Stimulation of plant germination

Abstract

The present document is directed to the field of agriculture and to a method for stimulating plant germination. More specifically, the present document is directed to a method for steeping seeds in order to stimulate their germination, compositions for such use and steeped seeds. The method comprises steeping the seed in an aqueous solution comprising oxytocin, a fragment or a variant thereof, wherein the seeds are not allowed to germinate in the oxytocin solution. This was shown to lead to a stronger growth enhancing effect on the seeds.

Description

[0001]P32148PC 1 STIMULATION OF PLANT GERMINATION TECHNICAL FIELD The present document is directed to the field of agriculture and to a method for stimulating plant germination. BACKGROUND OF THE INVENTION Successful germination of seeds is crucial in the agriculture in order to obtain large harvests. Successful seed germination not only results in a larger number of plants but may also be of importance for combating weeds competing with the seeds for space and nutrients. A seed contains an embryo, one end of which will form the root, the radicle, and other end the stem and leaves, the plumule. A monocotyledoneous plant contains one cotyledon, i.e. one seed leaf occupying a small part of the seed. A dicotyledoneous plant contains two cotyledons. Upon germination of a monocotyledoneous plant, the radicle grows downwards through the split seed coat to produce the primary root, and the shoot, encased in its protective sheath, the coleoptile, grows upwards. In dicots, a primary root emerges from the seed allowing water absorption by the new plant. An apical meristem develops from therefrom and produces the plant’s root system. The shoot then comes forth from the seed. The shoot in a dicotyledoneous plant consists of the cotyledons, hypocotyl and epicotyl. In gymnosperms, such as conifers, after the embryo absorbs water, first a seedling root breaks emerges through the seed coat and turns down into the soil. Thereafter, the stem below the cotyledons elongates. When the cotyledons start to photosynthesize, the shoots start to grow. P32148PC 2 Plant germination and growth can be stimulated by the use of plant hormones, such as auxins, gibberelins, cytokinins and abscisic acid. However, such agents are expensive and may also be toxic when used in larger amounts. WO02 / 102160 discloses the use of a human hormone, oxytocin, to stimulate the germination of plants by germinating the seeds in a solution containing oxytocin. However, germinating seeds in a solution containing oxytocin may both be expensive and impractical if larger amounts of seeds are to be germinated. In contrast to plant germination, seed steeping represents a cost efficient alternative to germination of plants for plant producers and growers. An object of the present invention is therefore to provide improved methods and means for stimulating plant germination and / or growth by specifically treating the seeds before germination and proving a novel and feasible alternative to growers. SUMMARY The present document is directed to a method for steeping a seed such as a seed from a monocotyledoneous or dicotyledoneous plant or a gymnosperm (conifers), said method comprising the steps of: a) providing a seed; b) providing an aqueous solution comprising oxytocin, and / or a variant and / or fragment thereof, typically having a concentration of 0.5-50 ^M oxytocin; c) steeping said seed in said aqueous solution comprising oxytocin; d) removing said seed from said aqueous solution comprising oxytocin, wherein said fragment and / or variant of oxytocin is a fragment and / or variant according to SEQ ID NO:2 possessing oxytocin activity, wherein SEQ ID NO:2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 wherein X1 is selected from the group consisting of Cys and nothing; X2 is selected from the group consisting of Tyr, Phe, Leu and nothing; X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and nothing; 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 nothing; X6 is selected from the group consisting of Ile, Leu, nothing, Val, Hos, Daba, Thr, Arg, and Cit; P32148PC 3 X7is selected from the group consisting of Gly, nothing, and Ala; X8is selected from the group consisting of Gly and nothing. The present document also provides a seed obtainable or obtained by the above method. The steeping in step c) is typically carried out for a time period of from a couple of minutes (such as about 1-5 minutes) to about 72 hours, such as from about 5 minutes to about 72 hours, such as from about 15 minutes to about 72 hours, such as from about 0.5 hours to about 72 hours, such as 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.. Optionally, the method may further comprise a step e) of drying the seed. The seed may be any kind of seed. Non-limiting types of seeds are seeds from monocotyledoneous or dicotyledoneous plants or seeds from gymnosperms. A monocotyledoneous seed may for example be a cereal grain, such as a wheat, rye, oat, barley, millet, and / or dinkel seed. A dicotyledoneous seed may for example be a quinoa seed, seed from legumes, such as peas, sunflower seed, dill seed, and / or birch seed. A gymnosperm seed may for example be a seed from a conifer, such as pine, cedar, larch or spruce. The concentration of oxytocin is typically 0.5-50 ^M, such as 1-50 ^M, 1-30 ^M, 1-20 ^M, 1-10 ^M, 1-8 ^M, 2-8 ^M, 1-6 ^M, 1-5 ^M, 2-6 ^M, 3-5 ^M or 2-4 ^M. The aqueous solution of oxytocin may further comprise one or more of a liquid and / or solid carrier, excipient, organic fertilizer and / or agricultural agent, such as a plant hormone. The present document also provides a method for stimulating the germination and / or growth of the root and / or coleoptile / shoot of a seed, said method comprising the steps of: P32148PC 4 i) steeping said seed by a method comprising the steps of: a) providing a seed, such as a seed from a monocotyledoneous or dicotyledoneous plant or a gymnosperm; b) providing an aqueous solution comprising oxytocin and / or a variant and / or fragment thereof as defined herein, typically having a concentration of 0.5-50 ^M oxytocin; c) steeping said seed in said aqueous solution comprising oxytocin; d) removing said seed from said aqueous solution comprising oxytocin and optionally rinsing and / or drying said seed; and ii) allowing said seed to germinate. There is also provided the use of an aqueous solution comprising oxytocin and / or a variant and / or fragment thereof as herein for steeping seeds and / or stimulating the germination and / or growth of the root and / or coleoptile of a seed, such as a conifer, monocotyledoneous or dicotyledoneous seed. Other features and advantages of the invention will be apparent from the following detailed description, drawings, examples, and from the claims. DEFINITIONS By “steeping”, “to steep” and the like is in the context of the present document intended to soak a seed in a solution to allow uptake of water and / or allow a substance in the solution to be absorbed by the seed, before allowing germination of the seed. Herein the term “soaking” and the like may be used instead of “steeping” and the like. Oxytocin (SEQ ID NO: 1) is understood the have the following chemical structure. Whenever “oxytocin” is mentioned in the present document, this term is also understood to encompass a fragment, variant or homologue of an oxytocin molecule / peptide that encompasses a biological activity comparable to the oxytocin molecule itself (SEQ ID NO:1), or a plant cyclotide. The variant and / or fragment of oxytocin may e.g. have P32148PC 5 substantially the same stimulating effects on germination of a seed and / or growth of a plant after steeping as when oxytocin as such is used for steeping. Accordingly, a “variant” of oxytocin as referred to herein, refers to a peptide which has been varied in its amino acid structure as compared to the oxytocin molecule in that some amino acid positions may have been altered by introducing other amino acids in such positions, such as natural or unnatural amino acids as exemplified herein, or it may have been extended by adding one or more natural or unnatural amino acid(s) to either ends of the peptide. In addition, other structural variations may also have been performed to the present peptides as referred to herein, such as synthetic modifications. Said “variant” still maintains a biological activity similar to oxytocin and said oxytocin variant is also stabilized by being present in a pharmaceutical composition according to the present invention. A “variant” of oxytocin as referred to herein may not include vasopressin (SEQ ID NO:7). Vasopressin is a nonapeptide with the sequence Cys-Tyr-Phe-Gln-Asn-Cys- Pro-Arg-Gly, wherein the cysteine residues form a disulfide bond. Furthermore, a “fragment” of oxytocin, as referred to herein is a peptide which comprises a part of the amino acid sequence of oxytocin, but wherein one or more amino acids may have been removed from one or both of the amino acid terminal end(s). This term also refers to a fragment of an oxytocin variant as defined in SEQ ID NO:2, hence meaning that also encompassed by the present invention is any fragment of a peptide as presented by SEQ ID NO:2. As used herein, a fragment and / or variant of oxytocin according to SEQ ID NO:2 is understood to be X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2 wherein X1 is selected from the group consisting of Cys and nothing; X2 is selected from the group consisting of Tyr, Phe, Leu, and nothing; X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and nothing; 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 nothing; X6 is selected from the group consisting of Ile, Leu, nothing, Val, Hos, Daba, Thr, Arg, and Cit; X7is selected from the group consisting of Gly, nothing, and Ala; P32148PC 6 X8is selected from the group consisting of Gly and nothing. However, it will be appreciated that vasopressin is typically not included in the scope of SEQ ID NO:2 although it may be. In a fragment and / or variant of oxytocin according to SEQ ID NO: 2, a cysteine bridge may be formed between X1, when X1is Cys, and the Cys in position 6 (as counted from the N-terminal in the formula above). BRIEF DESCRIPTION OF DRAWINGS Figure 1: Shows the growth of the root and coleoptile at different doses of oxytocin (oxytocin dissolved in tap water) of spring wheat soaked in tap water or in 2 μM and 4 μM oxytocin solution for 24 hours (1:0, 1:2 and 1:4 respectively) or 48 hours (2:0, 2:1 and 2:4). The diagrams on the left indicate the effect of germination of wet seeds after 24 or 48 hours of soaking. The right charts show the effect of seeds that first have been soaked for 24 or 48 hours after which they have been dried in room temperature for 3 days. The values indicate length growth (upper charts) and weight gain (lower diagrams) as a percentage of control (0 μM oxytocin) growth after 3 days. The values are based on average values from five petri dishes with 8-10 sprouted seeds / petri dish.1: 0, 1: 2, 1: 4, 2: 0, 2: 2 and 2: 4 indicates "days of soaking:concentration of oxytocin". Figure 2: Shows the effect as a percentage of control (0 μM) of different oxytocin (0, 2 and 4 μM) doses on root and shoots length in sprouts of spring wheat. Spring wheat was grown in petri dishes in the dark at 18 °C and 70% RH for 14 days. Figure 3: Sprout weight of wheat, without washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 4: Sprout weight of sunflower, without washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 5: Sprout weight of pak choi, without washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 6: Photograph of roots of pak choi after steeping in 2 ^M and 4 ^M oxytocin or water. Both steeping treatments resulted in more root development. P32148PC 7 Figure 7: Sprout weight of red radish, without washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 8: Sprout weight of pea, without washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 9: Photograph of roots of pea after steeping in 2 ^M oxytocin (right) or water (left). Steeping resulted in the development of more root and shoot biomass. Figure 10: Sprout weight of crisp salad, without washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 11: Sprout weight of wheat, with washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 12: Sprout weight of sunflower, with washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 13: Sprout weight of pea, with washing after steeping in 2 ^M and 4 ^M oxytocin or water. Figure 14: Number of sprouted seeds of dill after steeping in 2 ^M and 4 ^M oxytocin or water.0:0 means unsteeped seeds; 0:24 / 0:48 means seeds steeped in water for 24 and 48 hours respectively, 2:24 / 2:48 means seeds steeped in 2 ^M oxytocin for 24 and 48 hours, respectively, and 4:24 / 4:48 means seeds steeped in 4 ^M oxytocin for 24 and 48 hours, respectively. Figure 15: Length of whole plant (from root tip to shoot tip), shoots and roots, respectively, of spruce steeped in water (control), or 2 ^M or 4 ^M oxytocin. Figure 16: Length of whole plant (from root tip to shoot tip), shoots and roots, respectively, of pine steeped in water (control), or 2 ^M or 4 ^M oxytocin. P32148PC 8 DETAILED DESCRIPTION Methods for steeping and / or stimulating the germination of seeds The present inventors have surprisingly found that the germination and / or growth of a seed of a plant can be enhanced by steeping the seed in an oxytocin containing solution before the seed is allowed to germinate. Further, steeping seeds according to the present document may lead to a larger number of seeds germinating and / or a faster germination. The present document thus discloses a method for steeping a seed, said method comprising the steps of: a) providing a seed; b) providing an aqueous solution comprising oxytocin; c) steeping said seed in said aqueous solution comprising oxytocin; and d) removing said seed from said aqueous solution comprising oxytocin. The present document is also directed to a seed obtainable or obtained by the herein disclosed steeping method. The steeping in step c) is typically carried out for a time period of from a couple of minutes (such as about 1-5 minutes) to about 72 hours, such as from about 5 minutes to about 72 hours, such as from about 15 minutes to about 72 hours, such as from about 0.5 hours to about 72 hours, such as 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 steeping time may be adjusted depending on the shell of the seed with shorter times being used for seeds with no shell or a shell that is easily penetrable by the oxytocin solution and longer steeping times for seeds with shells that are less penetrable. The steeping is typically stopped before emergence of the radicle and / or coleoptile. In monocot seeds, the embryo's radicle and cotyledon are covered by a coleorhiza and coleoptile, respectively. When a monocot seed germinates, the coleorhiza first grows out of the seed, thereafter the radicle. When a dicot seed germinates, the radicle first emerges, thereafter the shoot comprising the cotyledon. As used herein, germination refers to the time point when the coleoptile and / or radicle emerges. Accordingly, in the method of the present document, the seed is removed from the oxytocin solution before P32148PC 9 the emergence of the radicle and / or the coleoptile. After removing the seed from the oxytocin solution, the seed may be rinsed (such as in an aqueous solution) and / or dried. The method for steeping a seed according to the present document may therefore comprise the steps of: a) providing a seed; b) providing an aqueous solution comprising oxytocin; c) steeping said seed in said aqueous solution comprising oxytocin; d) removing said seed from said aqueous solution comprising oxytocin before said seed has started to germinate. The steeping may take place at any temperature that does not harm the seeds. Typically, the steeping is performed at a temperature above 0oC but below 40oC. For example, the steeping may be performed at a temperature of from about 5oC to about 30oC, such as from about 15oC to about 25oC, from about 20oC to about 25oC, such as at room temperature, from about 2oC to about 10oC, or from about 3oC to about 8oC, such as at about 4oC. A colder temperature during steeping may help in preventing germination during the steeping for rapidly germinating seeds. The steeping method of the present document may further comprise a step of drying the seeds after the steeping and before germination / planting. Such a drying step may be performed for a time period of at least 6 hours to 5 days, such as 1, 2, 3, 4, or 5 days. The drying may e.g. be performed at about 15-25oC, such as about 20oC. The drying may e.g. be performed by drying the seeds in an oven set at such temperatures. Alternatively, the seeds may be freeze-dried. As is demonstrated in the experimental section, such drying may be used for stimulating the growth of the shoot more than the root, i.e. the growth stimulating effect may be directed to the shoot rather than the root. The steeping method of the present document may comprise a step of rinsing (washing) the seeds after the steeping and before germination / planting. Such rinsing is typically performed before a drying step, if such is included in the method. The rinsing may be performed with an aqueous solution, such as water or a salt solution. The rinsing may remove some or substantially all the oxytocin solution from the seed. Surprisingly, as is demonstrated in the experimental section, rinsing the seed after steeping does not prevent the beneficial effects of the steeping on germination and / or growth. P32148PC 10 The seeds steeped with the method for steeping a seed according to the present document may be planted immediately after the steeping. Alternatively, the seed may be stored for a period of time before it is allowed to germinate. Thus, the steeped seed may be stored for a period of time before e.g. being planted in soil or put in a nutrient solution for growth. Typically, such storage lasts for at least one day up to several months, such as 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, the steeped seeds are stored for less than a year, although it may be longer. The seed that may be used in accordance with the present document is any seed. The seed can thus be a seed of a monocotyledoneous plant, dicotyledoneous plant or a gymnosperm (such as a conifer). Non-limiting examples of monocotyledoneous seeds are cereal grains, such as wheat, rye, oat, barley, millet, and / or dinkel. Monocotyledoneous plants, commonly called monocots or monocotyledons, have a single cotyledon, (the embryonic leaf) in their seeds. Non-limiting examples of dicotyledoneous seeds are quinoa seed, seeds from legumes, such as peas, sunflower seeds, dill seed, and birch seeds. Further, the seed may be a seed of a gymnosperm, such as a conifer, such as pine, cedar, larch or spruce. When a conifer seed is treated with a method according to the present document, a shorter steeping time may be used as the seed lacks an outer shell. Thus, steeping times as low as from about 0.5 hours to about 6 hours, such as from about 0.5 hours to about 3 hours, such as from about 1 to about 2 hours may be used for conifer seeds. The steeping of the seeds may take place in any manner that allows the seed to take up the oxytocin solution. For example, the seeds may be soaked in an oxytocin solution with or without agitation. It is also possible to provide the oxytocin solution to the seed, such as by dipping in the oxytocin containing solution or spraying the seeds with the oxytocin solution, to allow the seeds to absorb the oxytocin solution. Typically, the seeds are P32148PC 11 soaked in a volume of oxytocin solution enough to essentially cover the seeds taking the swelling of the seeds (imbibition) into account. The aqueous solution of oxytocin may further comprise a liquid and / or solid carrier(s), excipient(s), organic fertilizer(s) and / or agricultural agent(s) in order to e.g. stimulate plant growth or protect the seed or plant. One example of such agents is plant hormones. The present document is also directed to a method for stimulating the germination and / or growth of the root and / or coleoptile / shoot of a seed, said method comprising the steps of: i) steeping a seed by the herein described method of steeping and optionally rinsing and / or drying the steeped seed; ii) allowing said seed to germinate. Preferably, such germination does not take place in the presence of oxytocin. An example of a method for stimulating the germination of a seed in accordance with the present document is malting of a seed in the presence of oxytocin, wherein the seed is steeped in an aqueous solution comprising oxytocin, removed from the aqueous solution comprising oxytocin and then allowed to germinate before the germinated seed is dried. The methods disclosed herein may also be used to stimulate the germination and / or growth of a seed of a seed with impaired ability to germinate. A seed may have an impaired ability to germinate due to e.g. age or infection. Herein is also provided the use of an aqueous solution comprising oxytocin and / or a variant and / or fragment thereof as herein for steeping seeds and / or stimulating the germination and / or growth of the root and / or coleoptile of a seed, such as a seed from a monocotyledoneous or dicotyledoneous plant or a gymnosperm, such as a conifer. A seed steeped according to the method for steeping according to the present document may be germinated and / or grown in the presence and / or absence of oxytocin. Typically, the steeped seed is germinated and / or grown in the absence of oxytocin, though. As is demonstrated in the experimental section, surprisingly, the growth enhancing effect of the oxytocin may be higher if the seeds are only steed and not geminated / grown in oxytocin. However, also germinating and / or growing the seed in the presence of oxytocin may in some instances have a beneficial effect on germination and / or growth. P32148PC 12 As is demonstrated in the experimental section, steeping of seeds in oxytocin solution provided a strong growth enhancing effect, despite the fact that the seeds were not grown in oxytocin. The effect of oxytocin when a seed is steeped in the presence of oxytocin is therefore long-lasting and remains even if the seed is e.g. dried or washed after the steeping in oxytocin. This is unexpected and has large implications on the usability of oxytocin for stimulating plant growth as is it much more practical to pre-treat seeds with oxytocin than to provide it during germination and / or growth, in particular for crops that are to be sowed outside in soil. By treating the seed as such, instead of e.g. treating the soil or plant, is beneficial as it e.g. allows a better control of the amount and timing of the application of the oxytocin than if oxytocin is provided at later stages. The present method for steeping a seed may therefore be importance for the purposes of precision agriculture. Oxytocin containing compositions The solution of oxytocin which is used in the present document is preferably an aqueous solution. The concentration of oxytocin in such an aqueous solution is typically 0.5-50 ^M, such as 1-50 ^M, 1-30 ^M, 1-20 ^M, 1-10 ^M, 1-8 ^M, 2-8 ^M, 1-6 ^M, 1-5 ^M, 2-6 ^M, 3-5 ^M or 2-4 ^M. The aqueous solution may consist of water and oxytocin. In addition to oxytocin, the composition may comprise one or more of a liquid and / or solid carrier(s), excipient(s), organic fertilizer(s) and / or agricultural agent(s). Oxytocin and fragments / variants thereof In accordance with the present document oxytocin is a peptide according to SEQ ID NO:1 (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly, i.e. human oxytocin). Further, instead of, or in addition to oxytocin, one or more fragment(s) and / or variant(s) of oxytocin according to according to SEQ ID NO:2, and which possesses oxytocin activity, may be used. SEQ ID NO:2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2(Formula (I)) wherein X1is selected from the group consisting of Cys and nothing; P32148PC 13 X2is selected from the group consisting of Tyr, Phe, Leu, and nothing; X3is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and nothing; X4is selected from the group consisting of Gln, Ser, Thr, Cit, Arg, and Daba; X5is selected from the group consisting of Pro and nothing; X6is selected from the group consisting of Ile, Leu, nothing, Val, Hos, Daba, Thr, Arg, and Cit; X7is selected from the group consisting of Gly, nothing, and Ala; X8is selected from the group consisting of Gly and nothing. SEQ ID NO: 2 may not comprise vasopressin. Alternatively, it is possible to use one or more plant cyclotides as the oxytocin. The variant and / or fragment of oxytocin as defined herein may thus be comprised in a plant cyclotide. Accordingly, it is to be understood that when X1in formula (I) is cysteine (Cys) then the thiol group of X1 may form a disulfide with the thiol group of the cysteine that is located between the asparagine (Asn) and X5 thereby forming a cyclic structure of formula (Ia): (Formula (Ia)) Such a cysteine bond is present in oxytocin and may also be present in a fragment and / or variant of SEQ ID NO:2 possessing oxytocin activity, such as in the below exemplified fragments and variants. Examples of substances in accordance with SEQ ID NO:2 possessing oxytocin activity include mesotocin, isotocin, annetocin and vasotocin. Further examples may be found in WO 2012 / 140216. A further substance possessing oxytocin activity is the nonapeptide with the sequence tyrosine-isoleucine-glutamine-asparagine-cysteine- proline-leucine-glycine-amide and a cystein on the cystein in position 6, i.e. a substance having the same chemical structure as oxytocin with the exception that the bond between the cystein in position 1 and tyrosine in position 2 is broken and said cystein in position 1 instead forms a branch on the cystein in position 6. P32148PC 14 The one or more variant(s) and / or fragment(s) of oxytocin (SEQ ID NO:2) possessing oxytocin activity may also be selected from the group consisting of the following compounds, as well as pharmaceutically acceptable salts thereof: Mesotocin: Cys-Tyr-Ile-Gln -Asn-Cys-Pro-Ile-Gly-NH2(SEQ ID NO:3) X1is Cys, X2is Tyr, X3is Ile, X4is Gln, X5is Pro, X6is Ile, X7is Gly, and X8is nothing; Isotocin: Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly-NH2(SEQ ID NO:4) X1is Cys, X2is Tyr, X3is Ile, X4is Ser, X5is Pro, X6is Ile, X7is Gly, and X8is nothing; Annetocin: Cys-Phe-Val-Arg- Asn-Cys-Pro-Thr-Gly-NH2(SEQ ID NO:5) X1is Cys, X2is Phe, X3is Val, X4is Arg, X5is Pro, X6is Thr, X7is Gly, and X8is nothing; 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 nothing; 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, X8 is nothing 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 is nothing; 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 X7-8 is nothing; Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO:11) X1 is nothing, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nothing; Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO:12) X1-X2 is nothing, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nothing; Gln-Asn-Cys-Pro-Leu-Gly-NH2 (SEQ ID NO:13) X1-X3 is nothing, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, and X8 is nothing; P32148PC 15 Ile-Gln-Asn-Cys-Pro-NH2(SEQ ID NO:14) X1-X2is nothing, X3is Ile, X4is Gln, X5is Pro, and X6-X8is nothing; Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-Gly-NH2(SEQ ID NO:15) X1is Cys, X2is Tyr, X3is Ile, X4is Gln, X5is Pro, X6is Leu, X7is Gly, and X8is Gly; Gln-Asn-Cys-Pro-Leu-Leu-NH2(SEQ ID NO:16) X1-X3is nothing, X4is Gln, X5is Pro, X6is Leu, X7is Leu, and X8is nothing; Cys-Tyr-Val-Thr-Asn-Cys-Pro-Leu-Gly-NH2(SEQ ID NO:17) X1is Cys, X2is Tyr, X3is Val, X4is Thr, X5is Pro, X6is Leu, X7is Gly, and X8is nothing; Cys-Tyr-Hoph-Thr-Asn-Cys-Pro-Val-Gly-NH2(SEQ ID NO:18) X1is Cys, X2is Tyr, X3is Hoph, X4is Thr, X5is Pro, X6is Val, X7is Gly, X8is nothing; 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 is nothing; 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 is nothing; 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 is nothing; Cys-Tyr-Hoph-Daba-Asn-Cys-Pro-Cit-Ala-NH2 (SEQ ID NO:22) X1 is Cys, X2 is Tyr, X3 is Hoph, X4 is Daba, X5 is Pro, X6 is Cit, X7 is Ala, and X8 is nothing; 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 is nothing. The unnatural amino acids in said substances have the following structures: Cyclohexylalanine, herein referred to as Cha, P32148PC 16 Homophenylalanine, herein referred to as Hoph, Citrulline, herein referred to as Cit, Diaminobutyric acid, herein referred to as Daba, and to as Hos, . Further, a variant and / or fragment of oxytocin according to SEQ ID NO:2 possessing oxytocin activity may be an oxytocin analogue from fish, such as a sequence according to SEQ ID NO: 24 (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH2) or insects, such as a sequence according to SEQ ID NO: 25 (Cys- Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly). P32148PC 17 When a position in SEQ ID NO:2 is stated as being “nothing" it means that it represents a single bond between the items (letter, atom or group). Metabolic derivatives or metabolic degradation products may be oxytocin like peptides, e.g. with nine amino acids such as oxytocin, mesotocin, isotocin, and annetocin from which one or more amino acids has been deleted from either the carboxyl terminal end or the amino terminal end or both the carboxyl terminal and the amino terminal end, such as 1-3 amino acids from each terminal. In certain aspects, one, two or three amino acids may have been deleted from the carboxy terminal end i. e. Gly only, Gly and Leu, or Gly, Leu, and Pro. For example, two or three amino may have been deleted from the amino terminal i.e. Cys only, Cys and Tyr, or Cys, Tyr, and Ile. In certain aspects one, two or three amino acids may have been deleted both from carboxy terminal end i.e. Gly only, Gly and Leu, or Gly, Leu, and Pro, and one, two or three amino acids may have been deleted from the amino end i. e. Cys only, Cys and Tyr, or Cys, Tyr, and Ile. It could be ascertained that these variants are analogues of oxytocin, mesotocin, isotocin or annetocin by immunological methods, e.g. RIA (radioimmunoassay), IRMA (radiometric methods), RIST (radioimmunosorbent test), and RAST (radioallergosorbent test). The present document also includes variants of oxytocin having at least 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99 % sequence identity to oxytocin, said variants showing an oxytocin activity, as defined herein. The oxytocin or a variant thereof may be present in both D- and L-form, as well as racemates thereof. By inversion of the peptide sequence thereof, the D-form could be converted to the L-form. These and the peptides above can be produced by methods known to a person skilled in the art, e.g. according to Merrifield, P. B., "Solid Phase Synthesis", Angew. Chemie, 1985, No.97, p.801. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. P32148PC 18 EXPERIMENTAL SECTION Example 1: Steeping of wheat seeds Seeds of spring wheat (Diskett) were steeped in tap water with or without oxytocin for 24 or 48 hours at 18oC. The concentration of oxytocin was 2 or 4 ^M. For each treatment 150 seeds were used. The seeds were covered with the solution during steeping. After steeping for 24 or 48 hours, 50 seeds per treatment were placed in Petri dishes (-10 seeds per Petri dish) with tap water and sealed with parafilm before being placed in a growth chamber in the dark with a temperature of 18oC and a relative humidity of 70%. After 72 hours, when the seeds were germinated and root and shoots developed, the root and shoot lengths for each germ was measured and the combined weight of the germs’ roots and shoots measured. After steeping for 24 or 48 hours, 50 seeds per treatment were also allowed to dry for three days before being germinated as described above for the wet seeds. Statistics Analysis of variance was performed for each parameter regarding effect of the dose tested. If the analysis of variance showed a significant difference between tested doses, the significance between two doses was tested with the Mann-Whitney pairwise test. In the statistics, all germinated seeds were included. Results Steeping for 24 hours in oxytocin (2 or 4 ^M) significantly stimulated the root and shoot (the coleoptile) growth when the seeds were germinated in Petri dishes directly after steeping, i.e. as still wet seeds, as compared to seeds steeped in water only, see Figure 1. When the seeds were dried before being place in the Petri dishes, the positive effect on root and shoot growth was higher for seeds steeped for 48 hours than 24 hours, Figure 1. In this case, the growth stimulating effect was more pronounced for the shoot (the coleoptile) than for the root (Figure 1). P32148PC 19 Example 2: Germination in oxytocin solution (continuous exposure during germination) Filter papers were placed in Petri dishes and 4 ml of water with (2 or 4 ^M) or without oxytocin before adding seeds of spring wheat. The Petri dishes were sealed with parafilm before being placed in the dark in a growth chamber at a temperature of 18oC and 70% relative humidity. When the seeds had germinated, the length of the root and shoot was measured. In comparison with Example 1, the seeds in Example 2 were allowed to germinate in the oxytocin solution (or the negative control without oxytocin). Results As can be seen in Figure 2, the length of the root was positively affected by the germination in oxytocin solution, while no or a small negative effect on the length of the shoot (the coleoptile) was observed. However, the growth stimulating effect was not as high when the seeds were allowed to germinate in oxytocin solution as if the seeds were steeped in oxytocin solution. Conclusions Examples 1 and 2 Steeping of seeds in oxytocin solution provided a much stronger growth enhancing effect than if seeds were allowed to germinate in oxytocin solution. This is unexpected and has large implications on the usability of oxytocin for stimulating plant growth as is it much more practical to pre-treat seeds with oxytocin than to provide it during germination and / or growth, in particular for crops that are to be sowed outside in soil. Example 3: Steeping of seeds from wheat, crisp salad, sunflower, pak choi, red radish, and pea in oxytocin solution The seeds from the respective plants were steeped for 12 hours in an aqueous solution of oxytocin (water and oxytocin) at a concentration of 2 micromolar and 4 micromolar, and in water (control). The seeds were steeped at 4° (1-3 dl seeds depending on seed size). It was ensured that all the seeds were fully covered by the oxytocin solution / water during the whole steeping period. Importantly, no sprouting of the seeds was observed for the 12 P32148PC 20 hours’ steeping period. None of the seeds did therefore germinate during the steeping period. Hemp mats was cut to A4 size and soaked thoroughly in water whereafter excess water was removed. Excess oxytocin solution / water was removed from the seeds after the steeping and the seeds were transferred to the hemp mats and spread on these. The seeds were allowed to grow for 7-12 days at room temperature (ca 20-25oC) depending on the seed type. The seeds were regularly sprayed with water to keep the seeds moist / wet. The seeds were subsequently harvested, and the sprout weight was determined. Results The results of the different tests are shown in Figures 3-10. The weight of the sprouts of the control treated with water only (control) is set at 100. A comparison of the growth of seeds steeped in 2 micromolar or 4 micromolar oxytocin solution with seeds steeped only in water shows a clear growth enhancing effect of the oxytocin at both tested concentrations. Further, as can be seen from Figures 6 and 9, oxytocin treatment had a visible, positive effect on root development. Also, the positive effects of oxytocin on sprouting were visible by the eye as illustrated in Figure 9. Example 4: Steeping of seeds from wheat, sunflower, and pea in oxytocin solution The seeds from the respective plants were steeped for 12 hours in an aqueous solution of oxytocin (water and oxytocin) at a concentration of 2 micromolar (sunflower and pea) or 4 micromolar (wheat) depending on which concentration was determined optimal in Example 3, and in water (control). The seeds were steeped at 4° (1-3 dl seeds depending on seed size). It was ensured that all the seeds were fully covered by the oxytocin solution / water during the whole steeping period. Importantly, no sprouting of the seeds P32148PC 21 was observed for the 12 hours’ steeping period. None of the seeds did therefore germinate during the steeping period. The oxytocin treated seeds were then thoroughly washed in water (10-15 liters per seed type) to remove oxytocin solution from the seeds. Thereafter, excess moisture was removed from the seeds by placing the seeds on paper (filter paper or household paper) until the seed had a dry appearance (ca 3 hours). Hemp mats was cut to A4 size and soaked thoroughly in water whereafter excess water was removed. The seeds were allowed to grow for 7-12 days at room temperature (ca 20-25oC) depending on the seed type. The seeds were sprayed with water to keep the seeds moist / wet. The seeds were then harvested, and their sprout weight determined, see Figures 11-13. Example 4: Steeping of dill seeds Dill seeds were steeped in 0 μM, 2 μM, or 4 μM oxytocin solution (oxytocin and water) at 4oC. The dill seeds were soaked for 24 and 48 hours before drying in circulating air at 30°C. After the seeds had been dried, 25 seeds from each treatment were placed in petri dishes with 2 filter paper soaked with 4.5 ml tap water. As a control, 25 untreated (not steeped in either water or oxytocin solution) seeds were added. Germinated seeds were counted every day until the final measurement of the seeds' roots and shoots. The final measurement was made after 10 days. Results As is demonstrated in Figure 14, steeping of dill seeds for 24 or 48 the seeds germinated faster in the presence of oxytocin as compared to non-steeped seeds or seeds steeped in water (as measured by counting the number of seeds which had sprouted). P32148PC 22 Example 5: Steeping of gymnosperms (spruce and pine) Seeds from gymnosperms (spruce and pine) were soaked in oxytocin solution (2 ^M or 4 ^M) or in water (control) for 1-2 hours at about 20oC. Thereafter the seeds were transferred to pots containing 90 ml peat and covered with sawdust. During the first and second week after sowing, during which period sprouting occurred, the seeds were watered 3-5 times each day. After two weeks, the pots were transferred to a greenhouse where growth of the plants was continued for 6 weeks. The pots were then moved outside for further growth. The seeds were treated with oxytocin on 2024-05-25 and were harvested on 2024-09-01. Figures 15 (spruce) and 16 (pine) show the difference in length of the whole plant (from root tip to shoot tip, left), length of the root (middle) and length of the shoot (right) between the oxytocin treated seeds and the control. As can be seen in Figures 15 and 16, steeping in oxytocin gave a robust growth enhancing effect on both the root and the shoot. This was particularly surprising given the short steeping time used in this experiment. It is to be understood that while the invention has been described in conjunction with the detailed description thereof, 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. Unless expressly described to the contrary, each of the preferred features described herein can be used in combination with any and all of the other herein described preferred features.

Claims

P32148PC 23 CLAIMS 1. A method for steeping a seed, such as a seed from a monocotyledoneous or dicotyledoneous plant or a gymnosperm, such as a conifer, said method comprising the steps of: a) providing a seed; b) providing an aqueous solution comprising oxytocin and / or a variant and / or fragment thereof; c) steeping said seed in said aqueous solution comprising oxytocin; d) removing said seed from said aqueous solution comprising oxytocin, wherein said fragment and / or variant of oxytocin is a fragment and / or variant according to SEQ ID NO:2 possessing oxytocin activity, wherein SEQ ID NO:2 is X1-X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2wherein X1 is selected from the group consisting of Cys and nothing; X2 is selected from the group consisting of Tyr, Phe, Leu, and nothing; X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and nothing; 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 nothing; X6 is selected from the group consisting of Ile, Leu, nothing, Val, Hos, Daba, Thr, Arg, and Cit; X7 is selected from the group consisting of Gly, nothing, and Ala; X8 is selected from the group consisting of Gly and nothing.

2. The method according to claim 1, wherein said steeping in step c) is carried out for a time period of from about 1 minute to about 72 hours, such as from about 5 minutes to about 72 hours, such as from about 15 minutes to about 72 hours, such as from about 0.5 hours to about 72 hours, such as 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 a step e) of rinsing and / or drying the seed.

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

5. The method according to any one of the preceding claims, wherein said monocotyledoneous seed is a wheat, rye, oat, barley, millet, and / or dinkel seed.P32148PC 24 6. The method according to any one of claims 1 to 3, wherein said dicotyledoneous seeds is a quinoa seed, seed from legumes, such as peas, sunflower seed, dill seed, and / or birch seed.

7. The method according to any one of claims 1 to 3, wherein said 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 ^M, such as 1-50 ^M, 1-30 ^M, 1-20 ^M, 1-10 ^M, 1-8 ^M, 2-8 ^M, 1-6 ^M, 1-5 ^M, 2-6 ^M, 3-5 ^M or 2-4 ^M.

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

10. A method for stimulating the germination and / or growth of the root and / or coleoptile / shoot of a seed, such as a monocotyledoneous or dicotyledoneous seed or a gymnosperm, said method comprising the steps of: i) steeping a seed by the method disclosed in any one of claims 1-9; ii) allowing said seed to germinate.

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

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

13. A seed obtainable or obtained by the method as defined in any one of claims 1-12.

14. Use of an aqueous solution comprising oxytocin and / or a variant and / or fragment thereof as defined in any one of claims 1 and 8-9 for steeping a seed, and / or stimulating the germination, and / or growth of the root and / or coleoptile of a seed, such as a seed from a monocotyledoneous or dicotyledoneous plant or a seed from a gymnosperm, such as a conifer.

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