Stimulation of plant germination

Abstract

This specification relates to the field of agriculture and to methods for stimulating plant germination. More specifically, this specification relates to a method for immersing seeds to stimulate seed germination, compositions for such use, and immersed seeds. The method comprises immersing seeds in an aqueous solution containing oxytocin, a fragment thereof, or a variant thereof, wherein the seeds are not germinated in the oxytocin solution. This has been shown to produce a more potent growth-promoting effect on the seeds.

Description

Technical Field

[0001] The present specification relates to the field of agriculture and to a method for stimulating the germination of plants.

Background Art

[0002] Good seed germination is important in agriculture to obtain large-scale harvests. Good seed germination not only results in a greater number of plants, but can also be important for combating weeds that compete with seeds for space and nutrients.

[0003] A seed contains an embryo, one end of which forms the root, that is, the radicle, and the other end forms the stem and leaves, that is, the plumule. Monocotyledons contain one cotyledon, that is, one seed leaf that occupies a small part of the seed. Dicotyledons contain two cotyledons.

[0004] During germination of monocotyledons, the radicle grows downward through the split seed coat to form the primary root, and the shoot enclosed in the coleoptile, a protective sheath, grows upward.

[0005] In dicotyledons, the primary root emerges from the seed, allowing water absorption by this new plant. Apical meristem develops therefrom to form the root system of the plant. Then, the shoot emerges from the seed. The shoot in dicotyledons consists of the cotyledons, the hypocotyl, and the epicotyl.

[0006] In gymnosperms, for example conifers, after the embryo absorbs water, the taproot first breaks through the seed coat and emerges, then grows down into the soil. Thereafter, the stem below the cotyledons elongates. When the cotyledons start photosynthesis, the shoot begins to grow.

[0007] Plant germination and growth can be stimulated by the use of plant hormones such as auxins, gibberellins, cytokinins, and abscisic acid. However, such agents are expensive and may be toxic when used in larger amounts.

[0008] International Publication No. 02 / 102160 discloses the use of the human hormone oxytocin to stimulate plant germination by germinating seeds in an oxytocin-containing solution. However, germinating seeds in an oxytocin-containing solution can be expensive and impractical when germinating large quantities of seeds. In contrast to plant germination, seed immersion offers a cost-effective alternative to plant germination for plant producers and growers.

[0009] Therefore, an object of the present invention is to provide improved methods and means for stimulating plant germination and / or growth by treating seeds in a specific manner before germination, thereby demonstrating novel and viable alternatives for growers. [Overview of the project]

[0010] This specification is, a) The process of providing seeds; b) A step of providing an aqueous solution containing oxytocin and / or its variants and / or fragments, typically having a concentration of oxytocin of 0.5 to 50 μM; c) The step of immersing the seeds in the aqueous solution containing oxytocin; d) The step of removing the seeds from the aqueous solution containing oxytocin; Includes, The oxytocin fragment and / or variant is the fragment and / or variant according to SEQ ID NO: 2 having oxytocin activity. Sequence ID 2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 (Here, X1 is selected from the group consisting of Cys and non-existence; X2 is selected from the group consisting of Tyr, Phe, Leu, and non-existence; X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and non-existence; X4 is selected from the group consisting of Gln, Ser, Thr, Cit, Arg, and Daba; X5 is selected from the group consisting of Pro and non-existent; X6 is selected from the group consisting of Ile, Leu, non-existence, Val, Hos, Daba, Thr, Arg, and Cit; X7 is selected from the group consisting of Gly, absence, and Ala; X8 is selected from the group consisting of Gly and non-existence. The present invention relates to a method for immersing seeds, such as seeds from monocots, dicots, or gymnosperms (conifers).

[0011] This specification also provides seeds that can be obtained or are obtained by the above method.

[0012] The immersion in step c) is typically carried out over a period of several minutes (e.g., about 1 to 5 minutes) to about 72 hours, e.g., about 5 minutes to about 72 hours, e.g., about 15 minutes to about 72 hours, e.g., about 0.5 hours to about 72 hours, e.g., about 1 hour to about 48 hours, e.g., about 1 hour to about 24 hours, e.g., about 12 hours to about 48 hours, or e.g., about 12 hours to about 24 hours.

[0013] Optionally, this method may further include step e) drying the seeds.

[0014] The seeds may be of any kind. Non-specific types of seeds include seeds from monocots or dicots, or seeds from gymnosperms.

[0015] The seeds of monocotyledonous plants may be grains such as wheat, rye, oats, barley, millet, and / or spelt.

[0016] Seeds of dicotyledonous plants may include, for example, quinoa seeds, seeds from leguminous plants such as peas, sunflower seeds, dill seeds, and / or birch seeds.

[0017] Gymnosperm seeds can be, for example, seeds from coniferous trees such as pine, cedar, larch, or spruce.

[0018] The concentration of oxytocin is typically 0.5 to 50 µM, for example 1 to 50 µM, 1 to 30 µM, 1 to 20 µM, 1 to 10 µM, 1 to 8 µM, 2 to 8 µM, 1 to 6 µM, 1 to 5 µM, 2 to 6 µM, 3 to 5 µM, or 2 to 4 µM.

[0019] The aqueous solution of oxytocin may further comprise one or more of liquid and / or solid carriers, excipients, organic fertilizers, and / or agricultural chemicals such as plant hormones.

[0020] The present specification also provides i) a) providing a seed, for example a seed from a monocotyledonous plant, a dicotyledonous plant or a gymnosperm; b) providing an aqueous solution comprising oxytocin as defined herein and / or a variant and / or fragment thereof, the concentration of oxytocin in the aqueous solution typically being 0.5 to 50 µM; c) immersing the seed in said aqueous solution comprising oxytocin; d) removing the seed from said aqueous solution comprising oxytocin; and optionally, rinsing and / or drying said seed, immersing said seed by a method comprising the above steps, and ii) germinating said seed, there is also provided a method for stimulating germination and / or growth of roots and / or coleoptiles / shoots of a seed, comprising the above steps.

[0021] There is also provided use of an aqueous solution comprising oxytocin and / or a variant and / or fragment thereof as described herein for immersing seeds such as conifer, monocotyledonous or dicotyledonous seeds, and / or for stimulating germination and / or growth of roots and / or coleoptiles of seeds.

[0022] Other features and advantages of the present invention will be apparent from the following detailed description, the drawings, the examples, and the claims.

[0023] definition In the context of this specification, "steeping," "to steep," etc., refers to soaking seeds in a solution to allow them to absorb water and / or to allow substances in the solution to be absorbed by the seeds before germination. In this specification, terms such as "soaking" may be used instead of "steeping," etc.

[0024] Oxytocin (SEQ ID NO: 1) is understood to have the following chemical structure.

[0025] [ka]

[0026] Wherever “oxytocin” is referred to herein, this term is understood to also include oxytocin molecule / peptide fragments, variants, or homologs, or plant cyclotides, that possess biological activity equivalent to that of the oxytocin molecule itself (SEQ ID NO: 1). Oxytocin variants and / or fragments may have substantially the same stimulating effect on seed germination and / or plant growth after immersion as oxytocin itself would have had with immersion.

[0027] Therefore, the “variants” of oxytocin referred to herein refer to peptides in which the amino acid structure is modified compared to the oxytocin molecule, where some amino acid positions may be altered by introducing other amino acids, such as natural or unnatural amino acids as exemplified herein, into those positions, or by extending the peptide by adding one or more natural or unnatural amino acids to any end. Furthermore, other structural modifications may be made to the peptide referred to herein, such as synthetic modifications. The “variants” still maintain the same biological activity as oxytocin, and the oxytocin variants are also stabilized by their presence in the pharmaceutical composition according to the present invention. The “variants” of oxytocin referred to herein may not include vasopressin (SEQ ID NO: 7). Vasopressin is a nonapeptide having the sequence Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly, where the cysteine ​​residues form disulfide bonds.

[0028] Furthermore, the “fragments” of oxytocin referred to herein are peptides that contain a portion of the amino acid sequence of oxytocin, but in which case one or more amino acids may have been removed from one or both of the amino acid terminals. This term also refers to fragments of oxytocin variants as defined in SEQ ID NO: 2, and therefore any fragment of the peptide shown in SEQ ID NO: 2 is also included in the present invention.

[0029] When used herein, the oxytocin fragment and / or variant according to SEQ ID NO: 2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 (Here, X1 is selected from the group consisting of Cys and non-existence; X2 is selected from the group consisting of Tyr, Phe, Leu, and non-existence; X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and non-existence; X4 is selected from the group consisting of Gln, Ser, Thr, Cit, Arg, and Daba; X5 is selected from the group consisting of Pro and non-existent; X6 is selected from the group consisting of Ile, Leu, non-existence, Val, Hos, Daba, Thr, Arg, and Cit; X7 is selected from the group consisting of Gly, absence, and Ala; X8 is selected from the group consisting of Gly and non-existence. It is understood that this is the case. However, it will be understood that vasopressin is not typically included in the range of SEQ ID NO: 2, although it may be included in some cases. In the oxytocin fragment and / or variant according to SEQ ID NO: 2, a cysteine ​​crosslink may be formed between X1, where X1 is Cys, and the Cys at position 6 (counting from the N-terminus) of the above formula. [Brief explanation of the drawing]

[0030] [Figure 1] This study shows the root and cotyledon sheath growth of spring wheat seeds soaked 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) with different doses of oxytocin (oxytocin dissolved in tap water). The left graph shows the effect on germination of moist seeds after 24 or 48 hours of soaking. The right graph shows the effect on seeds that were initially soaked for 24 or 48 hours and then dried at room temperature for 3 days. The numbers show length growth (upper graph) and weight increase (lower graph) as a percentage of control (0 μM oxytocin) growth after 3 days. The numbers are based on the average from 5 petri dishes, each containing 8–10 germinated seeds. 1:0, 1:2, 1:4, 2:0, 2:2, and 2:4 indicate "Immersion days:Oxytocin concentration". [Figure 2] This study demonstrates the effects of different oxytocin doses (0, 2, and 4 μM) as percentages of control (0 μM) on root and shoot length in spring wheat sprouts. Spring wheat was grown for 14 days in a dark petri dish at 18°C ​​and 70% relative humidity (RH). [Figure 3] The weight of wheat sprouts that were not washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 4] The weight of sunflower sprouts that were not washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 5] The weight of bok choy (pak choi) sprouts that were not washed after being soaked in 2 μM and 4 μM oxytocin or water. [Figure 6] Photographs of bok choy roots after immersion in 2 μM and 4 μM oxytocin or water. Both immersion treatments resulted in greater root development. [Figure 7] The weight of radish sprouts that were not washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 8] The weight of pea sprouts that were not washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 9] Photographs of pea roots after immersion in 2 μM oxytocin (right) or water (left). Immersion resulted in the development of more root and sprout biomass. [Figure 10] The weight of crisp salad sprouts that were not washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 11] The weight of wheat sprouts washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 12] The weight of sunflower sprouts after being immersed in 2 μM and 4 μM oxytocin or water, followed by washing. [Figure 13] The weight of pea sprouts washed after being immersed in 2 μM and 4 μM oxytocin or water. [Figure 14] The number of germinated dill seeds after immersion in 2 μM and 4 μM oxytocin or water. 0:0 means unimmersed seeds; 0:24 / 0:48 means seeds immersed in water for 24 hours and 48 hours, respectively; 2:24 / 2:48 means seeds immersed in 2 μM oxytocin for 24 hours and 48 hours, respectively; and 4:24 / 4:48 means seeds immersed in 4 μM oxytocin for 24 hours and 48 hours, respectively. [Figure 15] The lengths of the entire spruce plant (from the tip of the root to the tip of the bud), the bud, and the root, respectively, when immersed in water (control) or 2 μM or 4 μM oxytocin. [Figure 16] The lengths of the entire plant (from the tip of the root to the tip of the new shoot), the new shoot, and the roots of pine trees immersed in water (control) or 2 μM or 4 μM oxytocin. [Modes for carrying out the invention]

[0031] Methods for soaking seeds and / or stimulating seed germination The inventors have surprisingly discovered that immersing plant seeds in an oxytocin-containing solution before germination can enhance seed germination and / or growth. Furthermore, seed immersion according to this specification may lead to the germination of a larger number of seeds and / or faster germination.

[0032] Therefore, this specification, a) The process of providing seeds; b) A step of providing an aqueous solution containing oxytocin; c) the step of immersing the seeds in the aqueous solution containing oxytocin; and d) A step of removing the seeds from the aqueous solution containing oxytocin, A method for soaking seeds, including the following, is disclosed.

[0033] This specification also covers seeds that can be obtained or obtained by the immersion methods disclosed herein.

[0034] Immersion in step c) is typically carried out over a period of several minutes (e.g., about 1-5 minutes) to about 72 hours, e.g., about 5 minutes to about 72 hours, e.g., about 15 minutes to about 72 hours, e.g., about 0.5 hours to about 72 hours, e.g., about 1 hour to about 48 hours, e.g., about 1 hour to about 24 hours, e.g., about 12 hours to about 48 hours, or e.g., about 12 hours to about 24 hours. Immersion time may be adjusted depending on the seed shell, with shorter times used for shell-less seeds or seeds with shells that allow oxytocin solution to penetrate easily, and longer immersion times used for seeds with shells that do not allow penetration easily. Immersion is typically stopped before the emergence of the radicle and / or cotyledon sheath. In monocotyledonous plant seeds, the radicle and cotyledons of the embryo are covered by the radicular sheath and cotyledon sheath, respectively. When a monocotyledonous plant seed germinates, the radicular sheath grows from the seed first, followed by the growth of the radicle. When dicotyledonous plant seeds germinate, the radicle first appears, followed by a shoot containing cotyledons. As used herein, germination refers to the point in time when the cotyledon sheath and / or radicle appear. Therefore, in the method herein, the seeds are removed from the oxytocin solution before the appearance of the radicle and / or cotyledon sheath. After removal from the oxytocin solution, the seeds may be rinsed (in an aqueous solution, for example) and / or dried.

[0035] Therefore, the method for immersing seeds according to this specification is a) The process of providing seeds; b) A step of providing an aqueous solution containing oxytocin; c) The step of immersing the seeds in the aqueous solution containing oxytocin; d) The step of removing the seeds from the aqueous solution containing oxytocin before the seeds begin to germinate. It may include.

[0036] Soaking can be done at any temperature that does not harm the seeds. Typically, soaking is done at temperatures above 0°C but below 40°C. For example, soaking can be done at temperatures of about 5°C to about 30°C, e.g., about 15°C to about 25°C, about 20°C to about 25°C, e.g., room temperature, about 2°C to about 10°C, or about 3°C ​​to about 8°C, e.g., about 4°C. Lower temperatures during soaking may help prevent rapid germination of seeds during soaking.

[0037] The immersion method described herein may further include a step of drying the seeds after immersion, before germination / planting. Such a drying step may be carried out over a period of 6 hours to 5 days, for example, 1, 2, 3, 4, or 5 days. Drying may be carried out at, for example, about 15–25°C, for example, about 20°C. Drying may be carried out, for example, by drying the seeds in an oven set to such a temperature. Alternatively, the seeds may be freeze-dried. As demonstrated in the experimental section, such drying may be used to stimulate shoot growth rather than root growth, i.e., the growth stimulating effect may be directed more towards shoots than roots.

[0038] The immersion methods described herein may include a step of rinsing (washing) the seeds after immersion and before germination / planting. Such rinsing is typically performed before a drying step, if one is included in the method. Rinsing may be performed with water or an aqueous solution such as a saline solution. Rinsing may remove some or substantially all of the oxytocin solution from the seeds. Surprisingly, as demonstrated in the experimental section, rinsing the seeds after immersion does not interfere with the beneficial effects of immersion on germination and / or growth.

[0039] Seeds immersed by the method for immersion described herein may be planted immediately after immersion. Alternatively, seeds may be stored for a period of time before germination. Thus, immersed seeds may be stored for a period of time before being planted in soil, for example, or placed in a nutrient solution for growth. Typically, such storage can last from one day to several months, for example, about one day to about twelve months, about one day to about six months, about one day to about three months, about seven days to about three months, or about one day to about seven days. Typically, immersed seeds are stored for less than one year, but this can be longer.

[0040] The seeds that may be used in accordance with this specification are any seeds. Therefore, the seeds may be those of monocots, dicots, or gymnosperms (e.g., conifers).

[0041] Non-exclusive examples of monocotyledonous plant seeds include grains such as wheat, rye, oats, barley, millet, and / or spelt. Monocotyledonous plants (commonly called monocots or monocotyledons) have a single cotyledon (embryonic leaf) in their seed.

[0042] Non-exclusive examples of dicotyledonous plant seeds include quinoa seeds, seeds from legumes such as peas, sunflower seeds, dill seeds, and birch seeds.

[0043] Furthermore, the seeds may be gymnosperm seeds, such as those of coniferous trees like pine, cedar, larch, or spruce. When coniferous seeds are treated by the method specified herein, shorter immersion times can be used because the seeds do not have an outer shell. Thus, short immersion times such as about 0.5 hours to about 6 hours, for example, about 0.5 hours to about 3 hours, or for example, about 1 hour to about 2 hours, can be used for coniferous seeds.

[0044] Seed immersion can be carried out in any manner that allows the seeds to absorb the oxytocin solution. For example, seeds may be immersed in the oxytocin solution with or without agitation. It is also possible to provide the seeds with the oxytocin solution and allow them to absorb it by dipping them in the oxytocin solution or by spraying them with the oxytocin solution. Typically, the seeds are immersed in an amount of oxytocin solution sufficient to substantially cover them, taking into account the swelling (water absorption) of the seeds.

[0045] Aqueous solutions of oxytocin may further contain liquid and / or solid carriers, excipients, organic fertilizers, and / or agricultural agents, for example, to stimulate plant growth or protect seeds and plants. An example of such agents is a plant hormone.

[0046] This specification also states, i) Immersing the seeds in the immersion method described herein, and optionally rinsing and / or drying the immersed seeds; ii) The process of germinating the seeds, This includes methods for stimulating the germination and / or growth of seed roots and / or cotyledon sheaths / shoots. Preferably, such germination does not occur in the presence of oxytocin.

[0047] One example of a method for stimulating seed germination according to this specification is seed malting in the presence of oxytocin, where the seeds are immersed in an aqueous solution containing oxytocin, removed from the aqueous solution containing oxytocin, germinated, and then dried.

[0048] The methods disclosed herein may also be used to stimulate the germination and / or growth of seeds with reduced germination capacity. Seeds may have reduced germination capacity, for example, due to aging or infection.

[0049] This specification also provides the use of aqueous solutions containing oxytocin and / or its variants and / or fragments for immersing seeds and / or stimulating the germination and / or growth of the roots and / or cotyledon sheaths of seeds, such as monocotyledonous or dicotyledonous plants or gymnosperms, such as seeds from conifers.

[0050] Seeds immersed according to the immersion method described herein may germinate and / or grow in the presence and / or absence of oxytocin. However, typically, immersed seeds germinate and / or grow in the absence of oxytocin. Surprisingly, as demonstrated in the experimental section, if seeds are simply immersed without germination / growth in oxytocin, the growth-promoting effect of oxytocin may be greater. However, germinating and / or growing seeds in the presence of oxytocin may also, in some cases, have a beneficial effect on germination and / or growth.

[0051] As demonstrated in the experimental section, immersion of seeds in an oxytocin solution produced a potent growth-promoting effect, even though the seeds were not grown in oxytocin. Therefore, the effect of oxytocin when seeds are immersed in its presence is long-lasting and persists even after the seeds are dried or washed, for example. This is unexpected and has a significant impact on the usefulness of oxytocin for stimulating plant growth, as it is far more practical to pre-treat seeds with oxytocin than to provide oxytocin during germination and / or growth, especially in crops sown in outdoor soil. For example, treating seeds in this way instead of the soil or plants is beneficial because it allows for better control of the amount and timing of oxytocin application than when oxytocin is provided at a later stage. Therefore, this method for seed immersion may be important for precision agricultural purposes.

[0052] Oxytocin-containing composition The oxytocin solutions used herein are preferably aqueous solutions. 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.

[0053] In addition to oxytocin, the composition may include one or more liquid and / or solid carriers, excipients, organic fertilizers, and / or agricultural agents.

[0054] Oxytocin and its fragments / variants According to this specification, oxytocin is the peptide represented by SEQ ID NO: 1 (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly, i.e., human oxytocin). Furthermore, one or more oxytocin fragments and / or variants having oxytocin activity, represented by SEQ ID NO: 2, may be used instead of, or in addition to, oxytocin.

[0055] Sequence ID 2 is, X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 (Formula (I)) And here, X1 is selected from the group consisting of Cys and non-existence; X2 is selected from the group consisting of Tyr, Phe, Leu, and non-existence; X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and non-existence; X4 is selected from the group consisting of Gln, Ser, Thr, Cit, Arg, and Daba; X5 is selected from the group consisting of Pro and non-existent; X6 is selected from the group consisting of Ile, Leu, non-existence, Val, Hos, Daba, Thr, Arg, and Cit; X7 is selected from the group consisting of Gly, absence, and Ala; X8 is selected from the group consisting of Gly and non-existence. Sequence ID No. 2 may not contain vasopressin.

[0056] Alternatively, one or more plant cyclotides can be used as oxytocin. Therefore, variants and / or fragments of oxytocin as defined herein may be contained in plant cyclotides.

[0057] Therefore, it should be understood that when X1 is cysteine ​​(Cys) in formula (I), the thiol group of X1 forms a disulfide with the cysteine ​​thiol group located between asparagine (Asn) and X5, thereby forming the cyclic structure of formula (Ia).

[0058] [ka]

[0059] Such cysteine ​​bonds are present in oxytocin and may also be present in oxytocin-active fragments and / or variants of SEQ ID NO: 2, as exemplified below.

[0060] Substances possessing oxytocin activity according to Sequence ID No. 2 include mesotocin, isotocin, annetosine, and vasotocin. Further examples can be found in International Publication No. 2012 / 140216. Further substances possessing oxytocin activity are nonapeptides having the sequence tyrosine-isoleucine-glutamine-asparagine-cysteine-proline-leucine-glycine-amide, with a cysteine ​​on the cysteine ​​at position 6. That is, substances that have the same chemical structure as oxytocin, except that the bond between the cysteine ​​at position 1 and the tyrosine at position 2 is cleaved, and the cysteine ​​at position 1 instead forms a branch on the cysteine ​​at position 6.

[0061] The one or more variants and / or fragments of oxytocin (SEQ ID NO: 2) having oxytocin activity may also be selected from the group consisting of the following compounds and their pharmaceutically acceptable salts.

[0062] Mesotocin: 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 is non-existence;

[0063] Isotocin: 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 is nonexistence;

[0064] Annetosine: 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 is nonexistent;

[0065] Vasotocin: 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 is nonexistence;

[0066] 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 is nonexistence;

[0067] Cys-Tyr-Ile-Gln-Asn-Cys-Pro-NH2 (Sequence ID 9) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, and X6-X8 do not exist;

[0068] Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-NH2 (Sequence ID 10) X1 is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X 7-8 It does not exist;

[0069] Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (Sequence ID 11) X1 is nonexistent, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nonexistent;

[0070] Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (Sequence ID 12) X1-X2 are nonexistent, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nonexistent;

[0071] Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO: 13) X1-X3 are nonexistent, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nonexistent;

[0072] Ile-Gln-Asn-Cys-Pro-NH2 (Sequence ID 14) X1-X2 are nonexistent, X3 is Ile, X4 is Gln, X5 is Pro, and X6-X8 are nonexistent;

[0073] Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-Gly-NH2 (Sequence ID 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;

[0074] Gln-Asn-Cys-Pro-Leu-Leu-NH2 (Sequence ID 16) X1-X3 are nonexistent, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Leu, and X8 is nonexistent;

[0075] Cys-Tyr-Val-Thr-Asn-Cys-Pro-Leu-Gly-NH2 (Sequence ID 17) X1 is Cys, X2 is Tyr, X3 is Val, X4 is Thr, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nonexistent;

[0076] Cys-Tyr-Hoph-Thr-Asn-Cys-Pro-Val-Gly-NH2 (SEQ ID NO: 18) X1 is Cys, X2 is Tyr, X3 is Hoph, X4 is Thr, X5 is Pro, X6 is Val, X7 is Gly, and X8 is nonexistence;

[0077] Cys-Tyr-Phe-Cit-Asn-Cys-Pro-Leu-Gly-NH2 (Sequence ID 19) X1 is Cys, X2 is Tyr, X3 is Phe, X4 is Cit, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nonexistence;

[0078] Cys-Tyr-Cha-Arg-Asn-Cys-Pro-Hos-Ala-NH2 (Sequence ID 20) X1 is Cys, X2 is Tyr, X3 is Cha, X4 is Arg, X5 is Pro, X6 is Hos, X7 is Ala, and X8 is nonexistence;

[0079] Cys-Tyr-Val-Daba-Asn-Cys-Pro-Daba-Ala-NH2 (Sequence ID 21) X1 is Cys, X2 is Tyr, X3 is Val, X4 is Daba, X5 is Pro, X6 is Cit, X7 is Ala, and X8 is nonexistence;

[0080] Cys-Tyr-Hoph-Daba-Asn-Cys-Pro-Cit-Ala-NH2 (Sequence ID 22) X1 is Cys, X2 is Tyr, X3 is Hoph, X4 is Daba, X5 is Pro, X6 is Cit, X7 is Ala, X8 is nonexistent; and

[0081] Cys-Tyr-Phe-Arg-Asn-Cys-Pro-Val-Ala-NH2 (Sequence ID 23) X1 is Cys, X2 is Tyr, X3 is Phe, X4 is Arg, X5 is Pro, X6 is Val, X7 is Ala, and X8 is nonexistence.

[0082] The unnatural amino acids in the aforementioned substance have the following structure.

[0083] Cyclohexylalanine (referred to as Cha in this specification), [ka]

[0084] Homophenylalanine (referred to as Hoph in this specification), [ka]

[0085] Citrulline (referred to as Cit herein), [ka]

[0086] Diaminobutyric acid (referred to as Daba in this specification), and [ka]

[0087] Homoserine (referred to as Hos in this specification). [ka]

[0088] Furthermore, the oxytocin variants and / or fragments of oxytocin represented by SEQ ID NO: 2, which possess oxytocin activity, may be oxytocin analogs from fish, such as the sequence represented by SEQ ID NO: 24 (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH2), or oxytocin analogs from insects, such as the sequence represented by SEQ ID NO: 25 (Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly).

[0089] When a position in Sequence ID No. 2 is described as "non-existent," it means that it represents a single bond between items (letters, atoms, or groups).

[0090] Metabolic derivatives or metabolic degradation products may be oxytocin-like peptides, such as 9-amino acid peptides including oxytocin, mesotocin, isotocin, and annetosine, which have one or more amino acids (e.g., 1 to 3 amino acids from each end) deleted from the carboxyl terminus, the amino terminus, or both. In certain embodiments, one, two, or three amino acids may be deleted from the carboxyl terminus (i.e., Gly only, Gly and Leu, or Gly, Leu, and Pro). For example, two or three amino acids may be deleted from the amino terminus (i.e., Cys only, Cys and Tyr, or Cys, Tyr, and Ile).

[0091] In certain embodiments, one, two, or three amino acids may be deleted from the carboxyl terminus (i.e., Gly only, Gly and Leu, or Gly, Leu, and Pro), and one, two, or three amino acids may be deleted from the amino terminus (i.e., Cys only, Cys and Tyr, or Cys, Tyr, and Ile). The fact that these variants are analogs of oxytocin, mesotocin, isotocin, or annetosine can be confirmed by immunological methods, such as RIA (radioimmunoassay), IRMA (radioanalytic analysis), RIST (radioimmunoadsorption), and RAST (radioallergen adsorption). This specification also includes variants of oxytocin that have at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to oxytocin and exhibit oxytocin activity as defined herein.

[0092] Oxytocin or its variants may exist as both D- and L-isomers, as well as in racemic forms thereof. The D-isomer can be converted to the L-isomer by inversion of its peptide sequence. These and the peptides described above can be prepared 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.

[0093] The present invention will be further described in the following embodiments, but these will not limit the scope of the present invention as described in the claims. [Examples]

[0094] Experiment section Example 1: Soaking of wheat seeds Spring wheat (Diskett) seeds were soaked in tap water containing or without oxytocin at 18°C ​​for 24 or 48 hours. The oxytocin concentration was 2 μM or 4 μM. 150 seeds were used in each treatment. The seeds were covered with the solution during soaking.

[0095] After soaking for 24 or 48 hours, 50 seeds per treatment were placed in Petri dishes filled with tap water (approximately 10 seeds per Petri dish), sealed with Parafilm, and then placed in a dark growth chamber at 18°C ​​and 70% relative humidity. After 72 hours, when the seeds had germinated and roots and shoots had developed, the length of the roots and shoots of each germination was measured, and the total weight of the roots and shoots of each germination was measured.

[0096] After soaking for 24 or 48 hours, 50 seeds per treatment were dried for 3 days, and then the moist seeds were germinated as described above.

[0097] statistics Analysis of variance (ANOVA) was performed on each parameter related to the effect of the tested doses. If the ANOVA showed a significant difference between the tested doses, the significance between the two doses was tested using the Mann-Whitney Pairwise assay.

[0098] The statistics included all seeds that germinated.

[0099] result Soaking seeds in oxytocin (2 or 4 μM) for 24 hours significantly stimulated root and shoot (cotyledon sheath) growth when germinated immediately after soaking, i.e., while the seeds were still moist, in a petri dish, compared to seeds soaked in water alone (see Figure 1).

[0100] When the seeds were dried before being placed in the petri dish, the positive effect on root and shoot growth was greater for seeds soaked for 48 hours than for seeds soaked for 24 hours (Figure 1). In this case, the growth-stimulating effect was more pronounced in the shoots (cotyledon sheaths) than in the roots (Figure 1).

[0101] Example 2: Germination in oxytocin solution (continuous exposure during germination) A filter paper was placed in a Petri dish, and 4 ml of water containing (2 or 4 μM) or not containing oxytocin was added, followed by the addition of spring wheat seeds. The Petri dish was sealed with Parafilm and placed in a dark place in a growth chamber at 18°C ​​and 70% relative humidity. When the seeds germinated, the length of the roots and shoots was measured. In contrast to Example 1, the seeds in Example 2 were germinated in an oxytocin solution (or in a negative control without oxytocin).

[0102] result As shown in Figure 2, root length was positively affected by germination in oxytocin solution, but the length of the shoot (cotyledon sheath) was either not affected at all or slightly negatively affected. However, when seeds were germinated in oxytocin solution, the growth-stimulating effect was not as high as when the seeds were immersed in oxytocin solution.

[0103] Conclusion: Examples 1 and 2 Soaking seeds in an oxytocin solution produced a far more potent growth-promoting effect than germinating the seeds in an oxytocin solution. This was unexpected and significantly impacts the usefulness of oxytocin for stimulating plant growth, especially for crops sown in outdoor soil, where pre-treating seeds with oxytocin is far more practical than providing oxytocin during germination and / or growth.

[0104] Example 3: Immersion of wheat, crisp salad, sunflower, bok choy, radish, and pea seeds in oxytocin solution. Seeds of each plant were immersed for 12 hours in aqueous solutions of oxytocin at concentrations of 2 μM and 4 μM (water and oxytocin), as well as in water (control). Seeds were immersed at 4°C (1-3 dl of seeds depending on seed size). Throughout the entire immersion period, it was ensured that all seeds were completely covered with the oxytocin solution / water. Importantly, no seed germination was observed during the 12-hour immersion period. Therefore, none of the seeds germinated during the immersion period.

[0105] I cut the hemp mat to A4 size, soaked it thoroughly in water, and then removed the excess water.

[0106] After soaking, the excess oxytocin solution / water was removed from the seeds, and the seeds were transferred to a hemp mat and spread out on top of it.

[0107] Depending on the type of seed, the seeds were grown at room temperature (approximately 20-25°C) for 7-12 days. To keep the seeds moist, they were regularly sprayed with water. The seeds were then harvested, and the weight of the sprouts was determined.

[0108] result The results of different tests are shown in Figures 3-10. The weight of the control sprouts treated with water only is set to 100. A comparison of seed growth immersed in 2 μM or 4 μM oxytocin solutions with seed growth immersed in water alone demonstrates a clear growth-promoting effect of oxytocin at both concentrations tested. Furthermore, as can be seen from Figures 6 and 9, oxytocin treatment had a visible positive effect on root development. Also, as shown in Figure 9, the positive effect of oxytocin on germination was visible.

[0109] Example 4: Immersion of wheat, sunflower, and pea seeds in oxytocin solution Seeds of each plant were immersed for 12 hours in an aqueous solution of oxytocin (water and oxytocin) at a concentration of 2 μM (sunflower and pea) or 4 μM (wheat), according to the concentration determined to be optimal in Example 3, and in water (control). Seeds were immersed at 4°C (1-3 dl of seeds depending on seed size). Throughout the entire immersion period, it was ensured that all seeds were completely covered with the oxytocin solution / water. Importantly, no seed germination was observed during the 12-hour immersion period. Therefore, none of the seeds germinated during the immersion period.

[0110] The seeds treated with oxytocin were then thoroughly washed with water (10-15 liters per seed type) to remove the oxytocin solution. The seeds were then placed on paper (filter paper or household paper) for approximately 3 hours until they appeared dry, removing any excess moisture.

[0111] I cut the hemp mat to A4 size, soaked it thoroughly in water, and then removed the excess water.

[0112] Depending on the type of seed, the seeds were grown at room temperature (approximately 20-25°C) for 7-12 days. To keep the seeds moist, they were sprayed with water. The seeds were then harvested, and the weight of the sprouts was determined (see Figures 11-13).

[0113] Example 4: Soaking dill seeds Dill seeds were immersed in 0 μM, 2 μM, or 4 μM oxytocin solutions (oxytocin and water) at 4°C.

[0114] Dill seeds were soaked for 24 and 48 hours, then dried in circulating air at 30°C. After drying, 25 seeds from each treatment were placed in a petri dish containing two pieces of filter paper soaked in 4.5 ml of tap water. For control, 25 untreated seeds (not soaked in water or oxytocin solution) were added.

[0115] We counted germinated seeds daily until the final measurement of the roots and sprouts.

[0116] The final measurement was taken 10 days later.

[0117] result As shown in Figure 14, when dill seeds were soaked for 24 or 48 hours, they germinated faster in the presence of oxytocin compared to unsoaked seeds or seeds soaked in water (measured by counting the number of germinated seeds).

[0118] Example 5: Immersion of gymnosperms (spruce and pine) Gymnosperm seeds (spruce and pine) were soaked in oxytocin solution (2 μM or 4 μM) or water (control) at approximately 20°C for 1-2 hours. The seeds were then transferred to a pot containing 90 ml of peat and covered with sawdust.

[0119] During the first one and two weeks after sowing (when germination occurred), the seeds were watered 3-5 times daily. After two weeks, the pots were moved to a greenhouse where the plants continued to grow for six weeks. After that, the pots were moved outdoors for further growth. The seeds were treated with oxytocin on May 25, 2024, and harvested on September 1, 2024.

[0120] Figures 15 (Spear) and 16 (Pine) show the differences in overall plant length (from root tip to shoot tip, left), root length (center), and shoot length (right) between oxytocin-treated seeds and a control. As can be seen from Figures 15 and 16, immersion in oxytocin produced a potent growth-promoting effect on both roots and shoots. This was particularly surprising considering the short immersion time used in this experiment.

[0121] Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to describe, and not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0122] Unless otherwise expressly stated, each of the preferred features described herein may be used in combination with any and all other preferred features described herein.

Claims

1. a) The process of providing seeds; b) A step of providing an aqueous solution containing oxytocin and / or its variants and / or fragments; c) The step of immersing the seeds in the aqueous solution containing oxytocin; d) The step of removing the seeds from the aqueous solution containing oxytocin; Includes, The oxytocin fragment and / or variant is the fragment and / or variant according to SEQ ID NO: 2 having oxytocin activity. SEQ ID NO: 2 is X １ -X ２ -X ３ -X ４ -Asn-Cys-X ５ -X ６ -X ７ -X ８ -NH ２ (Here, X １ This is selected from the group consisting of Cys and non-existence; X ２ is selected from the group consisting of Tyr, Phe, Leu, and non-existence; X ３ This is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and non-existence; X ４ It is selected from the group consisting of Gln, Ser, Thr, Cit, Arg, and Daba; X ５ This is selected from the group consisting of Pro and non-existence; X ６ This is selected from the group consisting of Ile, Leu, non-existence, Val, Hos, Daba, Thr, Arg, and Cit; X ７ This is selected from the group consisting of Gly, non-existence, and Ala; X ８ (This is selected from the group consisting of Gly and non-existence.) A method for immersing seeds, such as seeds from monocots or dicots or gymnosperms, such as seeds from conifers.

2. The method according to claim 1, wherein the immersion in step c) is carried out over a period of about 1 minute to about 72 hours, for example, about 5 minutes to about 72 hours, for example, about 15 minutes to about 72 hours, for example, about 0.5 hours to about 72 hours, for example, about 1 hour to about 48 hours, about 1 hour to about 24 hours, about 12 hours to about 48 hours, or 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 claims 1 to 3, wherein the seeds of the monocotyledonous plant are grains.

5. The method according to any one of claims 1 to 4, wherein the seeds of the monocotyledonous plant are the seeds of wheat, rye, oats, barley, millet, and / or spelt.

6. The method according to any one of claims 1 to 3, wherein the seeds of the dicotyledonous plant are quinoa seeds, seeds from leguminous plants 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 claims 1 to 7, wherein the concentration of oxytocin is 0.5 to 50 μM, for example, 1 to 50 μM, 1 to 30 μM, 1 to 20 μM, 1 to 10 μM, 1 to 8 μM, 2 to 8 μM, 1 to 6 μM, 1 to 5 μM, 2 to 6 μM, 3 to 5 μM, or 2 to 4 μM.

9. The method according to any one of claims 1 to 8, wherein the aqueous solution of oxytocin further comprises a liquid and / or solid carrier, an excipient, an organic fertilizer, and / or an agricultural agent.

10. i) A step of immersing seeds by a method disclosed in any one of claims 1 to 9; ii) The process of germinating the aforementioned seeds, A method for stimulating the germination and / or growth of the roots and / or cotyledon sheaths / shoots of seeds, such as monocotyledonous or dicotyledonous plant seeds or gymnosperms, including the following:

11. The method according to any one of claims 1 to 10, wherein the seeds are seeds with reduced germination ability.

12. The method according to any one of claims 1 to 11, wherein the oxytocin is a plant cyclotide.

13. Seeds that can be obtained or obtained by a method defined in any one of claims 1 to 12.

14. Use of an aqueous solution containing oxytocin as defined in any one of claims 1 and 8 to 9, and / or its variants and / or fragments, for soaking seeds and / or stimulating the germination and / or growth of the roots and / or cotyledon sheaths of seeds, such as seeds from monocotyledonous or dicotyledonous plants or seeds from gymnosperms such as conifers.

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