantage of maintaining its natural growth orientation, preserving polarity and vascular alignment of the stem sections. This alignment supports more efficient nutrient transport and increases the likelihood of a successful graft union formation.

[0013] The graft union may be wounded at or near a graft junction. Optionally, wounding may involve complete separation of the grafted scion from the rootstock. Advantageously, the intergrafting tool facilities access of, e.g., a scalpel, to make a cut in the intergrafted material near the graft junction to achieve said separation. The dissected stems can then be transferred to a culture medium for shoot growth. The benefit of the design of the intergrafting tool, is that the resilience coefficient of the retaining portion and the body may be similar, preferably the resilience coefficient of the retaining portion and the body are identical. Hence, the retaining portion and the body may be made of the same material. Preferably, said material is a flexible polymer or an elastomer. The skilled person will recognize that even with similar or identical materials, differences in geometry, thickness, and shape can affect the elasticity of the body and / or the retaining portion. For example, the retaining portion may be configured to have a desired elasticity, making it easier to insert the plant stem.

[0014] In a further preferred embodiment, the retaining portion and the body may be integrally connected. In other words, the retaining portion and the body may be fabricated as a single piece.

[0015] . The retaining elements may be independent of each other and / or may only connect indirectly through the body. In some embodiments, where the retaining portion has a different resilience coefficient from the body, partial deformation of one of the retaining elements does not affect the other, allowing for a more controlled placement and removal of the plant in and out of the retaining portion. In a further embodiment, each of the two parallel bars has a cross section with a substantial quadrilateral shape. The quadrilateral shape of the cross section provides enhanced stability and strength, ensuring that the retaining elements can perform their function effectively. The quadrilateral shape offers increased structural integrity and resistance to deformation, which is beneficial for maintaining the positioning and securement of the plant stem during the intergrafting process.

[0016] In an embodiment, the retaining portion may further comprise an elongated cavity extending in the first direction and configured for receiving the stem. The elongated cavity can more appropriately accommodate the stem of the plant, particularly the scion and rootstock of the plant. The length of the elongated cavity may vary depending on the plant, for example, it may have a length of 10.0 mm to 30.0 mm, preferably of 15.0 mm to 25.0 mm.

[0017] In an embodiment, the elongated cavity may have a cross-section with a substantial trapezoidal shape, wherein preferably an opening of the elongated cavity corresponds to the small base of the trapezoidal shape. This configuration provides secure retention with a simple shape that is easy to manufacture using various techniques such as 3D printing, moulding, and similar methods. The trapezoidal shape further provides an increasing grip at the opening, which holds the plant securely while preventing damage and allows for securing stems that may vary in diameter while still providing a secure and gentle grip. The dimensions of the trapezoidal shape may also vary depending on the plant. In a further embodiment, the large base of the trapezoidal shape of the elongated cavity may have a width of 0.5 mm to 4.0 mm, preferably of 0.8 mm to 1 .2 mm and / or the small base of the trapezoidal shape of the elongated cavity may have a width of 0.2 mm to 2.0 mm, preferably of 0.1 mm to 0.5 mm.

[0018] In an embodiment, the retaining portion may extend in the first direction, and the intergraft seat may comprise a slot crossing the central region of the retaining portion, preferably extending in a second direction substantially perpendicular to the first direction. The intergraft seat comprising a slot in the second direction is particularly advantageous as it provides lateral space and access for introducing the intergrafted plant material while securing the other sections of the stem held by the retaining portion. Additionally, a slot is easy and cost-effective to manufacture with known processes like the ones previously mentioned.

[0019] In an embodiment, the intergraft seat may have a length of 0.5 mm to 10.0 mm, preferably of 1 .0 mm to 3.0 mm. The length of the intergraft seat refers to the dimension that runs in the first direction (X), which is appropriate for receiving the intergrafted plant material and allowing proper manipulations during the intergrafting process. The length of the intergraft seat may also vary depending on the plant and / or the intergrafting material.

[0020] In an embodiment, the retaining portion may comprise a resilient material, such as silicone. Alternatively, the retaining portion may consist of silicone. Other materials with similar resilience coefficients may be used, for example, thermoplastic elastomers (TPE), natural rubber, (elasticated) polyurethane, or nitrile rubber, either alone or in combination. Similarly, the body may comprise or consist of a similar material, including silicone, thermoplastic elastomers (TPE), natural rubber, polyurethane, or nitrile rubber. Preferably, the material is platinum cured silicone. In an embodiment, the interg rafting tool may be autoclavable. This allows the intergrafting tool to be reusable. In addition, sterilization in some cases could be critical in maintaining plant health and maintaining the success rate of grafting procedures, particularly in environments where multiple plants are handled.

[0021] According to the invention, there is further provided a mould for forming an intergrafting tool according to the first aspect. The mould allows for precise control over the dimensions and geometry of the tool, ensuring consistency and accuracy in manufacturing. The use of a mould enables efficient mass production, reducing manufacturing costs and time. Additionally, the mould can accommodate multi-material or co-moulding processes, allowing the creation of integrally connected components with differing resilience coefficients.

[0022] According to the invention, there is further provided an intergrafting tool array comprising a plurality of intergrafting tools according to the first aspect, wherein the intergrafting tools are arranged in series in the second direction, with their bodies connected adjacent to each other, and with their retaining portions spaced apart from one another and aligned parallel to the first direction, thereby enabling support of a plurality of plants with their stems aligned in the first direction, wherein preferably the bodies of the plurality of intergrafting tools are integrally connected to each other.

[0023] According to another aspect of the invention there is provided a method of generating an graft union using the intergrafting tool according to the first aspect, the method comprising:

[0024] (a) inserting a stem of a plant into the retaining portion of the intergrafting tool;

[0025] (b) removing or separating a stem segment located in the graft seat, thereby forming two stem sections and a gap in the stem therebetween,

[0026] (c) placing plant material in the intergraft seat and within the gap between the two stem sections; and

[0027] (d) allowing for a graft junction to be formed between the inserted plant material of step c and each of the two stem sections of step (b).

[0028] The stem of step (a) may be a full plant, preferably a seedling, and the two stem sections formed in step (b) may be a scion and a rootstock. The method involves using the intergrafting tool to generate a shoot of a plant. A stem of the plant can be inserted into the retaining portion of the intergrafting tool, which securely holds the stem in place during the intergrafting process. The retaining portion is configured to hold the stem in any desired orientation without causing damage.

[0029] Next, a stem segment located in the intergraft seat is removed and / or separated, creating two sections of the stem (scion and rootstock) and a gap between them. The stem segment to be removed may have substantially the same length as the intergraft seat. The intergraft seat is a designated area in the tool where the plant material will be placed. The removal of the stem segment prepares the remaining of the plant for intergrafting by creating space for the new plant material.

[0030] Finally, plant material, such as a callus, is placed into the intergraft seat and positioned within the gap between the two stem sections. This allows the grafting material to come into contact with the vascular system of the stem sections held by the intergrafting tool and create a graft junction. A graft union is formed as soon as graft junctions are formed between the two sections of the stem and the intergrafted plant material. In subsequent steps, the graft union can be wounded at or near at least one of the graft junctions. Optionally, said wounding entails complete separation of the grafted scion from the rootstock, preferably near one of the graft junctions formed. For this, a cut may be made in the ingrafted plant material near the side of the junction. The intergrafting tool allows for the access of a scalpel to the intergrafting seat to make such cut. Subsequently, the dissected stems can be easily taken out of the intergrafting tool and be transferred to culture medium for shoots to grow.

[0031] In an embodiment, the stem sections respectively may correspond to a scion and a rootstock of the plant and the plant material in the intergraft seat comprises a intergrafted plant material of another plant. Advantageously, the intergraft seat holds the scion and the rootstock of the plant ensuring proper alignment for graft junctions to be formed between the intergrafted plant material and each one of the scion and the rootstock, to form a grafted union.

[0032] Definitions

[0033] 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.

[0034] It is clear for the skilled person that any methods and materials similar or equivalent to those described herein can be used for practising the present invention.

[0035] The singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. The indefinite article "a" or "an" thus usually means "at least one".

[0036] The term “comprising” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0037] "Plant" refers to either the whole plant or to parts of a plant tissue or organs (e.g. pollen, seeds, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progeny derived from such a plant by selfing or crossing or apomictic reproduction. Non-limiting examples of plants include crop plants and cultivated plants, such as African eggplant, alliums, artichoke, asparagus, barley, beet, bell pepper, bitter gourd, bladder cherry, bottle gourd, cabbage, canola, carrot, cassava, cauliflower, celery, chicory, common bean, corn salad, cotton, cucumber, eggplant, endive, fennel, gherkin, grape, hot pepper, lettuce, maize, melon, oilseed rape, okra, parsley, parsnip, pepino, pepper, potato, pumpkin, radish, rice, ridge gourd, rocket, rye, snake gourd, sorghum, spinach, sponge gourd, squash, sugar beet, sugar cane, sunflower, tomatillo, tomato, tomato rootstock, vegetable Brassica, watermelon, wax gourd, wheat and zucchini.

[0038] A “scion” is a shoot of a plant, preferably a young shoot preferably of a young plant, cut for grafting. A scion preferably has a functional shoot apical meristem. A “rootstock” is a stem of a plant, preferably of a young plant, with a functional root system preferably comprising a functional root apical meristem.

[0039] Brief description of the drawings

[0040] Embodiments of the present invention will be described hereinafter, by way of example only, with reference to the accompanying drawings which are schematic in nature and therefore not necessarily drawn to scale.

[0041] In the drawings, identical or similar elements are indicated by the same reference sign.

[0042] Figure 1 schematically shows a front view of an intergrafting tool according to an embodiment of the invention;

[0043] Figure 2A schematically shows an upper view of the intergrafting tool of Figure 1 ;

[0044] Figure 2B schematically shows a side view of the intergrafting tool of Figure 1 ;

[0045] Figure 3 schematically shows an intergrafting tool array with four adjacent intergrafting tools connected in series;

[0046] Figure 4A and 4B schematically show a perspective view and a section of a mould for forming the intergrafting tool array of Figure 3.

[0047] Detailed description of embodiments

[0048] The following is a description of certain embodiments of the invention, given by way of example only and with reference to the figures. In the drawings, identical or similar elements are indicated by the same reference sign.

[0049] Figure 1 schematically shows a front view of an intergrafting tool according to an embodiment of the invention. The intergrafting tool 1 is shown in use supporting the stem of a plant P. The intergrafting tool 1 comprises a body 12 that extends in a first direction X. The body 12 has a length (L) along the first direction X and provides a stable base. In the depicted embodiment the body 12 has a squared or prismatic shape.

[0050] A retaining portion 14 is projecting from a front of the body 12 and is configured to surround and secure the stem of the plant P. The retaining portion 14 serves as the primary structure for supporting the plant during the intergrafting process and it is arranged to engage the stem of the plant P by partially surrounding it. An intergraft seat 16 is located at a central region of the retaining portion 14 which is configured for providing access to a stem segment of the plant P. The intergraft seat 16 allows for frontal and lateral access to the interior of the retaining portion 14, and thereby exposing the stem segment. The retaining portion 14 includes two opposing retaining elements 15a and 15b extending parallel to each other along the first direction X and positioned next to each other in the first direction X. An elongated cavity 17 within the retaining elements 15a and 15b also extends along the first direction X and is configured to receive the stem while the retaining elements 15a and 15b provide a gripping force to the stem. The body may have a length (first direction) of 10.0 mm to 30.0 mm, preferably of 15.0 mm to 25.0 mm, more preferably of 18.0 mm to 22.0 mm and / or a width (second direction) of 6.0 mm to 20.0 mm, preferably of 10.0 mm to 15.0 mm. The body may have a depth or thickness (frontal direction) of 3.0 mm to 10.0 mm, preferably of 4.0 mm to 6.0 mm. The retaining portion may have the same length as the body or may be different, for example, the retaining portion may have a length (first direction) of 10.0 mm to 30.0 mm, preferably of 15.0 mm to 25.0 mm, more preferably of 18.0 mm to 22.0 mm. The retaining portion may project 1 .0 mm to 10.0 mm frontally from the body, more preferably 2.0 mm to 4.0 mm.

[0051] Figure 2A schematically shows an upper view of the intergrafting tool 1 of Figure 1 . This view shows the retaining portion 14 projecting from the body 12. The retaining elements 15a and 15b are parallel bars with a quadrilateral cross-section having the elongated cavity 17 therebetween. The elongated cavity 17 has a trapezoidal cross-section, with the small base positioned at the front of the retaining portion 14 and the large base located at the rear, where the cavity 17 connects to the body 12. Each of the retaining elements 15a and 15b has a width of 3.0 mm to 8.0 mm, preferably of 5.0 mm to 6.0 mm.

[0052] Figure 2B schematically shows a side view of the intergrafting tool 1 of Figure 1 . The intergraft seat 16 includes a slot extending in the second direction Y, crossing the central region of the retaining portion 14. In the depicted embodiment, the slot of the intergraft seat 16 has a squared cross-section; however, alternative cross-sectional shapes, such as rounded or trapezoidal, may also be used to achieve the same functionality.

[0053] Figure 3 schematically shows an intergrafting tool array 2 with five intergrafting tools 1 connected in series along the second direction Y. The bodies 12 of the intergrafting tools 1 are connected adjacent to each other and with their retaining portions 14 spaced apart from one another and aligned parallel to the first direction X. The intergrafting tool array 2 enables support of a plurality of plants with their stems aligned in the first direction X. In the depicted embodiment, wherein preferably the bodies 12 of the plurality of intergrafting tools 1 are integrally connected to each other Multiple intergrafting tools 1 can be arranged in a side-by-side configuration with their retaining portions 14 facing frontally. The bodies 12 of each intergrafting tool 1 are aligned in the first direction X, such as plant stems can be supported parallel to one another. A space between the retaining portions 14 of 5.0 mm to 15.0 mm, preferably of 7.0 mm to 11.0 mm.

[0054] In the depicted embodiments of Figures 1 to 3, the body 12 of the intergraft tool(s) 1 has a length of about 20.0 mm, a width of about16.0 mm, and a depth of about 5.0 mm; the retaining portion 14 has a length of about 20.0 mm; the intergraft seat 16 has a length of about 2.0 mm; the retaining portion 14 projects about 3.0 mm frontally from the body 12; the retaining elements 15a and 15b each has a width of about 5.25 mm; the length of the elongated cavity 17 is about 20.0 mm; the large base of the trapezoidal shape is 1 .0 mm and the small base of the trapezoidal shape is about 0.37 mm.

[0055] Figure 4A schematically shows a perspective view of a mold 3 for forming the intergrafting tool array 2 depicted in Figure 3. The mold is configured to create the intergrafting tool array 2 with precise dimensions, matching the shape and dimensions of the intergrafting tool 1 in a negative form. The mold 3 includes cavities corresponding to the body 12 and retaining portions 14. Figure 4B schematically shows a section of the mold, highlighting the detailed internal structure. The section illustrates the negative trapezoidal cross-section of the elongated cavity 17 and of the slot for forming the intergraft seat 16. The mold also includes cavities for shaping the body 12 and retaining elements 15a and 15b. The mold is constructed from any suitable durable and heat- resistant materials such as metal alloys (e.g., stainless steel or aluminum) or high-strength polymer composites, selected based on their compatibility with molding silicone or similar resilient materials used for the intergrafting tool 1. In the depicted embodiment, the mold has a total length of about 30.0 mm, a total width of about 110.0 mm and a depth of about 12.0 mm.

Claims

9Claims1. An intergrafting tool (1) for supporting a plant during an intergrafting process, the intergrafting tool comprising: a body (12) extending in a first direction (X); a retaining portion (14) projecting frontally from the body (12) and including at least two opposing retaining elements (15a, 15b) formed by two parallel bars extending in the first direction (X), and positioned next to each other in a second direction (Y) substantially perpendicular to the first direction (X), the at least two opposing retaining elements (15a, 15b) being configured to at least partially surrounding a stem of the plant and apply a gripping force thereto to secure the plant to the body (12); and an intergraft seat (16), located at a central region of the retaining portion (14) and providing frontal and lateral access to an interior of the retaining portion (14), thereby exposing a stem segment when the plant is secured to the body (12).

2. The intergrafting tool of claim 1 , wherein a resilience coefficient of the retaining portion (14) is identical to a resilience coefficient of the body (12), preferably wherein the retaining portion (14) and the body (12) are integrally connected.

3. The intergrafting tool of claim 1 or claim 2, wherein each of the two parallel bars has a cross section with a substantial quadrilateral shape.

4. The intergrafting tool of any preceding claim, wherein the retaining portion (14) further comprises an elongated cavity (17) extending in the first direction and configured for receiving the stem.

5. The intergrafting tool of claim 4, wherein the elongated cavity (17) has a cross section with a substantial trapezoidal shape, wherein an opening of the elongated cavity (17) corresponds to the small base of the trapezoidal shape.

6. The intergrafting tool of claim 5, wherein the large base of the trapezoidal shape of the elongated cavity (17) has a width (WL) of 0.5 mm to 4.0 mm and / or the small base of the trapezoidal shape of the elongated cavity (17) has a width (Ws) of 0.2 mm to 2.0 mm, preferably of 0.1 mm to 0.5 mm.

7. The intergrafting tool of any preceding claim, wherein the retaining portion (14) extends in the first direction (X), and the intergraft seat (16) comprises a slot crossing the central region of the retaining portion (14), preferably extending in a second direction (Y) substantially perpendicular to the first direction (X).

8. The intergrafting tool of any preceding claim, wherein the intergraft seat (16) has a length of 0.5 mm to 10.0 mm, preferably of 1 .0 mm to 3.0 mm.

9. The intergrafting tool of any preceding claim, wherein the retaining portion (14) comprises a resilient material, such as silicone.

10. The intergrafting tool of any preceding claim, wherein the intergrafting tool is autoclavable.

11. A mould for forming an intergrafting tool according to any one of the preceding claims 1- 10.

12. An intergrafting tool array (2) comprising a plurality of intergrafting tools (1) according to any one of the claims 1-10, wherein the intergrafting tools (1) are arranged in series in the second direction (Y), with their bodies (12) connected adjacent to each other, and with their retaining portions (14) spaced apart from one another and aligned parallel to the first direction (X), thereby enabling support of a plurality of plants with their stems aligned in the first direction (X), wherein preferably the bodies (12) of the plurality of intergrafting tools (1) are integrally connected to each other.

13. A method of generating a graft union using the intergrafting tool of any of the claims 1-10, the method comprising:(a) inserting a stem of a plant into the retaining portion (14) of the intergrafting tool (1);(b) removing or separating a stem segment located in the graft seat (16), thereby forming two stem sections and a gap in the stem therebetween,(c) placing plant material in the intergraft seat (16) and within the gap between the two stem sections,(d) allowing for a graft junction to be formed between the inserted plant material of step (c) and each of the two stem sections of step (b).

14. The method of claim 13, wherein the stem sections respectively correspond to a scion and a rootstock of the plant and the plant material in the intergraft seat comprises an intergrafted plant material of another plant.

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