阅读说明：本技术 用于改善种子萌发和/或植物对环境胁迫耐受性的方法 (Methods for improving seed germination and/or plant tolerance to environmental stress ) 是由 奥利维尔·科尔 于 2018-12-07 设计创作，主要内容包括：本公开涉及由与未处理对照组相比含有增加的甘氨酸–甜菜碱内部浓度的,植物种子、由所述种子衍生的幼苗或植物。本公开还涉及一种用于生产含有增加的甘氨酸–甜菜碱内部浓度的植物种子、由所述种子衍生的幼苗或植物的方法,与未处理对照组相比,所述甘氨酸–甜菜碱将赋予种子、从所述种子衍生的幼苗或植物增强的萌发潜力和/或增加的对各种环境胁迫的耐受性。本公开还涉及甘氨酸–甜菜碱用于增强种子的萌发潜力和/或用于增加种子对环境胁迫的耐受性的用途。本公开还涉及来源于本发明的具有增加的甘氨酸–甜菜碱的国际浓度的种子的,幼苗和植物或其部分。本公开还涉及可通过本发明的方法获得的幼苗或植物或其部分。(The present disclosure relates to plant seeds, seedlings or plants derived from said seeds containing an increased glycine-betaine internal concentration compared to an untreated control. The present disclosure also relates to a method for producing a plant seed, seedling or plant derived from the seed containing an increased internal concentration of glycine-betaine that will confer to the seed, seedling or plant derived from the seed an enhanced germination potential and/or increased tolerance to various environmental stresses as compared to an untreated control group. The present disclosure also relates to the use of glycine-betaine for enhancing the germination potential of a seed and/or for increasing the tolerance of a seed to an environmental stress. The disclosure also relates to seedlings and plants or parts thereof derived from the seeds of the invention with an increased international concentration of glycine-betaine. The present disclosure also relates to a seedling or a plant or a part thereof obtainable by the method of the invention.)

1. A method for producing seeds containing an increased internal concentration of glycine-betaine, the method comprising the steps of:

(a) contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

(b) collecting seeds of the plants treated with glycine-betaine obtained after step (a), wherein the seeds contain an increased internal concentration of glycine-betaine compared to seeds from untreated control plants.

2. The method of claim 1, further comprising the steps of:

(c) growing said seed containing an increased internal concentration of glycine-betaine to produce said seed-derived seedling or plant.

3. A method as claimed in claim 1 or 2 wherein the step of contacting the plant with glycine-betaine is carried out by applying glycine-betaine to the aerial parts of the plant, the floral parts or flowers of the plant, either before or after or at flowering, thereby directing glycine-betaine to the interior of the seed produced by the plant, optionally wherein glycine-betaine is applied by spraying.

4. A method as claimed in any one of claims 1 to 3, wherein said step of contacting aerial parts of a plant, flower parts of a plant or flowers of a plant with said effective amount of glycine-betaine is carried out by applying said glycine-betaine in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

5. The method of any one of claims 1 to 5, wherein the glycine-betaine internal concentration in the seed is increased by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750%, or 1000% as compared to seed from an untreated control plant.

6. The method of any one of claims 1-5, wherein the seed is corn seed, wheat seed, rice seed, soybean seed, or canola seed.

7. The method of any one of claims 2-6, wherein the seed, seedling or plant derived from the seed has enhanced germination potential and/or increased tolerance to environmental stress as compared to an untreated control group.

8. The method of claim 7, wherein the environmental stress is selected from the group consisting of cold temperatures, extremes, water deficit, drought, and combinations thereof.

9. The method of claim 8, wherein the environmental stress is cold temperature.

10. A method for enhancing the germination potential of a seed and/or increasing the tolerance of a seed to an environmental stress, the method comprising:

(a) contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

(b) collecting seeds produced by the glycine-betaine treated plant obtained after step (a),

wherein the seed has enhanced germination potential and/or increased tolerance to environmental stress as compared to seed from a control plant not treated with glycine-betaine.

11. A method for enhancing the germination potential of a seed and/or increasing the tolerance of a seed, or a seedling or plant derived from said seed, to an environmental stress, the method comprising the steps of:

(a) contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

(b) collecting seed produced from the plant treated with glycine-betaine obtained after step (a), wherein the seed has enhanced germination potential and/or increased tolerance to environmental stress compared to seed from an untreated control group of plants, or wherein the seedling or plant derived from the seed has increased tolerance to environmental stress compared to seedling or plant from untreated control group of seeds.

12. The method of claim 10 or 11, wherein the seed contains an increased glycine-betaine internal concentration as compared to seed from an untreated control plant.

13. The method of any one of claims 10-12, further comprising the step of (c) growing said seed to produce a seedling or plant derived from said seed.

14. A method as claimed in any one of claims 10 to 13, wherein the step of contacting the aerial parts of the plant, the flower parts of the plant or the flower plant with glycine-betaine is carried out by applying glycine-betaine to the aerial parts of the plant, the flower parts of the plant or the flower before or after flowering, whereby glycine-betaine is directed to the interior of the seeds produced by the plant, optionally wherein glycine-betaine is applied by spraying.

15. A method as claimed in any one of claims 10 to 14, wherein the step of contacting the aerial parts of the plant, the flower parts of the plant or the flowers of the plant with an effective amount of glycine-betaine is carried out by applying said glycine-betaine in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

16. A method according to any one of claims 10 to 15 for increasing the tolerance of seeds, seedlings or plants derived from said seeds to cold temperatures or extremes of temperature.

17. A method as claimed in any one of claims 10 to 16 for increasing the tolerance of a seed, seedling or plant derived from said seed to drought or water deficit conditions.

18. The method of claim 16, wherein the tolerance of a seed, seedling or plant derived from the seed to cold or extreme temperatures is increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to an untreated control.

19. The method of any one of claims 10 to 18 for enhancing seed germination potential.

20. The method of claim 19, wherein the seed germination potential is enhanced by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to seed from an untreated control plant.

21. The method of any one of claims 10-20, wherein the seed is a corn seed, a wheat seed, a rice seed, a soybean seed, or a canola seed.

22. Seed with an increased glycine-betaine internal concentration, obtainable by the method according to any of the preceding claims.

23. A seed comprising an increased internal concentration of glycine-betaine as compared to an untreated control, wherein said seed is obtainable by contacting an aerial part of a plant, a flower part of a plant or a flower of a plant with an effective amount of glycine-betaine.

24. The seed of claim 23, wherein the seed is obtainable by:

contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

seeds were collected from plants treated with glycine-betaine.

25. The seed of any one of claims 22-24, wherein the glycine-betaine internal concentration is increased by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30% or 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750%, or 1000% compared to an untreated control.

26. The seed of any one of claims 22-25, wherein the seed has enhanced germination potential and/or increased tolerance to environmental stress as compared to an untreated control group.

27. The seed of claim 26, wherein the tolerance of the seed to an environmental stress is increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to an untreated control.

28. The seed of any one of claims 21-27, wherein the environmental stress is selected from the group consisting of cold temperature, extreme temperature, water deficit, drought, and combinations thereof.

29. The seed of claim 28, wherein the environmental stress is cold temperature.

30. The seed of claim 26, wherein the germination potential of the seed is enhanced by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to an untreated control.

31. The seed of any one of claims 21-30, wherein the seed is a corn seed, a wheat seed, a rice seed, a soybean seed, or a canola seed.

32. Use of glycine betaine for the production of seeds having enhanced germination potential and/or increased tolerance to environmental stress compared to an untreated control group, wherein glycine betaine is applied to an aerial part of a plant, a flower part of a plant or a flower of a plant.

33. The use of claim 32, which increases the glycine-betaine internal concentration in said seed by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750%, or 1000% as compared to seed from an untreated control plant.

34. The use of claim 32 or 33, which increases the tolerance of said seed to an environmental stress by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to a seed from an untreated control plant.

35. The use of any one of claims 32-34, wherein the environmental stress is selected from the group consisting of cold temperatures, extremes of temperature, water deficit, drought, and combinations thereof.

36. The use of claim 35, wherein the environmental stress is cold temperature.

37. The use of any one of claims 32-36, which increases the germination potential of the seed by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to an untreated control.

38. Use as claimed in any one of claims 31 to 37, wherein glycine betaine is applied to the aerial parts of the plant, the floral parts of the plant or the flowers before and after or at the time of flowering, thereby directing glycine betaine to the interior of the seeds produced by the plant, optionally wherein glycine betaine is applied by spraying.

39. The use as claimed in any one of claims 32 to 38, wherein glycine-betaine is applied in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

40. The use according to any one of claims 31 to 39, wherein the seed is a maize seed, a wheat seed, a rice seed, a soybean seed or a rape seed.

41. A plant or seedling derived from or comprising a seed according to any one of claims 22 to 31, or a part of said plant or seedling.

42. A plant or seedling, or a part of said plant or seedling, obtainable by the method of claim 2 or 13.

Technical Field

The present disclosure relates to a seed from a plant containing an increased glycine-betaine internal concentration (internal concentration), seedlings or plants derived from the seed, and methods for producing the same. The present disclosure also relates to a method for providing a benefit to a plant seed or to a seedling or plant derived from said seed. More particularly, the present disclosure provides a method for producing a seed of a plant or a seedling or a plant derived from said seed having enhanced seed germination and/or increased tolerance to various environmental stresses. The present disclosure also relates to the use of glycine-betaine for enhancing the germination potential of a seed and/or for increasing the tolerance of a seed to an environmental stress. The present disclosure also relates to seedlings and plants or parts thereof derived from the seeds of the invention with an increased international concentration of glycine-betaine (international concentration). The present disclosure also relates to a seedling or a plant or a part thereof obtainable by the method of the invention.

Background

Plant growth and development are affected by various environmental stresses such as water deficit, drought, extreme humidity, salinity, nutrient deficiency, and suboptimal (subphylum) temperature. Indeed, stress affects plant performance, such as reduced yield, increased susceptibility to diseases and pests, reduced seed germination, reduced plant growth, and reproductive failure. It is at the seed germination and seedling growth stages that plants are most susceptible to environmental stress. For example, extreme temperatures, particularly cold and cold, can delay germination and emergence of seedlings and reduce the height of plants and the length of their roots. It is known that rapid and uniform seed germination helps plants establish healthy growth.

Glycine-betaine (N, N, N-trimethylglycine) is the most potent osmoprotectant produced in plants in response to drought, salinity and suboptimal temperature-induced dehydration (Ashraf and Foolad, 2007; Chen and Murata, 2008). The accumulation of glycine-betaine under stress environmental conditions has been well documented in many plants. Plants with no or low glycine-betaine accumulation show increased stress tolerance after modulation of the genes of the glycine-betaine synthesis pathway and transformation (Ashraf and Foolad, 2007). Thus, since glycine-betaine has been shown to provide some protection to plants from stressful environmental conditions, it has been used to treat soil, plants and seeds.

For example, WO 95/35022 discloses a method of treating seeds with betaine to enhance seedling growth and protect the seeds from adverse environmental conditions. The seeds may be soaked and dried or coated with betaine. The adverse conditions listed are water stress, excess NaCl, extreme temperatures or pH and heavy metal toxicity.

Seed coating is a common method used in agriculture for applying various chemical agents to enhance its performance and to optimize the growth and development of germinated plants. However, several problems are known to exist with seed coating formulation systems. For example, an active ingredient that may be used to coat a seed prior to germination may need to become effective only after germination of the seed. Alternatively, as another example, it may be contemplated that the components of the coating release their active ingredients over an extended period of time, possibly throughout germination. Thus, it is expected that during such extended periods of time during the life cycle of a seed, an effective active ingredient may be susceptible to environmental conditions of seed planting, such as rainfall, soil moisture conditions, temperature changes, sun exposure, and the like. Depending on the composition of the seed coating, the components or active ingredients are prone to loss or may be attacked by biological activities in the field (e.g. microorganisms or pests), which will result in a final loss of effectiveness of the coating treatment.

Thus, there remains a need to produce plant seeds, seedlings or plants derived from such seeds that contain an increased internal concentration of glycine-betaine that will confer to the plant seeds, seedlings or plants derived from such seeds an enhanced germination potential and/or tolerance to various environmental stresses. There is also a need to produce glycine-betaine seeds whose internal glycine-betaine content will be protected against total or partial degradation from glycine-betaine coated seeds due to environmental activity. Seed germination and/or yield is increased and seedlings and plants derived from glycine-betaine rich seeds are protected from various abiotic stresses.

Disclosure of Invention

The methods of the present disclosure provide for a plant seed, seedling or plant derived from the seed, containing an increased internal concentration of glycine-betaine, resulting in plant growth with desirable characteristics due to the presence of the internal glycine-betaine within the seed. The present disclosure relates to methods for producing seeds containing increased glycine-betaine internal concentrations having enhanced seed germination and/or tolerance to environmental stress as compared to untreated controls. In the methods of the present disclosure, the internal content of glycine-betaine (internal content) remains present over time and does not decompose, degrade, or otherwise lose its activity compared to glycine-betaine coated seeds that are sensitive to environmental perturbations.

Accordingly, the present invention provides a method for producing seeds containing an increased glycine-betaine internal concentration, comprising the steps of:

(a) contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

(b) collecting seeds of the plants treated with glycine-betaine obtained after step (a), wherein the seeds contain an increased internal concentration of glycine-betaine compared to seeds from untreated control plants. The method may further comprise the steps of:

(c) growing said seed containing an increased internal concentration of glycine-betaine to produce said seed-derived seedling or plant.

The step of contacting the plant with glycine-betaine may be performed by applying glycine-betaine to the aerial parts of the plant, the flower parts of the plant or the flowers before or after flowering or at the time of flowering, thereby introducing glycine-betaine to the inside of the seeds produced by the plant. Glycine-betaine may be applied by spraying. The step of contacting the aerial parts of the plant, the flower parts of the plant or the flowers of the plant with an effective amount of glycine-betaine may be performed by applying glycine-betaine in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

In any of the methods of the invention described herein, the glycine-betaine internal concentration in the seed may be increased by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750%, or 1000% as compared to seed from an untreated control plant.

In any of the methods of the invention described herein, the seed, the seedling or plant derived from the seed may have enhanced germination potential and/or increased tolerance to environmental stress as compared to an untreated control group. In any of the methods of the invention described herein, the environmental stress may be selected from the group consisting of cold temperature (cold temperature), extreme temperature, water deficit, drought, and combinations thereof. In any of the methods of the invention described herein, the environmental stress can be cold temperature.

The present invention also provides a method for enhancing the germination potential of a seed and/or increasing the tolerance of a seed to an environmental stress, the method comprising:

(a) contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

(b) collecting seeds produced by the glycine-betaine treated plant obtained after step (a),

wherein the seed has enhanced germination potential and/or increased tolerance to environmental stress as compared to seed from a control plant not treated with glycine-betaine. The seed may contain an increased glycine-betaine internal concentration as compared to seed from an untreated control plant. The method may further comprise the step of (c) growing the seed to produce a seedling or plant derived from the seed.

The step of contacting the aerial parts of the plant, the flower parts of the plant or the flower plant with glycine-betaine may be carried out by applying glycine-betaine to the aerial parts of the plant, the flower parts of the plant or the flowers before or after flowering or at the time of flowering, thereby directing glycine-betaine to the inside of the seeds produced by the plant. Glycine-betaine may be applied by spraying.

The step of contacting the aerial parts of the plant, the flower parts of the plant or the flowers of the plant with an effective amount of glycine-betaine may be performed by applying glycine-betaine in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

The methods of the invention described herein for enhancing the germination potential of a seed and/or increasing the tolerance of a seed to an environmental stress may be used to increase the tolerance of a seed, seedling or plant derived from said seed to cold temperatures or extremes of temperature. The tolerance of a seed, seedling or plant derived from the seed to cold temperatures or temperature extremes can be increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to an untreated control.

The methods of the invention described herein for enhancing the germination potential of a seed and/or increasing the tolerance of a seed to an environmental stress can be used to increase the tolerance of a seed, seedling or plant derived from the seed to drought or water deficit conditions. The methods of the invention described herein for enhancing the germination potential of a seed and/or increasing the tolerance of a seed to an environmental stress can be used to enhance the germination potential of a seed. The seed germination potential may be enhanced by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to seed from an untreated control plant.

The present invention also provides a method for enhancing the germination potential of a seed and/or increasing the tolerance of a seed or a seedling or plant derived from said seed to an environmental stress, comprising the steps of:

(a) contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

(b) collecting seed produced from the plant treated with glycine-betaine obtained after step (a), wherein the seed has enhanced germination potential and/or increased tolerance to environmental stress compared to seed from an untreated control group of plants, or wherein the seedling or plant derived from the seed has increased tolerance to environmental stress compared to seedling or plant from untreated control group of seeds. The seed may contain an increased glycine-betaine internal concentration as compared to seed from an untreated control plant. The method may further comprise the step of (c) growing the seed to produce a seedling or plant derived from the seed.

The step of contacting the aerial parts of the plant, the flower parts of the plant or the flower plant (flower plant) with glycine-betaine may be carried out by applying glycine-betaine to the aerial parts of the plant, the flower parts of the plant or the flower before or after or at the time of flowering, thereby directing glycine-betaine to the inside of the seed produced by the plant. Glycine-betaine may be applied by spraying.

The step of contacting the aerial parts of the plant, the flower parts of the plant or the flowers of the plant with an effective amount of glycine-betaine may be performed by applying glycine-betaine in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

The methods of the invention described herein for enhancing the germination potential of a seed and/or increasing the tolerance of a seed or a seedling or plant derived from said seed to an environmental stress may be used to increase the tolerance of a seed, a seedling or plant derived from said seed to cold temperatures or extremes of temperature. The tolerance of a seed, seedling or plant derived from the seed to cold temperatures or temperature extremes can be increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to an untreated control.

The methods of the invention for enhancing the germination potential of a seed and/or for increasing the tolerance of a seed, a seedling or a plant derived from said seed to an environmental stress can be used for increasing the tolerance of a seed, a seedling or a plant derived from said seed to drought or water deficit conditions. The method of enhancing seed germination potential and/or increasing tolerance to environmental stress of a seed, seedling or plant derived from said seed of the invention may be used to enhance seed germination potential. The seed germination potential may be enhanced by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to seed from an untreated control plant.

In any of the methods of the invention described herein, the seed may be a corn seed, a wheat seed, a rice seed, a soybean seed, or a canola seed.

The present invention also provides seeds containing an increased internal concentration of glycine-betaine obtainable by the method of the invention as described herein.

The present invention also provides seeds comprising an increased internal concentration of glycine-betaine as compared to an untreated control, wherein the seeds are obtainable by contacting an aerial part of a plant, a flower part of a plant or a flower of a plant with an effective amount of glycine-betaine. The seed may be obtained by:

contacting an aerial part of a plant, a flower part of a plant, or a flower of a plant with an effective amount of glycine-betaine; and

seeds were collected from plants treated with glycine-betaine.

The glycine-betaine internal concentration of the seed of the invention described herein or obtainable by the method of the invention described herein may be increased by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30% or 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750% or 1000% compared to an untreated control group. The glycine betaine internal concentration of the seeds of the invention described herein or obtainable by the methods of the invention described herein may have an enhanced germination potential and/or an increased tolerance to environmental stress compared to an untreated control group. The seed may have an increased tolerance to an environmental stress of at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to an untreated control. The environmental stress may be selected from the group consisting of cold temperatures, extreme temperatures, water deficit, drought, and combinations thereof. The environmental stress may be cold temperature. The germination potential of the seed may be increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% as compared to an untreated control. The seed may be a corn seed, a wheat seed, a rice seed, a soybean seed, or a canola seed.

The present invention also provides the use of glycine-betaine for producing seeds with enhanced germination potential and/or increased tolerance to environmental stress as compared to an untreated control group, wherein glycine-betaine is applied to an aerial part of a plant, a flower part of a plant or a flower of a plant. The use of glycine-betaine according to the invention as described herein may increase the internal concentration of glycine-betaine in the seed by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750% or 1000% compared to seed from untreated control plants. The use of glycine-betaine according to the invention described herein may increase the tolerance of the seed to environmental stress by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to seed from untreated control plants. The environmental stress may be selected from the group consisting of cold temperatures, extreme temperatures, water deficit, drought, and combinations thereof. The environmental stress may be cold temperature.

The use of glycine-betaine according to the invention as described herein may increase the germination potential of said seed by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to an untreated control group.

In any of the uses of glycine betaine provided by the invention described herein, the glycine betaine may be applied to the aerial parts of the plant, the floral parts of the plant or the flowers before and after or at the time of flowering, whereby the glycine betaine is directed to the interior of the seeds produced by the plant, optionally wherein the glycine betaine is applied by spraying. In any of the uses of the glycine-betaines provided by the invention described herein, the glycine-betaine may be applied in an amount of about 1 to 10 kg/ha, 2 to 8 kg/ha, 3 to 7 kg/ha or 4 to 6 kg/ha.

In any of the uses of the glycine-betaines provided by the invention described herein, the seed may be a corn seed, a wheat seed, a rice seed, a soybean seed, or a canola seed.

The present invention provides a plant or seedling derived from or comprising a seed provided by the invention as described herein or a part of said plant or seedling. The present invention also provides a plant or seedling derived from or comprising a seed, or a part of said plant or seedling, obtainable by the method of the invention as described herein.

The present invention also provides a plant or seedling, or part of said plant or seedling, obtainable by any of the methods of the invention described herein, comprising the step of (c) growing said seed containing an increased internal concentration of glycine-betaine to produce a seedling or plant derived from said seed.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

The method according to the present disclosure uses a novel approach to introduce glycine-betaine into plants without harming the plant seeds during or after application. The seeds produced by this method contain a higher or increased glycine betaine internal concentration compared to untreated control seeds. Glycine-betaine is applied to the aerial parts of plants, the flower parts of plants or the flowers of plants, which is used as a natural inlet for plants and allows glycine-betaine to be efficiently introduced into the next generation of plant seeds. More specifically, when glycine-betaine is applied to the aerial parts of a plant (e.g., by spraying) at or around the time of flowering, glycine-betaine is transferred, transported, or transported and directed to the inside of (or the emerging seed) the seed produced by the plant when grain formation begins. The methods of the present disclosure can aid crop productivity by enhancing seed germination and/or increasing tolerance or resistance to various environmental stresses as compared to an untreated control group. Furthermore, the transport or transfer of glycine-betaine within the seed rather than external application, e.g. as a seed coating, makes glycine-betaine less sensitive to environmental conditions and provides excellent compatibility to chemical seed coatings. Further, by planting the seeds produced by the methods of the present disclosure, glycine-betaine is carried or transported to subsequent generations of plants.

Flowering plants (angiosperms) allow glycine-betaine to be transferred to seeds during grain development (grain degradation) after the flowering phase. Suitable plants include monocots and dicots. Examples of monocotyledons include gramineous (Poaceae) plants. Examples of dicotyledonous plants include leguminous (Fabaceae) plants and cruciferous (Brassicaceae) plants. In one embodiment, the plant is a graminaceous plant. Examples of graminaceous plants herein include agriculturally important species such as rice, wheat, barley, rye, millet (millet), sorghum, maize and oats. In another embodiment, the plant is a legume, such as a pea, bean (bean), lupin, alfalfa, soybean, or lentil. In another embodiment, the plant is a crucifer, such as canola.

In the context of the present disclosure, glycine-betaine solution is contacted or applied to the aerial parts of plants, the flower parts of plants or the flowers of plants at or before or after flowering by, for example, spraying an effective amount of glycine-betaine solution. The term "contacted" as used herein refers to any manner of contacting the surface to be treated, e.g. the aerial parts of a plant, the flower parts of a plant or the flowers of a plant, with glycine-betaine solution. As used herein, the term "effective amount" is an amount sufficient to achieve a beneficial or desired result. The term "at flowering" as used in the context of the present disclosure is the period of flowering (blooms) of the host plant (host plant). The term "pre-and post-anthesis" refers to the period of time before or until the emergence of inflorescences (inflorescences) and after the end of flowering, and includes the different stages of kernel development. The method of contacting the plant with glycine-betaine is not limited to a specific method as long as it enables the glycine-betaine solution to be effectively contacted with the plant or plant part. Spraying is particularly useful in industrial processes. Spraying is recommended because of the protective effect on the favorable conditions of the glycine betaine used.

Glycine-betaines extracted from sugar beet are commercially available, for example under the trademark Glycine-betaine(Lallemand). Other betaine products, such as betaine monohydrate, betaine hydrochloride, and crude betaine liquids are also commercially available and may be used for the purposes of this disclosure.

Glycine betaine is usually applied in suspension at a suitable concentration. Thus, in one embodiment, the glycine-betaine suspension is applied to the aerial parts of the plant, to the flower parts of the plant or to the flowers of the plant in one or several successive treatments. The amount used varies depending on the plant species and the growth period. For example, at least, about 0.1 to 20kg per hectare can be used; about 0.5 to 15 kg; about 1 to 10kg or about 1 to 6kg of glycine-betaine. In another embodiment, glycine-betaine is applied or delivered directly to the aerial parts of plants, to the flower parts of plants or to the flowers of plants in an amount of at least about 1kg, 2kg, 3kg, 4kg, 5kg, 6kg, 7kg, 8kg, 9kg, 10kg, 11kg, 12kg, 13kg, 14kg, 15kg, 16kg, 17kg, 18kg, 19kg or 20kg dry matter per hectare. The amounts given herein are merely suggestive and one skilled in the art can readily determine effective concentrations to function in the manner described herein.

If desired, application can be carried out, for example, by spraying with a spray of some other fertilizer or insecticide. The glycine-betaine solution may also contain wetting agents and/or surfactants which aid in the penetration of the aerial parts of the plant, the flower parts of the plant or the flowers of the plant. Glycine-betaine utilized according to the present disclosure is transported to plant cells, actively regulating the osmotic balance of the cells, and is also involved in other processes of cellular metabolism. Cells treated with glycine-betaine are more viable even when subjected to exogenous stress factors.

The method of the present disclosure further comprises a collecting step. The "collection step" is a step of collecting plant seeds that contain an internally increased concentration of glycine-betaine as compared to untreated control seeds.

The seeds obtained by the method of the invention can be treated like ordinary seeds and can be further processed in a conventional manner. For example, coatings with growth promoters or other chemicals beneficial to plant growth and plant health, such as insecticides, fungicides, insect repellents, fertilizers, antibiotics, and the like, may be provided. Of course, the seeds may be coated with glycine betaine. The term "seed" as used in the context of the present disclosure refers to all kinds of seeds, plant propagation material and plant propagules (propagules), including but not limited to true seeds (true seeds), seed pieces (seed pieces), corms (corms), bulbs (bulbbs), fruits, tubers, grains (sprouts), cut shoots (cuts) and the like. As used herein, a seed is prepared for planting and growing.

Glycine-betaine (along with its beneficial properties) is protected inside the seed from exposure to environmental hazards. Thus, the seeds can be stored for a considerable period of time without significant loss of beneficial traits. For example, plant seeds obtained by the methods of the present disclosure can be stored for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 2 years, or at least 3 years.

The methods of the present disclosure provide seeds containing high internal concentrations of glycine betaine, which can be further transported or transferred in next generation plant material. Experimental results have found that next generation plant bodies such as seedlings can be occupied by glycine-betaine and/or receive the benefits of enhanced tolerance to various environmental stresses provided by glycine-betaine. In the context of the present disclosure, the term "stress" includes, but is not limited to, all biotic and abiotic stresses that may affect a plant or seed from infection to the environment. For example, cold, heat, water deficit, drought, salinity, chemicals, weather conditions, fungal or bacterial infections, insect infestations, soil nutrient deficiencies or excesses, soil compactness or density, light, shading, or soil pH, or any combination of these conditions, are the types of stress that a plant or seed may experience and respond to. Those physical or biochemical characteristics of a plant or seed that may be affected by stress include, for example, yield, height, color, vigor, root growth, shoot growth, flowering time and quality, seed quality, pollen quality, reproductive potential, germination or development, resistance to fungal disease, or any combination of these or other plant characteristics.

The glycine-betaine content of seeds produced by the methods of the present disclosure is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, more than 20%, more than 25%, more than 30% compared to that of untreated seeds.

The glycine-betaine internal concentration of seeds produced by the methods of the invention described herein may be increased by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, or 1100% as compared to seeds from untreated control plants. The glycine-betaine internal concentration of seeds produced by the methods of the invention described herein can be increased by about 2% to about 1100%, about 3% to about 1000%, about 5% to about 750%, about 4% to about 500%, about 5% to about 400%, about 10% to about 300%, about 15% to about 200%, about 25% to about 100%, or about 15% to about 50% compared to seeds from untreated control plants.

The invention provides seeds described herein that have an internal concentration of glycine-betaine that is increased by at least about 2%, 3%, 4%, 5%, 10%, 15%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, or 1100% as compared to seed from an untreated control plant. The invention provides seeds described herein that have an increase in glycine-betaine internal concentration of at least about 2% to about 1100%, about 3% to about 1000%, about 5% to about 750%, about 4% to about 500%, about 5% to about 400%, about 10% to about 300%, about 15% to about 200%, about 25% to about 100%, or about 15% to about 50% as compared to seed from untreated control plants.

The seeds, seedlings, or plants derived from the methods of the present disclosure have increased stress tolerance or resistance as compared to an untreated control (i.e., a control without the methods of the present disclosure), which is at least about a 5, 10, 25, 30, 35, 40, 45, or 50% change in temperature tolerance (chilling or freezing tolerance), or at least about a 5, 10, 20, 25, 30, 35, 40, 45, or 50% change in drought tolerance (or water deficit conditions).

When referring to a seed, seedling or plant obtainable by a method of the invention as described herein, or a seed, seedling or plant provided by the invention as described herein, the term relates to an increase in any useful property or characteristic of said seed, seedling or plant which may be adversely affected by exposure to an environmental stress. As noted above, useful characteristics or features of seeds, seedlings that may be adversely affected by exposure to environmental stress may be selected from yield, height, color, vigor, root growth, shoot growth, flowering time and quality, seed quality, pollen quality, reproductive potential, germination or development, germination potential, resistance to fungal disease, or any combination of these or other plant characteristics. When referring to "increased tolerance to environmental stress," an increase in a useful property or characteristic of a seed, seedling, or plant may be a significant increase in the useful property or characteristic. A significant increase can be a statistically significant increase as determined by an appropriate statistical test. For example, a statistical test, such as a t-test or any other suitable statistical test, for determining whether two data sets differ significantly. One skilled in the art will be readily able to evaluate useful characteristics or features of seeds, seedlings, or plants using any technique known in the art, and select and perform appropriate statistical tests.

The germination potential (an example of a useful property) of a seed may be adversely affected (i.e., reduced) by exposure to environmental stress (e.g., cold temperature). Thus, for example, to assess tolerance to environmental stress of glycine-betaine treated seeds, germination of glycine-betaine treated seeds can be compared to germination of untreated control seeds after exposure to environmental stress for an appropriate time. For example, the seeds may be exposed to cold temperatures (e.g., about 10 ℃ for 7 days). Following exposure to environmental stress, the proportion of seeds that successfully germinated on both glycine-betaine treated seeds and control seeds can be determined and the two values compared. When the proportion of germinated glycine-betaine-treated seeds is higher (e.g., significantly higher) than that of untreated control seeds, it can be said that the glycine-betaine-treated seeds have increased tolerance to environmental stress (in this case, increased tolerance to cold temperatures and/or increased cold tolerance).

The growth, survival and/or yield of the seeds/fruits of the seedlings or plants may be adversely affected by exposure to environmental stresses, such as cold temperatures. To assess the tolerance of the seedlings or plants of the invention described herein to environmental stress, the growth, survival and/or yield of the seeds/fruits (or any other useful characteristic or property described above) of a plurality of seedlings or plants treated with glycine-betaine may be compared to the growth, survival and/or yield of the seeds/fruits (or any other useful characteristic or property described above) of a plurality of untreated seedlings or plants. A glycine-betaine treated seedling or plant may be said to have increased tolerance to environmental stress, such as increased tolerance to cold temperatures and/or increased cold resistance, when the growth, survival and/or yield (or any other useful characteristic or property described above) of the seed/fruit of the glycine-betaine treated seedling or plant is higher (e.g., significantly higher) as compared to an untreated control seedling or plant.

The tolerance to environmental stress of the seed, seedling or plant obtainable by the method of the invention described herein, or provided by the invention described herein, may be increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to an untreated control. The tolerance to environmental stress of the seed, seedling or plant obtainable by the method of the invention described herein, or provided by the invention described herein, may be increased by about 0.2% to 50%, about 0.4% to about 45%, about 0.6% to about 40%, about 0.8% to about 35%, about 1% to about 30%, about 1.2% to about 25%, about 1.4% to about 20%, about 1.6% to about 10%, about 1.8% to about 5%, or about 2% to about 4% compared to an untreated control.

The tolerance to cold temperatures (e.g., cold or freezing tolerance) of the seeds, seedlings or plants obtainable by the methods of the invention described herein or provided by the invention described herein can be increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% as compared to an untreated control. The tolerance to cold temperatures (e.g., cold or freezing tolerance) of the seeds, seedlings or plants obtainable by the methods of the invention described herein or provided by the invention described herein can be increased by at least about 0.2% to 50%, about 0.4% to about 45%, about 0.6% to about 40%, about 0.8% to about 35%, about 1% to about 30%, about 1.2% to about 25%, about 1.4% to about 20%, about 1.6% to about 10%, about 1.8% to about 5%, or about 2% to about 4% as compared to an untreated control.

In addition, the methods of the present disclosure provide seeds with enhanced seed germination potential, seedling emergence, or plant growth, or more uniform germination compared to untreated controls. In the context of the present disclosure, the term "seed germination" relates to clear evidence of root growth developing from an embryo on a seed, especially after a resting period. The term "seed germination potential" relates to the ability of a seed to develop root growth from an embryo on the seed, e.g., after dormancy and/or exposure to an environmental stress for a period of time. When referring to "enhanced seed germination" and/or "enhanced germination potential" of a seed, these terms relate to an increase in seed germination or seed germination potential of a glycine-betaine treated seed as compared to an untreated control seed. For example, to assess seed germination or an increase in seed germination potential, the germination of multiple seeds treated with glycine-betaine may be compared to the germination of multiple untreated control seeds. The ratio of successfully germinated seeds of both glycine-betaine treated seeds and control seeds can be determined and compared. Glycine-betaine treated seeds can be said to have enhanced germination when the proportion of germinated glycine-betaine treated seeds is higher (e.g., significantly higher) than untreated control seeds. Such seeds may also be referred to as having "enhanced germination potential". The skilled person can use an appropriate statistical test, e.g. a t-test, to determine whether the proportion of glycine-betaine treated seeds that successfully germinated is significantly higher than the proportion of untreated control seeds that successfully germinated. In the context of the present disclosure, the term "seedling emergence" means the observable plant growth above the surface of a rooting medium (rooting medium). The methods of the present disclosure provide for greater seed germination, seedling emergence, or growth from at least about 5, 10, 20, 25, 30, 35, 40, 45, or 50% of the plants compared to an untreated control group.

The germination or germination potential of a seed obtainable by a method of the invention described herein or provided by the invention described herein may be increased by at least about 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% as compared to a seed from an untreated control plant. The germination or germination potential of a seed obtainable by a method of the invention described herein or provided by the invention described herein may be increased by at least about 0.2% to 50%, about 0.4% to about 45%, about 0.6% to about 40%, about 0.8% to about 35%, about 1% to about 30%, about 1.2% to about 25%, about 1.4% to about 20%, about 1.6% to about 10%, about 1.8% to about 5%, or about 2% to about 4% as compared to a seed from an untreated control plant.

The present disclosure will be more readily understood by reference to the following examples, which are given to illustrate the present disclosure and not to limit its scope.