Stimulating germination of plants

Abstract

The present document relates to the field of agriculture and a method for stimulating germination of plants. More specifically, the present document relates to a method for seed soaking of seeds to stimulate their germination, a composition for such use and seeds after seed soaking. The method comprises soaking the seeds in an aqueous solution comprising oxytocin, a fragment thereof or a variant thereof, wherein the seeds are not allowed to germinate in the oxytocin solution. The result shows that this results in a stronger growth enhancing effect on the seeds.

Description

Technical Field

[0001] This article relates to the agricultural field and a method for stimulating plant germination. Background Technology

[0002] Successful seed germination is crucial for a bumper agricultural harvest. Not only does successful germination result in a greater number of plants, but it can also be important for suppressing weeds that compete with seeds for space and nutrients.

[0003] A seed contains an embryo; one end of the embryo will form a root, called the radicle, and the other end will form a stem and leaves, called the plumule. Monocotyledonous plants contain one cotyledon, which is the seed leaf that occupies a small portion of the seed. Dicotyledonous plants contain two cotyledons.

[0004] When monocotyledonous plants germinate, the radicle grows downward through the split seed coat to produce primary roots, while the bud, which is wrapped in its protective sheath (coleoptile), grows upward.

[0005] In dicotyledonous plants, primary roots emerge from the seed, enabling the new plant to absorb water. Apical meristem develops from this root system, producing the plant's root system. Then, a bud emerges from the seed. A bud in a dicotyledonous plant consists of cotyledons, a hypocotyl, and an epicotyl.

[0006] In gymnosperms (such as conifers), after the embryo absorbs water, the seedling roots first break through the seed coat and descend into the soil. Subsequently, the stem below the cotyledons elongates. As the cotyledons begin photosynthesis, the buds begin to grow.

[0007] Plant germination and growth can be stimulated using plant hormones such as auxins, gibberellins, cytokinins, and abscisic acid. However, these agents are expensive and can be toxic when used in large quantities.

[0008] WO02 / 102160 discloses stimulating plant germination by inducing seed germination in a solution containing the human hormone oxytocin. However, germinating seeds in a solution containing oxytocin can be both expensive and impractical for germinating large quantities of seeds. Seed soaking is a more cost-effective alternative to germination for plant producers and growers compared to direct germination.

[0009] Therefore, the object of the present invention is to provide improved methods and means for stimulating plant germination and / or growth by specifically treating seeds before germination and providing growers with novel and feasible alternatives. Summary of the Invention

[0010] This article relates to a method for soaking seeds, such as seeds from monocotyledonous or dicotyledonous plants or gymnosperms (conifers), the method comprising the following steps:

[0011] a) Provide seeds; b) Provide an aqueous solution containing oxytocin and / or its variants and / or fragments, typically with a concentration of 0.5–50 μM oxytocin; c) Soak the seeds in the aqueous solution containing oxytocin; d) Remove the seeds from the aqueous solution containing oxytocin. The oxytocin fragments and / or variants thereof are oxytocin-active fragments and / or variants according to SEQ ID NO: 2. Where SEQ ID NO:2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 in X1 selects a group consisting of free Cys and non-existent groups; X2 selects a group consisting of Tyr, Phe, Leu and no other group; X3 selects a group consisting of Ile, Val, Hoph, Phe, Cha, and the group that does not exist; X4 can be selected from groups consisting of Gln, Ser, Thr, Cit, Arg, and Daba. X5 selects FreePro and the group that does not exist; X6 selects the group consisting of Ile, Leu, non-existent, Val, Hos, Daba, Thr, Arg, and Cit; X7 selects the group consisting of Gly, non-existent, and Ala; X8 selects groups consisting of Gly and non-existent elements.

[0012] This article also provides seeds that can be obtained or acquired using the methods described above.

[0013] The soaking in step c) usually takes a period of time, from a few minutes (e.g., about 1-5 minutes) to about 72 hours, for example from about 5 minutes to about 72 hours, for example from about 15 minutes to about 72 hours, for example from about 0.5 hours to about 72 hours, for example from about 1 hour to about 48 hours, from about 1 hour to about 24 hours, from about 12 hours to about 48 hours, or from about 12 hours to about 24 hours.

[0014] Optionally, the method may further include step e) drying the seeds.

[0015] The seeds can be of any kind. Non-restrictive types of seeds include seeds from monocotyledonous or dicotyledonous plants or seeds from gymnosperms.

[0016] Monocotyledonous seeds can be grains, such as wheat, rye, oats, barley, millet, and / or spelt wheat seeds.

[0017] Dicotyledonous seeds can be, for example, quinoa seeds, legume seeds such as peas, sunflower seeds, dill seeds, and / or birch seeds.

[0018] Gymnosperm seeds can be, for example, seeds from coniferous trees such as pine, cedar, larch, or spruce.

[0019] Oxytocin concentrations are typically 0.5-50 μM, such as 1-50 μM, 1-30 μM, 1-20 μM, 1-10 μM, 1-8 μM, 2-8 μM, 1-6 μM, 1-5 μM, 2-6 μM, 3-5 μM, or 2-4 μM.

[0020] The aqueous solution of oxytocin may further contain one or more liquid and / or solid carriers, excipients, organic fertilizers and / or agricultural agents, such as plant hormones.

[0021] This article also provides a method for stimulating the germination and / or growth of the roots and / or coleoptiles / buds of seeds, the method comprising the following steps: i) Soaking the seeds by a method including the following steps: a) Provide seeds, such as seeds from monocotyledonous or dicotyledonous plants or gymnosperms; b) Provide an aqueous solution containing oxytocin and / or its variants and / or fragments as defined herein, typically at a concentration of 0.5–50 μM oxytocin; c) Soak the seeds in the aqueous solution containing oxytocin; d) Remove the seeds from the aqueous solution containing oxytocin and optionally rinse and / or dry the seeds; and ii) Germinate the seed.

[0022] It is also provided that an aqueous solution containing oxytocin and / or its variants and / or fragments as described herein is used for soaking seeds and / or stimulating germination and / or growth of the roots and / or coleoptiles of seeds, such as coniferous, monocotyledonous or dicotyledonous seeds.

[0023] Other features and advantages of the present invention will become apparent from the following detailed description, drawings, embodiments and claims.

[0024] definition In this document, "soaking" and the like refer to immersing seeds in a solution to allow them to absorb water and / or allow substances in the solution to be absorbed by the seeds before allowing them to germinate. In this document, the term "soaking" and the like may be used in place of "soaking" and the like.

[0025] Oxytocin (SEQ ID NO: 1) is understood to have the following chemical structure.

[0026]

[0027] Whenever “oxytocin” is used herein, the term is also understood to encompass fragments, variants, or homologs of oxytocin molecules / peptides that contain biological activities equivalent to those of the oxytocin molecule itself (SEQ ID NO: 1) or plant cyclic peptides. Variants and / or fragments of oxytocin can, for example, have substantially the same stimulatory effect on seed germination and / or plant growth after soaking as when soaking with oxytocin itself.

[0028] Therefore, the term "variant" of oxytocin as used herein refers to a peptide whose amino acid structure has been altered compared to the oxytocin molecule. This is because certain amino acid positions may have been changed by introducing other amino acids, such as the natural or non-natural amino acids exemplified herein, or it may have been elongated by adding one or more natural or non-natural amino acids to either end of the peptide. Furthermore, other structural changes, such as synthetic modifications, may be made to the peptides of the invention as referred to herein. The "variant" retains biological activity similar to oxytocin, and the oxytocin variant is stabilized by its presence in the pharmaceutical compositions according to the invention. The term "variant" of oxytocin as used herein may exclude vasopressin (SEQ ID NO: 7). Vasopressin is a nonapeptide with the sequence Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly, wherein cysteine ​​residues form disulfide bonds.

[0029] Furthermore, the term "fragment" of oxytocin as used herein refers to a peptide containing a portion of the amino acid sequence of oxytocin, but in which one or more amino acids may have been removed from one or both amino terms. The term also refers to fragments of oxytocin variants as defined in SEQ ID NO: 2, and therefore means that any fragment of the peptide presented in SEQ ID NO: 2 is also included in this invention.

[0030] As used herein, fragments and / or variants of oxytocin according to SEQ ID NO: 2 are understood to be X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 in X1 selects a group consisting of free Cys and non-existent groups; X2 selects a group consisting of Tyr, Phe, Leu and no other group; X3 selects a group consisting of Ile, Val, Hoph, Phe, Cha, and the group that does not exist; X4 can be selected from groups consisting of Gln, Ser, Thr, Cit, Arg, and Daba. X5 selects FreePro and the group that does not exist; X6 selects the group consisting of Ile, Leu, non-existent, Val, Hos, Daba, Thr, Arg, and Cit; X7 selects the group consisting of Gly, non-existent, and Ala; X8 is selected from the group consisting of Gly and the absence of Gly. However, it should be understood that vasopressin is generally not included in the scope of SEQ ID NO: 2, although it may be included. In fragments and / or variants of oxytocin according to SEQ ID NO: 2, a cysteine ​​bridge may be formed between X1 (when X1 is Cys) and Cys at position 6 (counted from the N-terminus in the above formula). Attached Figure Description

[0031] Figure 1 The graph shows the growth of roots and coleoptiles of spring wheat after soaking in tap water or 2 μM and 4 μM oxytocin solutions for 24 hours (1:0, 1:2, and 1:4, respectively) or 48 hours (2:0, 2:1, and 2:4, respectively) at different doses of oxytocin (oxytocin dissolved in tap water). The graph on the left shows the germination effect of wet seeds after soaking for 24 or 48 hours. The graph on the right shows the effect after soaking seeds for 24 or 48 hours and then drying them at room temperature for 3 days. The values ​​represent the percentage growth in length (top) and weight (bottom) as a control (0 μM oxytocin) after 3 days. The values ​​are based on the average of five petri dishes, each with 8–10 germinated seeds. 1:0, 1:2, 1:4, 2:0, 2:2, and 2:4 represent "soaking days: oxytocin concentration".

[0032] Figure 2 The effects of different doses of oxytocin (0, 2, and 4 μM) on root and shoot length in spring wheat seedlings are shown, expressed as a percentage of the control (0 μM). Spring wheat was grown in petri dishes in the dark at 18°C ​​and 70% relative humidity for 14 days.

[0033] Figure 3 The weight of wheat sprouts after soaking in 2μM and 4μM oxytocin or water without washing.

[0034] Figure 4 The weight of sunflower seedlings after soaking in 2μM and 4μM oxytocin or water without washing.

[0035] Figure 5 The weight of the bok choy sprouts after soaking in 2μM and 4μM oxytocin or water without washing.

[0036] Figure 6Photos of bok choy roots after soaking in 2μM and 4μM oxytocin or in water. Both soaking treatments resulted in more root development.

[0037] Figure 7 The weight of carrot sprouts after soaking in 2μM and 4μM oxytocin or water without washing.

[0038] Figure 8 The weight of pea sprouts after soaking in 2μM and 4μM oxytocin or water without washing.

[0039] Figure 9 Photographs of pea roots after soaking in 2 μM oxytocin (right) or water (left). Soaking resulted in greater root and shoot biomass development.

[0040] Figure 10 The weight of crisp lettuce sprouts after soaking in 2μM and 4μM oxytocin or water without washing.

[0041] Figure 11 The weight of wheat sprouts after soaking in 2μM and 4μM oxytocin or water and then washing.

[0042] Figure 12 The weight of sunflower seedlings after soaking in 2μM and 4μM oxytocin or water and then washing.

[0043] Figure 13 The weight of pea sprouts, after soaking in 2μM and 4μM oxytocin or water and then washing.

[0044] Figure 14 : Number of germinating dill seeds after soaking in 2μM and 4μM oxytocin or in water. 0:0 indicates unsoaked seeds; 0:24 / 0:48 indicates seeds soaked in water for 24 and 48 hours respectively; 2:24 / 2:48 indicates seeds soaked in 2μM oxytocin for 24 and 48 hours respectively; 4:24 / 4:48 indicates seeds soaked in 4μM oxytocin for 24 and 48 hours respectively.

[0045] Figure 15 The length of the whole plant (from root tip to bud tip), buds and roots after spruce seeds were soaked in water (control), 2μM or 4μM oxytocin.

[0046] Figure 16 The length of the whole plant (from root tip to bud tip), buds and roots after pine trees were soaked in water (control), 2μM or 4μM oxytocin. Detailed Implementation

[0047] Methods for soaking seeds and / or stimulating seed germination The inventors have surprisingly discovered that soaking seeds in a solution containing oxytocin before allowing them to germinate can enhance seed germination and / or growth. Furthermore, the soaking method described herein results in a greater number of seeds germinating and / or germinating more rapidly.

[0048] Therefore, this paper discloses a method for soaking seeds, the method comprising the following steps: a) Provide seeds; b) Provide an aqueous solution containing oxytocin; c) Soaking the seeds in the aqueous solution containing oxytocin; and d) Remove the seeds from the aqueous solution containing oxytocin.

[0049] This article also relates to seeds that can be obtained or acquired through the soaking methods disclosed herein.

[0050] Soaking in step c) typically lasts for a period of time, ranging from a few minutes (e.g., about 1-5 minutes) to about 72 hours, for example, from about 5 minutes to about 72 hours, for example, from about 15 minutes to about 72 hours, for example, from about 0.5 hours to about 72 hours, for example, from about 1 hour to about 48 hours, from about 1 hour to about 24 hours, from about 12 hours to about 48 hours, or from about 12 hours to about 24 hours. The soaking time can be adjusted according to the seed coat type; shorter times are used for seeds without a seed coat or whose seed coat is easily penetrated by the oxytocin solution, and longer times are used for seeds whose seed coat is more difficult to penetrate. Soaking usually stops before the radicle and / or coleoptile emerge. In monocotyledonous seeds, the radicle and cotyledons of the embryo are covered by the radicle sheath and coleoptile, respectively. When monocotyledonous seeds germinate, the radicle sheath emerges first from the seed, followed by the radicle. When dicotyledonous seeds germinate, the radicle emerges first, followed by the bud containing the cotyledons. As used herein, germination refers to the point in time when the coleoptile and / or radicle emerges. Therefore, in the method of this invention, the seed is removed from the oxytocin solution before the radicle and / or coleoptile emerge. After removal from the oxytocin solution, the seed can be rinsed (e.g., in an aqueous solution) and / or dried.

[0051] Therefore, the method for soaking seeds according to the present invention may include the following steps: a) Provide seeds; b) Provide an aqueous solution containing oxytocin; c) Soak the seeds in the aqueous solution containing oxytocin; d) Before the seeds begin to germinate, remove the seeds from the aqueous solution containing oxytocin.

[0052] Soaking can be carried out at any temperature that will not damage the seeds. Generally, soaking is done at temperatures above 0°C but below 40°C. For example, soaking can be done at temperatures from about 5°C to about 30°C, such as from about 15°C to about 25°C, from about 20°C to about 25°C, such as room temperature, from about 2°C to about 10°C, or from about 3°C ​​to about 8°C, such as about 4°C. For seeds that germinate rapidly, lower temperatures during soaking may help prevent germination during the soaking process.

[0053] The seed soaking method of the present invention may further include a step of drying the seeds after soaking and before germination / planting. Such a drying step may be carried out over a period of at least 6 hours to 5 days, for example, 1, 2, 3, 4, or 5 days. Drying may be carried out, for example, at about 15-25°C, such as about 20°C. Drying may be carried out, for example, by drying the seeds in an oven set at such a temperature. Alternatively, the seeds may be freeze-dried. As shown in the experimental section, such drying can be used to stimulate bud growth more than root growth, i.e., the growth stimulation effect may be directed towards the bud rather than the root.

[0054] The seed soaking method of the present invention may include the step of rinsing (washing) the seeds after soaking and before germination / planting. If the method includes a drying step, such rinsing is typically performed before the drying step. Rinsing can be done with an aqueous solution, such as water or a salt solution. Rinsing can remove some or substantially all of the oxytocin solution. Surprisingly, as shown in the experimental section, rinsing the seeds after soaking does not impair the beneficial effects of soaking on germination and / or growth.

[0055] Seeds soaked using the soaking method of this invention can be planted immediately after soaking. Alternatively, the seeds can be stored for a period of time before being allowed to germinate. Thus, soaked seeds can be stored for a period of time, for example, before being planted in soil or placed in a nutrient solution for growth. Typically, such storage lasts from at least one day to several months, for example from about 1 day to about 12 months, from about 1 day to about 6 months, from about 1 day to about 3 months, from about 7 days to about 3 months, or from about 1 day to about 7 days. Typically, soaked seeds are stored for less than a year, although it may be longer.

[0056] The seeds that can be used according to the present invention are any seeds. Therefore, the seeds can be those of monocotyledons, dicotyledons, or gymnosperms (e.g., conifers).

[0057] Non-limiting examples of monocotyledonous seeds are grains such as wheat, rye, oats, barley, millet, and / or spelt wheat. Monocotyledonous plants, commonly referred to as monocotyledons or monocotyledons, have a single cotyledon (embryonic leaf) in their seeds.

[0058] Non-limiting examples of dicotyledonous seeds include quinoa seeds, legume seeds such as peas, sunflower seeds, dill seeds, and birch seeds.

[0059] Furthermore, the seeds can be those of gymnosperms, such as conifers, like pine, cedar, larch, or spruce. When treating conifer seeds using the method of the present invention, a shorter soaking time can be used because the seeds do not have a husk. Therefore, for conifer seeds, a soaking time of from about 0.5 hours to about 6 hours, for example from about 0.5 hours to about 3 hours, or for example from about 1 to about 2 hours, can be used.

[0060] Seed soaking can be carried out in any way that allows the seeds to absorb the oxytocin solution. For example, seeds can be soaked in the oxytocin solution with or without stirring. The oxytocin solution can also be provided to the seeds, for example, by immersing them in a solution containing oxytocin or by spraying them with the solution, allowing the seeds to absorb it. Typically, seeds are soaked in a sufficient amount of oxytocin solution to substantially cover them, while also taking into account seed swelling (imbiosis).

[0061] Aqueous solutions of oxytocin may further contain liquid and / or solid carriers, excipients, organic fertilizers, and / or agricultural reagents to, for example, stimulate plant growth or protect seeds or plants. An example of such reagents is plant hormones.

[0062] This article also relates to a method for stimulating the germination and / or growth of the roots and / or coleoptiles / buds of seeds, the method comprising the following steps: i) Soak the seeds using the soaking method described herein, and optionally rinse and / or dry the soaked seeds; ii) Germinate the seed.

[0063] Preferably, this type of germination does not occur in the presence of oxytocin.

[0064] One example of the method for stimulating seed germination according to the present invention is seed malting in the presence of oxytocin, wherein the seeds are soaked in an aqueous solution containing oxytocin, removed from the aqueous solution containing oxytocin, then germinated, and then the germinated seeds are dried.

[0065] The methods disclosed herein can also be used to stimulate germination and / or growth in seeds with impaired germination ability. Seeds may have impaired germination ability due to, for example, aging or infection.

[0066] This document also provides the use of aqueous solutions containing oxytocin and / or its variants and / or fragments as described herein for soaking seeds and / or stimulating germination and / or growth of the roots and / or coleoptiles of seeds, such as those from monocotyledonous or dicotyledonous plants or gymnosperms, such as conifers.

[0067] Seeds soaked according to the soaking method of the present invention can germinate and / or grow in the presence and / or absence of oxytocin. However, typically, soaked seeds germinate and / or grow in the absence of oxytocin. As shown in the experimental section, surprisingly, the growth-enhancing effect of oxytocin may be even greater if the seeds are soaked only without germination / growth in oxytocin. However, in some cases, germination and / or growth of seeds in the presence of oxytocin may also have beneficial effects on germination and / or growth.

[0068] As shown in the experimental section, although the seeds were not grown in oxytocin, soaking them in an oxytocin solution provided a strong growth-enhancing effect. Therefore, when seeds are soaked in the presence of oxytocin, the effect of oxytocin is persistent, and this effect persists even after soaking, such as after drying or washing the seeds. This is unexpected and has significant implications for the availability of using oxytocin to stimulate plant growth, as pretreating seeds with oxytocin is more practical than providing oxytocin during germination and / or growth, especially for crops to be sown in outdoor soil. Treating the seeds themselves in this way, rather than, for example, treating the soil or plants, is advantageous because it allows for better control over the dosage and timing of oxytocin application, for example, compared to providing it at a later stage. Therefore, the seed soaking method of the present invention may be important for the purposes of precision agriculture.

[0069] Compositions containing oxytocin The oxytocin solution used in this invention is preferably an aqueous solution. The concentration of oxytocin in such aqueous solutions is typically 0.5-50 μM, for example 1-50 μM, 1-30 μM, 1-20 μM, 1-10 μM, 1-8 μM, 2-8 μM, 1-6 μM, 1-5 μM, 2-6 μM, 3-5 μM, or 2-4 μM. The aqueous solution may consist of water and oxytocin.

[0070] In addition to oxytocin, the composition may contain one or more liquid and / or solid carriers, excipients, organic fertilizers and / or agricultural reagents.

[0071] Oxytocin and its fragments / variants According to the present invention, oxytocin is a peptide according to SEQ ID NO: 1 (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly, i.e., human oxytocin). Furthermore, in addition to or instead of oxytocin, one or more fragments and / or variants of oxytocin according to SEQ ID NO: 2 and having oxytocin activity may be used.

[0072] SEQ ID NO: 2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 (Formula (I)) in X1 selects a group consisting of free Cys and non-existent groups; X2 selects a group consisting of Tyr, Phe, Leu and no other group; X3 selects a group consisting of Ile, Val, Hoph, Phe, Cha, and the group that does not exist; X4 can be selected from groups consisting of Gln, Ser, Thr, Cit, Arg, and Daba. X5 selects FreePro and the group that does not exist; X6 selects the group consisting of Ile, Leu, non-existent, Val, Hos, Daba, Thr, Arg, and Cit; X7 selects the group consisting of Gly, non-existent, and Ala; X8 selects groups consisting of Gly and non-existent elements.

[0073] SEQ ID NO: 2 may not include vasopressin.

[0074] Alternatively, one or more plant cyclic peptides can be used as oxytocin. Therefore, variants and / or fragments of oxytocin as defined herein can be included in plant cyclic peptides.

[0075] Therefore, it should be understood that when X1 in formula (I) is cysteine ​​(Cys), the thiol group of X1 can form a disulfide bond with the thiol group of cysteine ​​located between asparagine (Asn) and X5, thereby forming the cyclic structure of formula (Ia): (Equation (Ia)) Such cysteine ​​bonds are present in oxytocin and may also be present in fragments and / or variants of SEQ ID NO: 2 that have oxytocin activity, such as those illustrated below.

[0076] Examples of substances with oxytocin activity according to SEQ ID NO: 2 include meso-oxytocin, iso-oxytocin, cyclophosphamide oxytocin, and angio-oxytocin. Other examples can be found in WO 2012 / 140216. Another substance with oxytocin activity is a nonapeptide with the sequence tyrosine-isoleucine-glutamine-asparagine-cysteine-proline-leucine-glycine-amide, and having one cysteine ​​residue at position 6, i.e., a substance with the same chemical structure as oxytocin, except that the bond between the cysteine ​​residue at position 1 and the tyrosine residue at position 2 is broken, and the cysteine ​​residue at position 1 forms a branch on the cysteine ​​residue at position 6.

[0077] One or more variants and / or fragments of oxytocin with oxytocin activity (SEQ ID NO: 2) may also be selected from the group consisting of the following compounds and their pharmaceutically acceptable salts: Oxytocin: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Ile-Gly-NH2 (SEQ ID NO: 3) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Ile, X7 is Gly and X8 does not exist; Isoxytocin: Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly-NH2 (SEQ ID NO: 4) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Ser, X5 is Pro, X6 is Ile, X7 is Gly and X8 does not exist; Cysticercus cyclops oxytocin: Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2 (SEQ ID NO: 5) X1 is Cys, X2 is Phe, X3 is Val, X4 is Arg, X5 is Pro, X6 is Thr, X7 is Gly and X8 does not exist; Oxytocin: Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH2 (SEQ ID NO: 6) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Arg, X7 is Gly and X8 does not exist; Cys-Tyr-Cha-Cit-Asn-Cys-Pro-Arg-Gly-NH2 (SEQ ID NO: 8) X1 is Cys, X2 is Tyr, X3 is Cha, X4 is Cit, X5 is Pro, X6 is Arg, X7 is Gly, and X8 does not exist. Cys-Tyr-Ile-Gln-Asn-Cys-Pro-NH2 (SEQ ID NO: 9) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, and X6-X8 do not exist; Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-NH2 (SEQ ID NO: 10) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, and X~7-8~ does not exist; Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 11) X1 does not exist, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly and X8 does not exist; Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 12) X1-X2 do not exist, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly and X8 does not exist; Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 13) X1-X3 do not exist, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly and X8 does not exist; Ile-Gln-Asn-Cys-Pro-NH2 (SEQ ID NO: 14) X1-X2 do not exist, X3 is Ile, X4 is Gln, X5 is Pro, and X6-X8 do not exist; Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-Gly-NH2 (SEQ ID NO: 15) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly and X8 is Gly; Gln-Asn-Cys-Pro-Leu-Leu-NH2 (SEQ ID NO: 16) X1-X3 do not exist, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Leu and X8 does not exist; Cys-Tyr-Val-Thr-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 17) X1 is Cys, X2 is Tyr, X3 is Val, X4 is Thr, X5 is Pro, X6 is Leu, X7 is Gly and X8 does not exist; Cys-Tyr-Hoph-Thr-Asn-Cys-Pro-Val-Gly-NH2 (SEQ ID NO: 18) X1 is Cys, X2 is Tyr, X3 is Hoh, X4 is Thr, X5 is Pro, X6 is Val, X7 is Gly, and X8 does not exist. Cys-Tyr-Phe-Cit-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 19) X1 is Cys, X2 is Tyr, X3 is Phe, X4 is Cit, X5 is Pro, X6 is Leu, X7 is Gly and X8 does not exist; Cys-Tyr-Cha-Arg-Asn-Cys-Pro-Hos-Ala-NH2 (SEQ ID NO: 20) X1 is Cys, X2 is Tyr, X3 is Cha, X4 is Arg, X5 is Pro, X6 is Hos, X7 is Ala and X8 does not exist; Cys-Tyr-Val-Daba-Asn-Cys-Pro-Daba-Ala-NH2 (SEQ ID NO: 21) X1 is Cys, X2 is Tyr, X3 is Val, X4 is Daba, X5 is Pro, X6 is Cit, X7 is Ala and X8 does not exist; Cys-Tyr-Hoph-Daba-Asn-Cys-Pro-Cit-Ala-NH2 (SEQ ID NO: 22) X1 is Cys, X2 is Tyr, X3 is Hoh, X4 is Daba, X5 is Pro, X6 is Cit, X7 is Ala and X8 does not exist; and Cys-Tyr-Phe-Arg-Asn-Cys-Pro-Val-Ala-NH2 (SEQ ID NO: 23) X1 is Cys, X2 is Tyr, X3 is Phe, X4 is Arg, X5 is Pro, X6 is Val, X7 is Ala, and X8 does not exist.

[0078] The non-natural amino acids in the substance have the following structures: Cyclohexylalanine, referred to as Cha in this article,

[0079] High phenylalanine, referred to as Hop in this article,

[0080] Citrulline, referred to as Cit in this article,

[0081] Diaminobutyric acid, referred to as Daba in this article, and

[0082] Homoserine, referred to as Hos in this article,

[0083] Furthermore, variants and / or fragments of oxytocin with oxytocin activity according to SEQ ID NO: 2 can be oxytocin analogs from fish, such as according to the sequence of SEQ ID NO: 24 (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH2), or oxytocin analogs from insects, such as according to the sequence of SEQ ID NO: 25 (Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly).

[0084] When a position in SEQ ID NO:2 is described as "not present", it means that it represents a single bond between items (letters, atoms, or groups).

[0085] Metabolic derivatives or metabolic degradation products can be oxytocin-like peptides, such as peptides with nine amino acids, like oxytocin, mesooxytocin, isooxytocin, and cyclophosphamide oxytocin, wherein one or more amino acids have been deleted from the carboxyl terminus or amino terminus, or simultaneously from both, for example, 1-3 amino acids deleted from each terminus. In some respects, one, two, or three amino acids may have been deleted from the carboxyl terminus, i.e., only Gly, Gly, and Leu, or Gly, Leu, and Pro. For example, two or three amino acids may have been deleted from the amino terminus, i.e., only Cys, Cys, and Tyr, or Cys, Tyr, and Ile.

[0086] In some respects, one, two, or three amino acids may have been deleted from the carboxyl terminus, i.e., only Gly, Gly, and Leu, or Gly, Leu, and Pro, and one, two, or three amino acids may have been deleted from the amino terminus, i.e. only Cys, Cys, and Tyr, or Cys, Tyr, and Ile. These variants can be identified as analogs of oxytocin, meso-oxytocin, iso-oxytocin, or vermicomytic oxytocin by immunological methods such as RIA (radioimmunoassay), IRMA (radiometry), RIST (radioimmunoadsorption assay), and RAST (radioallergen adsorption assay). The invention also includes oxytocin variants having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% sequence identity with oxytocin, said variants exhibiting oxytocin activity as defined herein.

[0087] Oxytocin or its variants can exist in D- and L-forms, as well as their racemic forms. The D-form can be converted to the L-form by reversing its peptide sequence. These and the aforementioned peptides can be produced by methods known to those skilled in the art, for example, according to Merrifield, PB, "Solid Phase Synthesis", Angew. Chemie, 1985, No. 97, p.801.

[0088] The present invention will be further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0089] Experimental Section Example 1: Soaking wheat seeds Spring wheat (Diskett) seeds were soaked at 18°C ​​in tap water with or without oxytocin for 24 or 48 hours. The oxytocin concentration was 2 or 4 μM. 150 seeds were used for each treatment. The seeds were covered with the solution during soaking.

[0090] After soaking for 24 or 48 hours, 50 seeds of each treatment were placed in petri dishes containing tap water (approximately 10 seeds per dish), sealed with sealing film, and then placed in a growth chamber at 18°C ​​and 70% relative humidity in the dark. After 72 hours, when the seeds germinated and roots and shoots developed, the root and shoot lengths of each germinated seed were measured, and the total weight of the roots and shoots of the germinated seeds was also measured.

[0091] After soaking for 24 or 48 hours, take 50 seeds for each treatment and allow them to dry for three days, then germinate the wet seeds as described above.

[0092] statistics An analysis of variance was performed on the effect of each parameter on the test dose. If the analysis of variance showed a significant difference between the test doses, the significance between the two doses was tested using a Mann-Whitney paired test.

[0093] All germinated seeds were included in the statistics.

[0094] result Soaking seeds in oxytocin (2 or 4 μM) for 24 hours significantly stimulated root and shoot (coleoptile) growth when the seeds germinated directly in petri dishes after soaking (i.e., still wet seeds) compared to seeds soaked only in water. See [link to relevant documentation]. Figure 1 .

[0095] When seeds were dried before being placed in petri dishes, seeds soaked for 48 hours had a greater positive effect on root and shoot growth than seeds soaked for 24 hours. Figure 1In this case, the growth stimulation effect is more significant for the bud (coleoptile) than for the root. Figure 1 ).

[0096] Example 2: Germination in oxytocin solution (continuous exposure during germination) Place filter paper in a petri dish, add 4 ml of water with or without oxytocin (2 or 4 μM), and then add spring wheat seeds. Seal the petri dish with sealing film and place it in a growth chamber at 18°C ​​and 70% relative humidity in the dark. After the seeds germinate, measure the length of the roots and shoots. Compared to Example 1, the seeds in Example 2 were allowed to germinate in an oxytocin solution (or a negative control without oxytocin).

[0097] result like Figure 2 As shown, root length was positively affected by germination in oxytocin solution, while no effect or a small negative effect was observed on bud (coleoptile) length. However, the growth-stimulating effect was not as high when seeds were allowed to germinate in oxytocin solution as when seeds were soaked in oxytocin solution.

[0098] Conclusions of Examples 1 and 2 Soaking seeds in an oxytocin solution provided a much stronger growth-enhancing effect than allowing the seeds to germinate in the oxytocin solution. This was unexpected and has significant implications for the availability of using oxytocin to stimulate plant growth, as pretreating seeds with oxytocin is more practical than providing oxytocin during germination and / or growth, especially for crops to be sown in outdoor soil.

[0099] Example 3: Soaking wheat, crisp lettuce, sunflower, bok choy, carrot, and pea seeds in an oxytocin solution. Seeds from the corresponding plants were soaked for 12 hours in aqueous solutions of oxytocin at concentrations of 2 μmol and 4 μmol (water and oxytocin), as well as in water (control). Seeds were soaked at 4°C (1–3 dL of seeds, depending on seed size). It was ensured that all seeds were completely covered by the oxytocin solution / water throughout the soaking period. Importantly, no seed germination was observed during the 12-hour soaking period. Therefore, no seeds germinated during the soaking period.

[0100] Cut the hemp mat into A4 size, soak it thoroughly in water, and then remove the excess water.

[0101] After soaking, remove excess oxytocin solution / water from the seeds, transfer the seeds to a hemp mat and spread them out.

[0102] Depending on the seed type, allow the seeds to germinate at room temperature (approximately 20-25°C) for 7-12 days. Regularly spray the seeds with water to keep them moist. Harvest the seeds afterwards and measure the weight of the sprouts.

[0103] result The results of different tests show Figure 3-10 In the middle, the weight of the control group treated with water alone was set at 100.

[0104] Comparison of the growth of seeds soaked in 2 μmol or 4 μmol oxytocin solutions with that of seeds soaked only in water showed that oxytocin had a significant growth-enhancing effect at both test concentrations.

[0105] In addition, from Figure 6 and 9 It can be seen that oxytocin treatment has a visible positive effect on root development. Furthermore, the positive effect of oxytocin on germination is visible to the naked eye, such as... Figure 9 As shown.

[0106] Example 4: Soaking wheat, sunflower, and pea seeds in oxytocin solution Seeds from the respective plants were soaked for 12 hours in an aqueous solution of oxytocin (water and oxytocin) at a concentration of 2 μmol (sunflower and pea) or 4 μmol (wheat), as well as in water (control). The specific concentration was selected according to the optimal concentration determined in Example 3. Seeds were soaked at 4°C (1-3 dL of seeds, depending on seed size). It was ensured that all seeds were completely covered by the oxytocin solution / water throughout the soaking period. Importantly, no seed germination was observed during the 12-hour soaking period. Therefore, no seeds germinated during the soaking period.

[0107] The oxytocin-treated seeds are then thoroughly washed in water (10-15 liters per seed type) to remove the oxytocin solution. After that, the seeds are placed on paper (filter paper or household paper) until they are outwardly dry (about 3 hours) to remove excess moisture.

[0108] Cut the hemp mat into A4 size, soak it thoroughly in water, and then remove the excess water.

[0109] Depending on the seed type, allow the seeds to germinate at room temperature (approximately 20-25°C) for 7-12 days. Spray the seeds with water regularly to keep them moist. Then harvest the seeds and measure the weight of the sprouts, see [link to relevant documentation]. Figure 11-13 .

[0110] Example 4: Soaking dill seeds Dill seeds were soaked in 0 μM, 2 μM or 4 μM oxytocin solution (oxytocin and water) at 4°C.

[0111] Dill seeds are soaked for 24 and 48 hours, then dried in circulating air at 30°C.

[0112] After the seeds were dried, 25 seeds from each treatment were placed in a petri dish with two sheets of filter paper moistened with 4.5 ml of tap water. As a control, 25 untreated seeds (neither soaked in water nor in oxytocin solution) were added.

[0113] Count the germinated seeds every day until finally measuring the roots and buds of the seeds.

[0114] The final measurement was taken 10 days later.

[0115] result like Figure 14 As shown, after soaking for 24 or 48 hours, dill seeds germinated faster in the presence of oxytocin compared to unsoaked or water-soaked seeds (measured by the number of germinated seeds).

[0116] Example 5: Soaking of gymnosperms (spruce and pine) Seeds from gymnosperms (spruce and pine) were soaked in an oxytocin solution (2 μM or 4 μM) or water (control) for 1–2 hours at a temperature of approximately 20°C. The seeds were then transferred to a pot containing 90 ml of peat moss and covered with sawdust.

[0117] Water 3-5 times daily during the first and second weeks after sowing (during which germination occurs). After two weeks, transfer the pots to a greenhouse where the plants will continue to grow for 6 weeks. Then move the pots outdoors for further growth. Seeds were treated with oxytocin on May 25, 2024, and harvested on September 1, 2024.

[0118] Figure 15 (Spruce) and Figure 16 (Pine tree) Shows the differences in whole plant (from root tip to shoot tip, left), root length (middle), and shoot length (right) between seeds treated with oxytocin and the control. From Figure 15 and Figure 16 It can be seen that oxytocin soaking produced a robust growth-enhancing effect on both roots and shoots. This is particularly surprising considering that a short soaking time was used in this experiment.

[0119] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0120] Unless otherwise stated, each preferred feature described herein may be used in combination with any and all other preferred features described herein.

Claims

1. A method for soaking seeds, said seeds being, for example, seeds from monocotyledonous or dicotyledonous plants or gymnosperms, such as conifers, said method comprising the following steps: a) Provide seeds; b) Provide an aqueous solution containing oxytocin and / or its variants and / or fragments; c) Soak the seeds in the aqueous solution containing oxytocin; d) Remove the seeds from the aqueous solution containing oxytocin, wherein the oxytocin fragment and / or variant is the oxytocin-active fragment and / or variant according to SEQ ID NO:

2. Where SEQ ID NO:2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 in X1 selects a group consisting of free Cys and non-existent groups; X2 selects a group consisting of Tyr, Phe, Leu and no other group; X3 selects a group consisting of Ile, Val, Hoph, Phe, Cha, and the group that does not exist; X4 can be selected from groups consisting of Gln, Ser, Thr, Cit, Arg, and Daba. X5 selects FreePro and the group that does not exist; X6 selects the group consisting of Ile, Leu, non-existent, Val, Hos, Daba, Thr, Arg, and Cit; X7 selects the group consisting of Gly, non-existent, and Ala; X8 selects groups consisting of Gly and non-existent elements.

2. The method according to claim 1, wherein the soaking time in step c) is from about 1 minute to about 72 hours, for example from about 5 minutes to about 72 hours, for example from about 15 minutes to about 72 hours, for example from about 0.5 hours to about 72 hours, for example from about 1 hour to about 48 hours, from about 1 hour to about 24 hours, from about 12 hours to about 48 hours, or from about 12 hours to about 24 hours.

3. The method according to claim 1 or 2, further comprising step e) rinsing and / or drying the seeds.

4. The method according to any one of the preceding claims, wherein the monocotyledonous seed is a grain.

5. The method according to any one of the preceding claims, wherein the monocotyledonous seed is wheat, rye, oats, barley, millet and / or spelt wheat seed.

6. The method according to any one of claims 1 to 3, wherein the dicotyledonous seeds are quinoa seeds, legume seeds such as peas, sunflower seeds, dill seeds, and / or birch seeds.

7. The method according to any one of claims 1 to 3, wherein the gymnosperm is a conifer, such as pine, cedar, larch or spruce.

8. The method according to any one of the preceding claims, wherein the concentration of oxytocin is 0.5-50 μM, for example 1-50 μM, 1-30 μM, 1-20 μM, 1-10 μM, 1-8 μM, 2-8 μM, 1-6 μM, 1-5 μM, 2-6 μM, 3-5 μM or 2-4 μM.

9. The method according to any one of the preceding claims, wherein the aqueous solution of oxytocin further comprises a liquid and / or solid carrier, an excipient, an organic fertilizer, and / or an agricultural reagent.

10. A method for stimulating the germination and / or growth of the roots and / or coleoptile / bud of a seed, said seed being, for example, a monocotyledonous or dicotyledonous seed or a gymnosperm, said method comprising the steps of: i) Soaking seeds by any one of claims 1-9; ii) to germinate the seed.

11. The method according to any one of claims 1-10, wherein the seed is a seed with impaired germination ability.

12. The method according to any one of the preceding claims, wherein the oxytocin is a plant cyclic peptide.

13. A seed that can be obtained or acquired by the method defined in any one of claims 1-12.

14. Use of an aqueous solution comprising oxytocin and / or variants and / or fragments thereof as defined in any one of claims 1 and 8-9 for soaking seeds and / or stimulating germination and / or growth of the roots and / or coleoptiles of seeds, said seeds being, for example, seeds from monocotyledonous or dicotyledonous plants or seeds from gymnosperms, such as conifers.

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