| US8748346B1 | 2014-06-10 | |||
| GB2520147A | 2015-05-13 | |||
| JPS5129229A | 1976-03-12 | |||
| BE596283A | 1961-04-21 |
| CLAIMS: 1. A seed treatment composition comprising one or more aromatic amino acids and one or more gibberellins. 2. Composition according to claim 1 , wherein said aromatic amino acid is one or more of the following: phenylalanine (Phe or F), tryptophan (Trp or W) and tyrosine (Tyr or Y), which amino acids may be in L- and/or D-form. 3. Composition according to claim 1 or 2, wherein said one or more gibberellin comprises gibberellic acid (GA3). 4. Composition according to any preceding claim, wherein the concentration of the aromatic amino acid is in the range of from about 0.0001 to about 10.0 mg/kg seed, or in the range of from about 0.001 to about 5.0 mg/kg seed, or in the range of from about 0.001 to about lmg/kg seed or in range of from about 0.001 to about O. lmg/kg seed. 5. Composition according to any preceding claim, wherein said gibberellin is preferably GAB and is present in the range of from about 0 to about 200.0 mg/kg seed, or in the range of from about 1.0 to about 120.0 mg/kg seed, or in the range of from about 4.0 to about 80.0 mg/kg seed, or preferably in the range of from about 8.0 to about 40.0 mg/kg seed. 6. Composition according to any preceding claim comprising the combination of tryptophan in the range of from about 0.005 to about O. lmg/kg seed and GA3 at a concentration in the range of from about 8.0 to about 40mg/kg seed. 7. Composition for the treatment or coating of seeds comprising tryptophan at a concentration in the range of from about 0.005 to about O. lmg/kg seed. 8. Composition according to any preceding claim further comprising one or more plant protection, plant health or plant growth products, such as one or more pesticide, herbicide, fertilizer, nutrient, adjuvant, vitamin, growth regulator, and dye. 9. A method for the preparation of a seed treatment composition, comprising mixing one or more aromatic amino acids with one or more gibberellins, in any order. 10. A method for the application of a seed treatment composition as defined in any of claims 1 to 8 or a method for coating or treating a seed with a seed treatment composition as defined in any of claims 1 to 8, comprising coating or directly or indirectly contacting said composition with a seed. 11. A treated or coated seed comprising the seed treatment composition according to any one of claims 1 to 8 or a seed obtainable from the method of claim 10. 12. Seed according to claim 11, wherein said seed is from a monocotyledonous plant or dicotyledonous plant, preferably cereals (for example, spring and winter wheat, maize, triticale, rice, barley, rye, oats, sorghum, millet); grasses (for example, turf grass, rye grass, fescue, dwarf perennial rye grass, strong creeping red fescue); commercially important crop plants (for example, sugar cane, sugar beet, cotton, canola, sunflower, mustard, corn, coffee, tea, tobacco); legumes (for example, soybeans or beans); members of the solanaceae family (for example, tomatoes, peppers and potatoes); greenhouse crops; tree crops (such as pome and stone fruit crops, and nut crops such as walnuts, pistachio and olives, coco pods, palms such as 011 palm and date palm; vegetables such as brassicas for instance cabbages and lettuces. 13. A plant obtainable from a seed according to claim 11 or 12. 14. A method for growing a plant from a treated or coated seed according to claim 11 or 12 or a method for obtaining plants having improved growth characteristics, comprising the steps of: a) sowing said coated seed or growing said treated seed; and b) growing a plant from said seed, optionally under abiotic and/or biotic stress conditions, c) obtaining plants having improved growth characteristics. 15. A kit comprising: a) one or more aromatic amino acids and one or more gibberellins; or b) a composition as defined in any of claims 1 to 8; and optionally c) an agriculturally acceptable carrier; and optionally d) an applicator for applying any of elements a), b) or c) to a seed. 16. Use of one or more aromatic amino acids and one or more gibberellins in improving seed performance and/or in improving the growth characteristics of plants resulting from treated or coated seeds. 17. Use of a composition as defined in any of claims 1 to 8 or use of a kit according to claim 15 in improving seed performance and/or in improving the growth characteristics of plants resulting from coated or treated seeds. 18. Use of a composition according to claim 6 for obtaining a synergistic effect, especially on aboveground plant biomass. 19. Use according to any one of claims 16 to 18, wherein said improved seed performance and/or improved growth characteristics may be manifested by, for example, any one or more of the following: increased percentage germination and/or emergence, increased speed of germination and/or emergence, improved uniformity of germination and/or emergence, increased plant establishment, increased percentage of seedlings that develop and survive, enhanced vigour, early vigour, increased fresh weight of seedlings and/or resultant plants, increased tillering, increased productive tillers, increased biomass (aboveground or belowground), increased yield (of aboveground parts, including seeds, or of belowground plant parts, including tubers, rhizomes, roots etc.), increased abiotic stress tolerance, increased biotic stress tolerance, reduction in crop losses. |
The present invention relates generally to the field of agriculture. In particular, the invention relates to seed treatment compositions, to a method for preparing the compositions and to their use. The invention also provides seeds coated in or treated with the compositions and to plants generated from the seeds.
Background
Plant protection products may be applied during the growth of a plant and/or added to the seed in the form of a seed treatment or seed dressing. Seed treatments or dressings are compositions applied to seeds prior to planting and typically comprise plant protection products serving to maximise the chances of successful establishment of healthy plants. Although foliar applications of plant protection products may be necessary at various stages of plant growth, they can prove quite wasteful with significant quantities of product being lost to the soil. The use of treated or dressed seeds is a more targeted and less wasteful approach, with plants that are established from such seeds requiring reduced subsequent applications of plant protection products, making seed treatments a cost effective and comparatively environmentally-friendly option for plant protection. Furthermore, due to the relatively small size of a seed compared to the plant derived therefrom, comparatively reduced quantities of any given plant protection product will be required, again reducing costs for the grower.
A wide variety of seed treatments are available. Seed treatments may comprise pesticides, such as insecticides or fungicides, which may be tailored according to the plant in question and growing region. Seed treatments may also comprise protective agents to prolong their shelf life during storage and distribution and to retain their viability. Seed treatments may also comprise agents to facilitate germination.
US patent number 8,748,346 B l describes a foliar treatment for enhancing plant growth and which is made from a mixture of gibberellic acid, triethanolamine, kinetin, isopropyl alcohol, tryptophan and humic acid. Published US patent application number 2004/006582 Al describes a seed treatment composition containing plant macronutrients, micronutrients, a pest inhibitor and at least one growth regulator and additionally a vitamin component, an amino acid component, a penetrant and an energy source.
Summary of the Invention
Surprisingly, it has now been found that the treatment of seeds with a composition comprising one or more aromatic amino acids and one or more gibberellins improves seed performance.
According to the present invention, there is therefore provided a seed treatment composition comprising one or more aromatic amino acids and one or more gibberellins. Also provided is a seed treatment composition comprising tryptophan at a particular concentration range as defined further herein.
A method for the preparation of a seed treatment composition is also provided.
The invention further provides a method for the application of a seed treatment composition to a seed, which method comprises coating (for example by spraying, dipping or brushing) a seed treatment composition of the invention onto the seed.
The present invention also provides a method for the treatment of seeds or a method for improving seed performance, comprising applying to a seed the composition of the invention. Also provided is a treated or coated seed comprising the seed treatment composition of the invention. Also provided by the invention are plants obtainable from such coated or treated seeds, which plants have improved growth characteristics relative to plants obtained from untreated seeds. A method for growing a plant from a coated or treated seed is also provided, as is a method for obtaining plants having improved growth characteristics, which method comprises the steps of:
a) sowing the coated or treated seed; and b) growing a plant from the seed, optionally under stress (biotic and/or abiotic) conditions, c) obtaining plants having improved growth characteristics.
The present invention also provides the use of one or more aromatic amino acids in combination with one or more gibberellins or the use of a composition as defined herein or the use of a kit according to the invention in improving seed performance and/or in providing plants with improved growth characteristics.
The improved seed performance or the improved growth characteristics exhibited by the resultant plants may be manifested by, for example, any one or more of the following: increased percentage germination and/or emergence, increased speed of germination and/or emergence, improved uniformity of germination and/or emergence, increased plant establishment, increased percentage of seedlings that develop and survive, enhanced vigour, early vigour, increased fresh weight of seedlings and/or resultant plants, increased tillering, increased productive tillers, increased biomass (aboveground or belowground), increased yield (of aboveground parts, including seeds, or of belowground plant parts, including tubers, rhizomes, roots etc.), increased abiotic stress tolerance, increased biotic stress tolerance, reduction in crop losses. Also provided is a kit comprising: a) one or more aromatic amino acids and one or more gibberellins; or
b) a composition as described herein; and optionally
c) an agriculturally acceptable carrier; and optionally
d) an applicator for applying any of elements a), b) or c) to a seed.
Detailed Description of the Invention
According to a first aspect of the present invention, there is provided a seed treatment composition comprising one or more aromatic amino acids and one or more gibberellins. An aromatic amino acid is any amino acid with an aromatic ring, examples of which include phenylalanine (Phe or F), tryptophan (Trp or W) and tyrosine (Tyr or Y). Phenylalanine is an essential amino acid and has the formula C9H11NO2. Tryptophan (IUPAC name: Tryptophan or (2S)-2-amino-3-(lH-indol-3-yl) propanoic acid) is also an essential amino acid. The aromatic amino acid comprised in the composition of the invention may be a D isomer or an L isomer or a combination of both isomers, in any proportion. In the case of tryptophan, the use of L-tryptophan is preferred. Aromatic amino acid analogues or derivatives may also be used in the composition of the invention.
Gibberellins are plant hormones that are involved in various growth and developmental processes throughout the life cycle of a plant. Although any gibberellin may be used in the compositions, kits and methods of the invention, the use of gibberellic acid (GA3) is preferred. The term "gibberellin" as used herein is taken to mean any class of gibberellin, for example GA1, GA3, GA4 and GA7. The terms "gibberellic acid", "GA3" and "gibberellic acid salt" are used interchangeably herein to mean a chemical of the formula C19H22O6 (or salts thereof) or having the IUPAC name (3S,3aS,4S,4aS,7S,9aR,9bR,12S)-7,12-dihydroxy- 3-methyl-6-methylene-2-oxoperhydro-4a,7-methano-9b,3-propeno azuleno[l,2-b]furan-4- carboxylic acid (or salts thereof). Gibberellic acid analogues, derivatives and gibberellic acid salts may also be used in the compositions of the invention.
The amount of aromatic amino acid in the seed treatment composition may be in the range of from about 0.0001 to about lOmg/kg seed, or in the range of from about 0.001 to about 5mg/kg seed, or in the range of from about 0.001 to about lmg/kg seed, or in the range of from about 0.001 to about 0. lmg/kg seed. A preferred range is from about 0.001 to about lmg/kg seed or from about 0.001 to about 0. lmg/kg seed.
Preferably, the aromatic amino acid is tryptophan, particularly L-tryptophan. Where the aromatic amino acid is tryptophan, the amount preferably ranges from about 0.001 to about lmg/kg seed, further preferably in the range of from about 0.005 to about 0. lmg/kg seed. A quantity of tryptophan in the range of from about 0.005 to about 0. lmg/kg seed is particularly preferred in combination with a gibberellin, particularly gibberellic acid, which combination gives a synergistic effect.
Although the combination of tryptophan and gibberellic acid is preferred, an improvement may also be realised with tryptophan alone, particularly when applied at a rate in the range of from about 0.005 to about 0. lmg/kg seed. Where the aromatic amino acid is phenylalanine, the amount preferably ranges from about 0.001 to about lOmg/kg seed, preferably from about 0.005 to 7mg/kg seed, more preferably from 0.007 to 4mg/kg seed, most preferably in the range of from about 0.01 to about lmg/kg seed.
The total amount of aromatic amino acid (whether tryptophan and/or phenylalanine and/or any other amino acid) in the seed treatment composition may be any concentration up to about 0.0001, or 0.0002, or 0.0003, or 0.0004, or 0.0005, or 0.0006, or 0.0007, or 0.0008, or 0.0009, or 0.001, or 0.002, or 0.003, or 0.004, or 0.005, or 0.006, or 0.007, or 0.008, or 0.009, or 0.0095, or 0.01, or 0.015, or 0.02, or 0.03, or 0.04, or 0.05, or 0.06, or 0.07, or 0.08, or 0.09, or 0.1, or 0.2, or 0.3, or 0.4, or 0.5, or 0.6, or 0.7, or 0.8, or 0.9, or 1.0, or 1.1, or 1.2, or 1.3, or 1.4, or 1.5, or 1.6, or 1.7, or 1.8, or 1.9, or 2.0, or 2.1, or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1, or 3.2, or 3.3, or 3.4, or 3.5, or 3.6, or 3.7, or 3.8, or 3.9, or 4.0, or 4.1, or 4.2, or 4.3, or 4.4, or 4.5, or 4.6, or 4.7, or 4.8, or 4.9, or 5.0, or 5.1, or 5.2, or 5.3, or 5.4, or 5.5, or 5.6, or 5.7, or 5.8, or 5.9, or 6.0, or 6.1, or 6.2, or 6.3, or 6.4, or 6.5, or 6.6, or 6.7, or 6.8, or 6.9, or 7.0, or 7.1, or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0, or 8.1, or 8.2, or 8.3, or 8.4, or 8.5, or 8.6, or 8.7, or 8.8, or 8.9, or 9.0, or 9.1, or 9.2, or 9.3, or 9.4, or 9.5, or 9.6, or 9.7, or 9.8, or 9.9, or up to about 10.0 mg/kg seed. The concentration of gibberellin, particularly gibberellic acid (GA3), in the seed treatment composition may be in the range of from about 0 to about 200.0mg/kg seed, or in the range of from about 1.0 to about 120.0 mg/kg seed, or in the range of from about 4.0 to about 80.0 mg/kg seed, or preferably in the range of from about 8.0 to about 40.0 mg/kg seed. The concentration of gibberellin if present, particularly gibberellic acid (GA3), in the seed treatment composition may be any concentration up to or 5.0, or 10.0, or 15.0, or 20.0, or 25.0, or 30.0, or 35.0, or 40.0, or 45.0, or 50.0, or 55.0, or 60.0, or 65.0, or 70.0, or 75.0, or 80.0, or 85.0, or 90.0, or 95.0, or 100.0, or 105.0, or 110.0, or 115.0, or 120.0, or 125.0, or 130.0, or 135.0, or 140.0, or 145.0, or 150.0, or 155.0, or 160.0, or 165.0, or 170.0, or 175.0, or 180.0, or 185.0, or 190.0, or 195.0 or up to about 200.0, mg/kg seed.
According to one embodiment, the composition comprises one or more aromatic amino acids and one or more gibberellins and an agriculturally acceptable carrier. The components of the compositions may be combined together to form a concentrate that is then optionally mixed with an agriculturally acceptable carrier such as water or a fertiliser or other suitable form for coating before use. Such concentrates form a further embodiment of the invention.
Agriculturally acceptable carriers used in the composition may be solid or liquid depending upon the nature of the formulation.
For instance, solid carriers and diluents may include natural clays, kaolin, pyrophyllite, bentonite, alumina, montmorillonite, kieselguhr, chalk, diatomaceous earths, calcium phosphates, pumice, attapulgite clays, fuller's earth, ground corn cobs, sands, silicates, sodium, calcium or magnesium carbonates, sodium bicarbonate, magnesium sulphate, lime, flours, talc, polysaccharides and other organic and inorganic solid carriers.
Liquid carriers and diluents may include water or organic solvents such as a ketone, alcohol or glycol ether. These solutions may contain a surface-active agent (for example to improve water dilution in a spray tank).
Compositions may additionally or alternatively include other types of reagent which are well known in the art, in particular, wetting agents, suspending agents and/or dispersing agents.
According to a preferred embodiment, there is provided a seed treatment composition comprising:
a) tryptophan in the range of from about 0.005 to about 0.1 mg/kg seed; or
b) phenylalanine in the range of from about 0.01 to about 1.0 mg/kg seed; and c) gibberellin, particularly gibberellic acid (GA3), at a concentration in the range of from about 8.0 to about 40.0 mg/kg seed; and optionally
d) an agriculturally acceptable carrier.
Surprisingly, the combination of tryptophan at a concentration in the range of from about 0.005 to about O. lmg/kg seed and gibberellic acid (GA3) at a concentration in the range of from about 8.0 to about 40.0mg/kg seed shows a synergistic effect. Advantageously, aboveground biomass (manifested by seedling shoot biomass) was increased by more than the sum of the individual parts when tryptophan and GA3 were combined at these concentrations and used to treat or coat seeds.
According to an embodiment of the invention, the composition may further comprise one or more plant protection, plant health or plant growth products, such as one or more pesticide (for example, fungicide, insecticide, nematicide), herbicide, fertilizers, nutrients, adjuvants, vitamins and growth regulators etc. The composition may also comprise a dye or other colourant so as to distinguish and identify seeds treated with the composition of the invention from non-treated seeds.
According to a second aspect of the present invention, there is provided a method for the preparation of a seed treatment composition, comprising mixing, in any order, one or more aromatic amino acids and one or more gibberellins. The components of the compositions may be combined together to form a concentrate.
According to a third aspect of the present invention, there is provided a method for the application of a seed treatment composition or a method for treating a seed, which method comprises coating the composition of the invention onto a seed (for example by spraying, dipping or brushing or any other conventional method). The method for application / method for treating may result in the seed being covered in whole (substantially in its entirety) or in part. The individual components making up the composition of the invention may be applied to the seed together, in combined form, or may be applied sequentially, in any order.
According to an alternative embodiment, the method for the application of a seed treatment or a method for the treatment of seeds comprises growing seeds in a growth medium with the seed treatment composition placed adjacent the seed, such that the composition is able to exert its effects at the seed-soil interface creating a microenvironment of sorts for each seed. According to this alternative embodiment, there is provided a method for the treatment of a seed, which method comprises growing the seeds in a plant growth medium and placing the seed treatment composition adjacent the seed in the same plant growth medium, such that the composition is able to exert its effects through the seed-soil interface. The term "coating" a seed refers to the more direct action of covering a seed with the composition of the invention (for example by spraying, dipping or brushing or any other conventional method); whereas the term "treating" a seed refers to a more indirect action, where, for example, the seed treatment composition is placed adjacent the seed, such that the composition is able to exert its effects at the seed-soil (or other growth medium) interface creating a microenvironment of sorts for each seed.
The application of a seed treatment composition to a seed, i.e. coating or treating a seed, results in improved seed performance. Therefore, according to a fourth aspect of the present invention, there is also provided a method for improving seed performance, comprising applying to a seed (for example by spraying, dipping or brushing or any other conventional method) the composition of the invention.
According to one embodiment, the individual components making up the composition (namely one or more aromatic amino acids and one or more gibberellins) may be applied together (combined application) or sequentially (in any order).
The composition applied to a seed may take any form suitable for coating or treating a seed, as would be known in the art. For example, including prills, dustable powders, soluble powders or tablets, water soluble granules, water dispersible granules, wettable powders, granules (slow or fast release), soluble concentrates, ultra-low volume liquids, emulsifiable concentrates, dispersible concentrates, emulsions (both oil in water and water in oil), micro- emulsions, suspension concentrates, aerosols, capsule suspensions and any other form suitable for coating or treating a seed. The form of the coating or treatment chosen in any instance may vary depending upon the seed in question, geographical and environmental considerations.
According to a fifth aspect of the invention, there is provided a treated or coated seed comprising the seed treatment composition of the invention. The seed may be covered, coated or treated in part or substantially in its entirety with the composition. The coated or treated seed advantageously has improved properties relative to untreated seeds. According to a sixth aspect of the invention, there is provided a plant obtainable from a treated or coated seed, which plants show improved growth characteristics relative to plants obtained from untreated seeds. According to a seventh aspect of the invention, there is provided, a method for growing a plant from a treated or coated seed or a method for obtaining plants having improved growth characteristics, which method comprises the steps of:
a) sowing a coated or growing a treated seed, as defined herein; and b) growing a plant from the seed, optionally under abiotic and/or biotic stress conditions, c) obtaining plants having improved growth characteristics.
Advantageously, the coated or treated seeds show improved seed performance and the resultant plants grown from such seeds show improved growth characteristics, which improved seed performance and/or improved growth characteristics may be manifested by, for example, any one or more of the following: increased percentage germination and/or emergence, increased speed of germination and/or emergence, improved uniformity of germination and/or emergence, increased plant establishment, increased percentage of seedlings that develop and survive, enhanced vigour, early vigour, increased fresh weight of seedlings and/or resultant plants, increased tillering, increased productive tillers, increased biomass (aboveground or belowground), increased yield (of aboveground parts, including seeds, or of belowground plant parts, including tubers, rhizomes, roots etc.), increased abiotic stress tolerance, increased biotic stress tolerance, reduction in crop losses.
According to an eighth aspect of the present invention, there is provided the use of one or more aromatic amino acids and one or more gibberellins in improving seed performance and/or in improving the growth characteristics of plants resulting from treating or coating seeds in the same. Also provided is the use of a composition or kit according to the invention for improving seed performance and/or improving the growth characteristics of plants resulting from coated or treated seeds. Advantageously, the use of such a seed treatment composition in comparison to foliar treatments represents a cost saving for the grower due to the relatively smaller quantities required. According to a ninth aspect of the present invention, there is provided a kit comprising: a) one or more aromatic amino acids and one or more gibberellins or tryptophan alone, particularly when applied at a rate in the range of from about 0.005 to about O. lmg/kg seed; or
b) a composition as described herein; and optionally
c) an agriculturally acceptable carrier; and optionally
d) an applicator for applying any of elements a), b) or c) to a seed.
The components making up the composition may be present in the amounts as described herein.
According to one embodiment, the components of a) may be applied to a seed either simultaneously or sequentially, in any order.
Advantageously, the compositions of the invention are equally useful under stress and non- stress conditions. According to an embodiment of the present invention, the improved effects of the composition of the invention (improved seed performance effects and improved plant growth characteristics) may be obtained under conditions of biotic and abiotic stress (this may be any stress caused by intense light, herbicide, ozone, oxidative stress, heat, chilling, freezing, drought, salinity, flooding, and heavy metal toxicity, for example, and in particular osmotic stress, such as salt stress.
According to another embodiment of the invention, the seed may be from a monocotyledonous plant or from a dicotyledonous plant. Particularly preferred are cereals (for example, spring and winter wheat, maize, triticale, rice, barley, rye, oats, sorghum, millet); grasses (for example, turf grass, rye grass, fescue); commercially important crop plants (for example, sugar cane, sugar beet, cotton, canola, sunflower, mustard, corn, coffee, tea, tobacco); legumes (for example, soybeans or beans); members of the soianaceae family (for example, tomatoes, peppers and potatoes); greenhouse crops; tree crops (such as pome and stone fruit crops, and nut crops such as walnuts, pistachio and olives, coco pods, palms such as oil palm and date palm; vegetables such as brassicas, for instance, cabbages and lettuces.
Preferred features of the second, third, fourth, fifth, sixth, seventh, eighth and ninth aspects of the invention may be as described above in connection with the first or any other aspect.
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Other features of the present invention will become apparent from the following examples. Generally speaking the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings). Thus features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. Figures
The present invention will now be described with reference to the following Figures which are by way of illustration alone, in which: Figure 1 shows the results of a petri dish-based germination assay of wheat seed coated in different concentrations of L-tryptophan, and germinated in 0 mM or 200 mM NaCl.
Figure 2 shows the results of a seedling assay in compost in the glasshouse in which wheat seeds were coated in 0 mg/kg or 0.01 mg/kg L-tryptophan and either 0 mg/kg, 8.7mg/kg or 17.3 mg/kg of gibberellic acid (GA3). Emergence is shown as a percentage of seeds sown. The experiment was conducted under both saline and non-saline conditions and the data presented is the combined means across salinity levels (as the performance of the coating solutions was consistent regardless of the stress level).
Figure 3 shows the percentage change in average seedling shoot fresh weight (FW) of wheat seed coated in different concentrations of L-tryptophan, both under saline (6.5 dS/m) and non-saline (0.7 dS/m) growing conditions. Figure 4 shows the percentage change in average seedling shoot fresh weight (FW) of wheat seed coated in different concentrations of L-tryptophan and either 0 or 17.3 mg/kg gibberellic acid (GA3), both under saline (6.5 dS/m) and non-saline (0.7/dS) growing conditions.
Figure 5 shows the percentage change in average seedling shoot fresh weight (FW) of wheat seed coated in L-tryptophan and/or gibberellic acid, under saline and non-saline conditions. Figures 4 and 5 show the results of the same experiment, but with a different presentation of the same data.
Figure 6 shows the percentage change in average seedling shoot fresh weight (FW) of wheat seed coated in L-tryptophan at various concentrations and gibberellic acid at 17.3 mg/kg. The experiment was conducted under both saline and non-saline growing conditions. Since the response to seed coating solution was similar regardless of stress level, the data presented has been combined across salinity levels. Figure 7 shows the pooled mean from three experiments in which the percentage change in average seedling shoot fresh weight (FW) is shown of wheat seed coated in 0 or 0.01 mg/kg of L- or D-tryptophan and with 0 or 17.3mg/kg GA3 and grown under non-saline conditions. Figure 8 shows the effect of phenylalanine on average seedling shoot fresh weight of wheat seedlings.
Examples
The present invention will now be described with reference to the following examples, which serve to illustrate the invention and which are not intended to limit the scope of the invention.
Example 1: Petri dish-based germination assay of wheat seed coated in L-tryptophan Seed coating solution:
L-tryptophan at 0.001, 0.01. 0.1, 1 or 10 mg/kg seed, applied at a total application volume of 5 ml/kg seed
Salinity levels:
0 mM or 200 mM NaCl Seed was treated and allowed to dry at room temperature overnight. Petri dishes were lined with a double layer of filter paper, moistened with either water (0 mM NaCl) or 200 mM NaCl. For each seed coating solution x salinity level combination, six replicate petri dishes (each containing 50 seeds) were prepared and left to germinate at 12°C in the dark. The number of germinated seeds was assessed daily, with seeds considered as having germinated when the radicle protruded by 1 mm. The final germination assessment was made 7 days after sowing.
The results are shown in Figure 1. As can be seen, the final germination percentage (expressed as a percentage of the number of seeds sown) showed a quadratic response to increasing L-tryptophan concentration, under both non-saline (0 mM NaCl) and saline (200mM NaCl) conditions. The final germination percentage increased with increasing L- tryptophan, reaching a plateau, before starting to decrease above around 1 mM.
Example 2: Greenhouse -based assay to determine final seedling, emergence in wheat
Seed coating solution: All combinations of L-tryptophan and GA 3 , at the following rates (applied at total application volumes of 5 ml/kg seed):
L-tryptophan: 0 or O.Olmg/kg seed
GA 3 : 0, 8.7 or 17.3 mg/kg seed
Irrigation salinity:
0.7 dS/m (tap water) (non-saline) or 6.5 dS/m (saline)
Seeds were treated and allowed to dry overnight at room temperature. Modular trays were filled with seed compost and watered to saturation with the respective irrigation solutions. Non-saline irrigation was tap water, with a conductivity of 0.7 dS/m, whilst the saline irrigation was a mixture of NaCl and MgS0 4 , resulting in a conductivity of 6.5 dS/m. Modular trays were allowed to drain overnight before sowing. Irrigation commenced 24 hours following sowing, and then as necessary until harvest. One experimental unit comprised 24 seeds (8 modular cells, each containing 3 seeds). The 0 mg/kg L-tryptophan and 0 mg/kg GA 3 control consisted of 24 replicate experimental units (576 seeds) at each salinity level, whilst the remaining seed coating solutions were replicated 8 times (192 seeds at each level of salinity). Seedlings were grown under glasshouse conditions at Rothamsted, Harpenden, UK.
Seedlings were harvested for shoot fresh weight 10 days after sowing. The final emergence percentage was also assessed at this time. Since the effect of the seed coating solutions was consistent across salinity levels, the data in Figure 2 has been presented as a mean under both non-saline and saline irrigation.
As can be seen in Figure 2, at 8.7 and 17.3 mg/kg gibberellic acid, GA 3 , (and 0 mg/kg L- tryptophan), there was a negative effect on seedling emergence. This negative effect was fully ameliorated by the addition of 0.01 mg/kg L-tryptophan. Example 3: Average seedling shoot fresh weight from wheat seeds coated in L-tryptophan and grown under saline and non-saline conditions
Seed coating solution: L-tryptophan at 0, 0.001, 0.01, 0.1 or 1 mg/kg seed, in a total application volume of 5 ml/kg seed
Irrigation salinity:
0.7 dS/m (tap water) (non-saline) or 6.5 dS/m (saline)
Each L-tryptophan concentration x salinity level combination consisted of 16 replicate units of 24 seeds (384 seeds). Harvest was 14 days after sowing. The method was otherwise as for Example 2. The results, shown in Figure 3, show that the addition of L-tryptophan increased the fresh weight of the shoots of two-week old seedlings, at both salinity levels. The most successful rate was 0.01 mg/kg L-tryptophan, with the beneficial effect diminishing at higher concentrations. Example 4: Percentage change in average seedling shoot fresh weight from wheat seeds coated in L-tryptophan and/or gibberellic acid under saline and non-saline conditions
Seed coating solution:
All combinations of L-tryptophan and GA 3 , at the following rates (applied in total application volumes of 5 ml/kg seed) concentrations:
L-tryptophan: 0, 0.005, 0.01 or 0.02 mg/kg seed
GA 3 : 0 or 17.3 mg/kg seed
Irrigation salinity:
0.7 dS/m (tap water) (non-saline) or 6.5 dS/m (saline) The 0 mg/kg L-tryptophan treatment (with either 0 or 17.3 mg/kg GA3) consisted of 16 replicate units (384 seeds) at each salinity level, whilst the remaining seed coating solution x salinity level combinations each comprised 8 replicate units (192 seeds). Harvest was 13 days after sowing. The method was otherwise as for Example 2. The results can be seen in Figure 4 and show that the addition of either L-tryptophan at a rate of 0.01 mg/kg, or gibberellic acid at a rate of 17.3 mg/kg, increased seedling shoot fresh weight. The combination of both 0.01 mg/kg L-tryptophan and 17.3 mg/kg GA 3 , however, was the most successful. Figure 5 plots the data from the same experiment but excludes the other rates of L- tryptophan, in order to clearly show the synergistic effect of these two compounds at these particular rates. In the case of the non-saline results, shoot FW increased by 2% and nearly 4% compared to the water-treated control in the L-tryptophan and GA3 treatments, respectively. However, in combination, L-tryptophan and GA3 increased shoot FW by 10%. Similarly, in the saline treatments, shoot FW increased by around 1% and 6% in the L- tryptophan and GA3 treatments, respectively, whilst in combination, the increase was around 13%.
Example 5: Percentage change in average seedling shoot fresh weight of wheat seeds coated in L-tryptophan at various rates and gibberellic acid at 17.3 mg kg
Seed coating solution:
0 mg/kg GA3 with 0 mg/kg L-tryptophan
17.3 mg/kg GA3 at each of the following L-tryptophan rates: 0, 0.007, 0.01, 0.015, 0.022, 0.033, 0.05 mg/kg
All applied in total application volumes of 5 ml/kg seed
Irrigation salinity:
0.7 dS/m (tap water) (non-saline) or 6.5 dS/m (saline)
Treatments involving 0 mg/kg L-tryptophan were replicated 16 times (384 seeds) at each salinity level. All other treatments consisted of 8 replicates (194 seeds) at each level of salinity. Harvest was 12 days after sowing. The method used was otherwise as for Example 2.
The results are presented in Figure 6 and have been combined across the two levels of irrigation salinity as the response of shoot FW to increasing L-tryptophan concentration was similar at both salinity levels. Previous experiments observed 0.01 mg/kg L-tryptophan to be the most successful concentration in terms of increasing shoot FW (both with and without GA3) (Examples 3 and 4). These experiments used dilution series with a factor of 5 or 10, and resulted in broadly bell- shaped curves. The current example seeks to investigate the relationship between L-tryptophan concentration and shoot FW at the top of the curve in order to provide more precise information on the most effective L-tryptophan concentration. Figure 6 shows that 0.01 mg/kg L-tryptophan (in combination with 17.3 mg/kg GA3), was indeed, the best concentration to be using.
Example 6: Percentage change in average seedling shoot fresh weight of seeds coated in L- tryptophan or D -tryptophan with either 0 or 17.3 mg kg GA3
Seed coating solution:
All combinations of tryptophan and GA 3 at the following rates, applied in total application volumes of 5 ml/kg seed:
Tryptophan: 0 or 0.01 mg/kg (in either the L- or D- form)
GA 3 : 0 or 17.3 mg/kg
Irrigation salinity:
0.7 dS/m (tap water) (non-saline) only
Example 6 presents the combined results of three experiments, all conducted under non- saline conditions. Each of the treatments involving 0 mg/kg tryptophan were replicated 24 times in each of the 3 experiments (total of 1728 seeds across the 3 experiments), whilst the remaining treatments were replicated 8 times. Harvest in each of the experiments was between 8 and 13 days after sowing. The methodologies were otherwise as for Example 2. The results are presented in Figure 7. As in Example 4, the synergistic effect between 17.3 mg/kg GA3 and 0.01 mg/kg tryptophan is again demonstrated. In the present example, it can be seen that the synergistic effect is evident using either the L- or the D- form of tryptophan. Across the 3 experiments, GA3 (in the absence of tryptophan) increased shoot FW by 1%. L-tryptophan, however, decreased shoot FW by nearly 1%, but in combination with GA3, the combined effect on shoot FW was an increase of 2.5%. The addition of D- tryptophan with GA3 also demonstrated a synergistic effect, and outperformed the L- tryptophan in the current example. The effect of D-tryptophan alone was a 1.5% increase in shoot FW, whereas in combination with GA3, the increase in shoot FW was nearly 5%. Example 7: The effect of phenylalanine on average seedling shoot fresh weight Seed coating solution: Phenylalanine at 0.0008, 0.008, 0.08, 0.8 and 8 mg/kg seed, in total application volumes of 5 ml/kg seed
Irrigation salinity:
0.7 dS/m (tap water) (non-saline) only Each experimental unit consisted of 36 seeds, with 8 replicate units per treatment. A single (non-saline) salinity level was used. The whole experiment was replicated three times, resulting in 864 seeds per treatment. Harvest was around 13 days after sowing. The methodologies were otherwise as for Example 2.
As can be seen in Figure 8, seed coating with all rates of phenylalanine resulted in larger seedling shoot fresh weights.
Results and Conclusions
The results show that when applied as a seed coating treatment at a commercial volume (5ml/kg seed), L- Tryptophan increases the final germination percentage of wheat seed.
After around two weeks, seedlings grown from seeds coated in either L-tryptophan, phenylalanine or gibberellic acid have significantly higher shoot fresh weights than the water- treated controls. The combination of L- or D-tryptophan and gibberellic acid consistently achieved higher seedling shoot fresh weights than either compound alone, especially with a concentration of 0.01 mM of L-tryptophan. The results also show that D-tryptophan has an equal and in some cases better effect than L-tryptophan, both alone and with GA3.