USE OF 6-OXO-1,4,5,6-TETRAHYDROPYRIDIZIN-3-CARBOXYLIC ACID, 1-METHYL 6-OXO-1,4,5,6-TETRAHYDROPYRIDIZIN-3-CARBOXYLIC ACID AND 1-AKLYL-6-OXO-1,4,5,6-TETRAHYDROPYRIDAZINE-3-CARBOXYLIC ACID FOR IMPROVING GROWTH PERFORMANCE IN PLANTS

Abstract

Contacting a plant with tetrahydropyridizin (THP), a compound including at least one of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or its salts or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or its salts, to plants can cause an increase in parameters such as germination, seedling emergence, growth, drought tolerance and recovery, yield, rhizobia induced root nodulation as compared to untreated wild type plants. 1-Methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or its salts is chemically the simplest 1-alkyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and as such is an example showing the effectiveness of a 1-alkyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid. Higher series derivatives such as 1-alkyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acids also induce beneficial effects on treated plants.

Claims

1. A method for improving plant performance, comprising applying tetrahydropyridizin (THP) carboxylic acid and derivates and salts thereof to a plant to enable an increase in performance in the plant to include its harvestable yield over that of untreated plants.

2. The method of claim 1, wherein the harvestable yield includes at least one of: weight and biomass of the foliar parts the plant over that for wild type plants; weight or biomass of the roots of the plant over that for wild type plants; seedling emergence of the plant over that for wild type plants; yield of the plant over that for wild type plants; agronomic performance of monocotyledonous and dicotyledonous plants over that for wild type plants; drought resistance over that for wild type plants; drought recovery over that for wild type plants; germination and seedling emergence rate of plants in cool and warm germination temperatures over that for wild type plants; root nodulation with a nitrogen fixing bacterium.

3. The method of claim 2, wherein said increasing weight further comprises increasing at least one of the dry weight or the fresh weight.

4. The method of claim 1, wherein THP is applied to the plant by at least one of folia application and seed treatment from a at least one of a solution, suspension, powder, spray or coating of the THP.

5. A method for improving plant performance, comprising: contacting a plant with a compound including at least one of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic salt to enable an increase in performance of the plant over that of untreated plants by increasing at least one of: weight and biomass of the foliar parts the plant over that for wild type plants; weight or biomass of the roots of the plant over that for wild type plants; seedling emergence of the plant over that for wild type plants; yield of the plant over that for wild type plants; agronomic performance of monocotyledonous and dicotyledonous plants over that for wild type plants; drought resistance over that for wild type plants; drought recovery over that for wild type plants; germination and seedling emergence rate of plants in cool and warm germination temperatures over that for wild type plants; root nodulation with a nitrogen fixing bacterium.

5. The method of claim 5, wherein said increasing weight further comprises increasing the dry weight.

6. The method of claim 5, wherein said increasing weight further comprises increasing the fresh weight.

7. The method of claim 4, wherein the plant is contacted with the compound by at least one of a solution, suspension, powder, spray, coating or injection of the at least one of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt.

8. The method of claim 4, further comprising adding 1-methyl to the at least one of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt, resulting in 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid contacting the plant.

9. The method of claim 8, wherein said increasing weight further comprises increasing the dry weight.

10. The method of claim 8, wherein said increasing weight further comprises increasing the fresh weight.

11. The method of claim 8, wherein the plant is contacted with the compound by at least one of a solution, suspension, powder, spray, coating or injection of the at least one of 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt.

12. The method of claim 4, further comprising adding 1-aklyl to the at least one of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt, resulting in 1-aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid when contacting the plant.

13. The method of claim 12, wherein said increasing weight further comprises increasing the dry weight.

14. The method of claim 12, wherein said increasing weight further comprises increasing the fresh weight.

15. The method of claim 12, wherein the plant is contacted with the compound by at least one of a solution, suspension, powder, spray, coating or injection of the at least one of 1-aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt.

16. A method for improving plant performance, comprising: contacting a plant with a compound including at least one of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic salt, 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt, 1-Aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, or 1-Aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic salt enabling an increase in performance of the plant over that of untreated plants by at least one of: weight and biomass of the foliar parts the plant over that for wild type plants; weight or biomass of the roots of the plant over that for wild type plants; seedling emergence of the plant over that for wild type plants; yield of the plant over that for wild type plants; agronomic performance of monocotyledonous and dicotyledonous plants over that for wild type plants; drought resistance over that for wild type plants; drought recovery over that for wild type plants; germination and seedling emergence rate of plants in cool and warm germination temperatures over that for wild type plants; root nodulation with a nitrogen fixing bacterium.

17. The method of claim 16, wherein said increasing weight further comprises increasing the dry weight.

18. The method of claim 16, wherein said increasing weight further comprises increasing the fresh weight.

19. The method of claim 16, wherein the plant is contacted with at least one of a solution, suspension, powder, spray, coating or injection of the at least one of 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxyalte salt.

20. The method of claim 16, wherein the compound can be applied to the plant in the form of at least one of a solution, a suspension and a powder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and form a part of the specification, present the chemical structures of the active compounds that can be applied to plants. They also illustrate the embodiments of the present invention and, along with the description, serve to explain the principles of the invention. In the drawings:

[0023] FIG. 1. labeled as prior art, illustrates a phenology diagram of the growth stages of wheat published by the University of Illinois.

[0024] FIG. 2. illustrates a phenology diagram of the growth stages of Maize.

[0025] FIGS. 3-4 illustrate structures of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, 1-methyl and 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, respectively.

[0026] FIG. 5 illustrates a schematic synthesis route description for higher series derivatives of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

[0027] FIG. 6 illustrates photographs of an In vitro assessment of the effects on the germination and early stage growth of wheat following treatment of naked wheat seed with of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

[0028] Referring to FIG. 7, illustrated is a schematic synthesis for production of alkyl THP's and their associated esters.

[0029] Referring to FIG. 8, illustrated is a schematic synthesis for production of aryl THP's and their associated esters.

[0030] Referring to FIG. 9, illustrated is a schematic synthesis for producing additional derivatives of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

DETAILED DESCRIPTION

[0031] Briefly, the present invention includes the application of tetrahydropyridizin (THP), which is an organic compound, in any of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-aklyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, or their salts, to plants (seeds, foliage, roots) to improve plant growth and other properties for plants that are so treated. 6-Oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid are sold commercially for use as reactants in commercial chemical syntheses. No other uses were found for these compounds. These compounds are the products of chemical syntheses and they are readily biodegraded by environmental microorganisms.

[0032] Referring to FIG. 1, illustrated is a phenology diagram 100 of wheat growth stages published by the University of Illinois. Although it does not teach embodiments of the present invention, it is useful as a reference for the application timings of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid described herein for the treatment of plants as further elaborated on in the examples. The growth stages of wheat as shown here are related to the two most commonly used methods of describing them (see Zadoks, J C, Chang T T, Konizak C F 1974, A decimal code for the growth stages of cereals. Weed Research 14(6): 415-421). Growth stages for a Feekes method are shown in the first row of diagram, while growth stages for a Zadoks method are shown in the second row of the diagram. Displayed on the X-axis are the Feekes method, which is given in the top row, and the Zadoks method, which is given in the bottom row.

[0033] Referring to FIG. 2, illustrated is a phenology diagram 200 of the growth stages of Maize. It is also useful as a reference to the application timings of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, which is further described in the examples that follow.

[0034] Referring to FIG. 3, illustrated is the structure 300 of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, and referring to FIG. 4 illustrated is the structure of 1-methyl and 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

[0035] Referring to FIG. 5, illustrated is a schematic synthesis route description 500 for higher series derivatives of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

[0036] Referring to FIG. 6, illustrated are three photographs 600 of an In vitro assessment of the effects on the germination and early stage growth of wheat following treatment of naked wheat seed with of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid. Untreated 610, THP 620 and a commercial seed treatment, Take Off® 630. The seed shown in 620 was grown under natural daylight conditions at 20-22 degrees Centigrade.

[0037] Referring to FIG. 7, illustrated is a schematic synthesis 700 for production of alkyl THP's and their associated ester. Starting with dialkyl-2-oxopentanedioate the methyl-6-oxo-1,4, 5,6-tetrahydropyridazine-3-carboxylate (THP) can be produced by reaction with hydrazine in the appropriate alcohol and acetic acid. The reaction of THP with alkyl bromides or alkyl iodides in dimethyl formamide produces alkyl THP's in an 80% yield over 2 steps. FIG. 7 shows the synthesis of starting from the dimethyl-2-oxopentanedioate to give alkyl 1-methyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-carboxylates.

[0038] Referring to FIG. 8, illustrated is a schematic synthesis 800 for production of aryl THP's and their associated esters. arylhydrazines can be produced by treatment of substituted anilines with sodium nitrite followed by reduction with tin chloride. The arylhydrazines can then be treated with 2-oxopentanedioic acid or esters to produce the aryl THP's as shown in FIG. 8.

[0039] Use of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-aklyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-carboxylic acids for improving performance of plants. 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid in the examples described in this document can be produced from the reaction of 2-ketoglutaric acid with hydrazine or monomethylhydrazine. Referring to FIG. 9, a method 900 is illustrated for producing additional derivatives of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl-6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

[0040] Reference will now be made in detail to the preferred embodiments of the present invention. It will be shown that when seeds or plants are treated with THP, such as 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and/or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid, the seedlings and growing plants outperform those from untreated seeds or plants. Alternatively, plants may be treated by application of a solution or solid or suspension of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid to the foliar or below ground portions thereof. Alternatively, the growing media in which the plants are growing could be treated with a solution or solid or suspension of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid.

[0041] 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid can be degraded by commonly occurring microbes into harmless derivatives, and as such their use is expected to be environmentally sound.

[0042] Wheat, corn, soybeans and peppers were chosen as representatives of monocotyledons and dicotyledons, respectively, the major groups of crop plants, and were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid. Germination, seedling emergence, root and shoot biomass accumulation, and yield were monitored.

[0043] In the practice of the present invention, plants were treated to initiate the beneficial responses. Seeds, root systems or foliar parts can be treated. Multiple treatments during the growing season can be used. The concentrations of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and/or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid are set forth in the examples herein below.

Turning Now to the Examples

Example 1

[0044] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) and treated plants to examine the impact on shoot and root biomass.

[0045] Winter wheat variety AWC 13 seeds were treated with either 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (MTHP) at an effective rate of 10 g diluted in 2 L water plus 0.25% Nouryon wetter and applied to 1 ton of seed. These seed were planted in coarse river sand and grown in a greenhouse under ambient light at the greenhouse at 21° C. Plants were irrigated every other day with water containing 50% strength Hoaglands solution. Plants were assessed for emergence and biomass accumulation 30 days after planting. The plants from seeds treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid had larger root biomasses (0.83, 0.85 g, dry weight/plant) compared with plants from untreated seeds (0.65 g dry weight/plant).

Example 2

[0046] Wheat, corn and pepper seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) using the same methodology and growth conditions as described in example 1 and the resulting plants were also treated with a light foliar cover spray using an electric hand-held sprayer. (100 g in 200 L water plus 0.25% Nouryon wetter) of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (30 days post emergence (wheat at Zadoks GS 14 (see FIG. 1), maize at V2, Pepper at 3-4 leaf stage) and then assessed for root and shoot biomass 45 days later. At 75 days post planting the plants were assessed for root and shoot biomass as measured with dry weight.

TABLE-US-00001 TABLE 1 % Difference vs Untreated Wheat Corn Pepper Root Dry Weight 12 8 19 Shoot Dry Weight 7 3 9 Emergence.sup.x 16 11 24 .sup.x= % Emergence at 10 days post planting

[0047] Seed treatment with THP increased emergence. The seed treatment plus a foliar application of THP both shoot and root biomass compared to the untreated cohort groups.

Example 3

[0048] Greenhouse grown wheat plants were given foliar treatment (using the same methodology and dosages as example 2) of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (MTHP) and their shoot biomass was determined. Plants were treated at Zadoks GS 20 (see FIG. 1). The plants were grown in coarse river sand in, 4 inch pots (10 plants per pot, 10 pots per treatment.) in a greenhouse and irrigated daily. They were provided 0.5 g/4″ pot of Osmaocte, a time-release complete nutrient mix per pot. There were 10 replicated per treatment.

TABLE-US-00002 Mean Shoot Dry Treatment mass(g) Con 1.28 a THP 1.35 a MTHP 1.34 a 10 plants assessed per pot. Mean of 10 pots taken. P = 0.05 Duncan Waller T-Test

Example 4

[0049] Greenhouse grown wheat plants (Variety Skyfall) were given foliar treatment of 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (MTHP) or 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP), utilizing the same methodology as in example 2. Foliar and root biomass were determined 30 days later at Zadocks growth stage 22-24.

TABLE-US-00003 Dry Weight Treatment Root (g) Shoot (g) Con 1.03 a 0.55 a THP 1.45 b 0.74 b MTHP 1.64 b 0.7 B P = 0.05 Duncan Waller T-Test

Example 5

[0050] Wheat plants (Variety AWC 13) were given a foliar treatment of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid(THP) or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (MTHP) and their foliar and root dry biomass determined 30 days after application. The foliar applications were applied at approximately at Zadoks GS 15-20 (see FIG. 1) and assessed 30 days later at Zadoks GS 22-24 (also shown in FIG. 1).

TABLE-US-00004 Dry Weight Treatment Root Shoot Con 0.65 a 0.47 a THP 1.5 b 1.01 b MTHP 2.06 b 0.98 b Each value is the mean of 10 plants P = 0.05 Duncan Waller T-Test

Example 6

[0051] Field grown wheat plants (Var: AWC 13) were given foliar treatment at Zadoks GS 25-26 (FIG. 1) of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) or 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (MTHP) both at 100 g in 200 L water plus 0.25% Nouryon wetter. Plants were grown under standard UK wheat growing cultural practice (https://ahdb.org.uk/wheargg). Yield was assessed at harvest.

TABLE-US-00005 Field Trial. Variety = AWC 13 Experimental Site = Woolpit, Suffolk, UK. Foliar Treatment Yield(T/ha) Con 9.6 a THP 10.25 b MTHP 10.35 b P = 0.16 Duncan Waller T-Test

Example 7

[0052] Wheat seeds were treated with 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid treated plants to examine the impact on root biomass for field grown plants.

[0053] Wheat seeds were treated with 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and field grown. The plants from seeds treated with 1-methyl 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid had larger root biomass (0.84 g dry weight/plant) compared with plants from untreated seeds (0.66 g dry weight/plant).

Example 8

[0054] Corn and wheat plants were given a foliar treatment Zadoks 15-20 (FIG. 1) and V1 respectively (as described in Example 2) with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) and then assessed for root and shoot biomass. Three treatment dosages were applied (Table 2). Plants were grown in a greenhouse with a diurnal cycle of 55-75 F with ambient light. The plants were grown in a coarse river sand in 4″ pots with 0.5 g of Osmacote/pot. The mean root and shoot fresh weights were assessed at 35 days post treatment.

TABLE-US-00006 TABLE 2 Dose, g/ha delivered in the equivalent of 200 L/ha Plant 0 50 100 200 Corn Shoot, FWt, g 6.1 7.8 7.2 5.1 Root, FWt, g 2.3 2.7 3.4 1.8 Wheat Shoot, FWt, g 3.2 4.4 5.2 5.8 Root, FWt, g 1.8 2.1 1.9 2.7

Example 9

[0055] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and the shoot and root fresh weights as shown in Table 1. Shoot and root fresh weights at this stage are components of early seedling emergence which is important for early establishment of a vigorous and productive crop.

[0056] Wheat seeds were treated with a solution of either 0.1 mM or 0.01 mM 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid in an aqueous solution at pH 5.7-5.8. The seeds were placed on moist germination paper and allowed to germinate at 75-85° F. The control seeds were imbibed in aqueous solution containing only the soft soap and glycerol.

TABLE-US-00007 TABLE 3 0.1 mM 6-oxo-1,4,5,6- 0.01 mM 6-oxo-1,4,5,6- tetrahydropyridizin-3- tetrahydropyridizin-3- Tissue Control carboxylic acid carboxylic acid Shoot, mg 27.4 42.1 44.7 Roots, mg 29.1 43.8 48.1

Example 10

[0057] Soybean seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and the weights of the shoots were determined as shown in Table 2. Early seedling emergence is important for early establishment of a vigorous and productive crop.

[0058] Soybean seeds (Var. Viking) were treated by imbibing them for three minutes with a solution of 0.01 mM 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid in an aqueous solution also containing soft soap and glycerol at pH 5.7-5.8. The control seeds were imbibed in aqueous solution containing only the soft soap and glycerol at pH 5.7-5.8. The seeds were placed on moist germination paper and allowed to germinate at 75-85° F. The fresh weights were taken to assess the shoot development.

TABLE-US-00008 TABLE 2 0.01 mM 6-oxo-1,4,5,6- Tissue Control tetrahydropyridizin-3-carboxylic acid Shoots, Fresh wt. mg 98.3 114.3 Shoots, Fresh Wt. mg 106.2 128.6

Example 11

[0059] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or a commercial seed treatment at labeled rates (Take Off ST) and allowed to germinate to examine and compare the effects of temperature on germination.

[0060] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid or Take Off, a commercial biostimulant (that claims faster germination and emergence). The treated seeds received the treatment compounds at the rate of 10 g per ton of seed utilizing the same dosing methodology as Example 1. However in this case seeds were germinated on moistened absorbent paper towel discs in closed petri dishes. The seeds were incubated at 20 C in darkness until germination commenced and then grown in natural diurnal conditions for a further 12 days. As shown in FIG. 6 seeds treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid generated greater root biomass and longer bright green shoots than the plants from either untreated seeds or Take Off treated seeds.

Example 12

[0061] Nine field grown wheat plots (20 m.sup.2) were established. In each plot, the area was split into two and one half treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) to assess for effects on yield. Plants were treated at Zadocks GS 30 as described in example 6 under standard cultural practices. The average yield response compared to the untreated plants across all sites was 0.596 tons per hectare.

TABLE-US-00009 TABLE 3 Yield as Percent Increase over Untreated Field Field Field Field Field Field Field Field Field Treatment 1 2 3 4 5 6 7 8 9 6-oxo-1,4,5,6- 6.25* 8.7* −0.3 13.9* 3.0 3.3 6.5 9.8* 8.1* tetrahydropyridizin- 3-carboxylic acid *Significant at P = 0.1 Duncan Waffler T-test.

Example 13

[0062] Wheat seedlings were given foliar treatments with a range of concentrations of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and the seedlings were grown and the numbers of tillers each plant produced was monitored as shown in Table 4. The number of tillers is considered positively related to grain yield.

[0063] Sets of wheat seedlings (Var. VitPro) were given foliar treatments of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid at concentrations of 0.1 mM, 0.01 mM or 0.001 mM *in a standard nutrient solution. Seedlings were given one foliar treatment at Zadoks GS 12 (FIG. 1); the plants grown in a greenhouse and fed a standard plant nutrient solution. The untreated control plants were sprayed with the nutrient solution containing sodium dodecyl sulfate, 0.1% and glycerol 1.2%. The number of tillers for each plant were counted at Zadoks GS 15 (FIG. 1) and the average number of tillers per plant calculated.

TABLE-US-00010 TABLE 4 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid concentration in treatment solutions Plant Parameter Tracked 0 0.1 mM 0.01 mM 0.001 mM Tillers per plant, average 3.7 7.4 9.5 4.1

Example 14

[0064] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid treated plants to examine the impact on shoot and root biomass for field grown plants.

[0065] Winter wheat variety AWC 13 seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and field grown. The plants from seeds treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid had larger root biomass (0.85 g dry weight/plant) compared with plants from untreated seeds (0.66 g dry weight/plant).

Example 15

[0066] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid at two different temperature regimes to investigate the affect on germination. Soil temperature impacts the percentage of seeds germinating and how fast seedlings emerge from the soil. Each of these parameters influences how well the plants develop and ultimately crop yield.

[0067] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (THP) at a rate of 10 g/ton of seeds in accordance with the methodology of example 1. Seeds were planted in 12″×12″ germination trays in a 1″ grid, 100 seeds per tray. Growth media was coarse river sand. Additionally, each cell in the grid received one granule of Osmacote approximately above where the seed was planted. Trays were irrigated every day. The seeds were then allowed to germinate at controlled environment chambers at either 10 C (50 F) or 20 C (68° F.) to examine the effects at different temperatures. The number of seedlings emerged was tracked daily.

TABLE-US-00011 TABLE 5 % Emergence Days after planting 5 6 7 8 9 10 11 10 C Untreated 0 0 0 4 14 38 45 Treated 0 0 11 19 27 60 68 20 C Untreated 11 28 54 72 97 97 97 Treated 14 48 80 100 100 100 100

Example 16

[0068] Wheat seeds were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic and leaf wilting was tracked to examine the resistance to drought in the seedlings as shown in Table 6.

[0069] Wheat seeds (Var. VitPro) were treated with an aqueous solution of either 0 mM, 1 mM, 0.5 mM,0.1 mM or 0.01 mM 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid at pH 5.7-5.8. The 0 mM 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid seeds were imbibed in aqueous solution containing only the soft soap and glycerol. The plants were allowed to grow to Zadoks GS13 (FIG. 1) when water was withheld until plants in all of the treatments had wilted leaves.

TABLE-US-00012 TABLE 6 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid concentration in treatment solutions Drought stress 0 mM 1 mM 0.5 mM 0.1 mM Wilted leaves 40 19 14 18 Total leaves 47 57 64 61 % Wilted leaves 85% 33% 22% 30%

Example 17

[0070] Wheat seedlings were given foliar treatments with a range of concentrations of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid and the seedlings were grown to near maturity and drought stressed. Their relative water contents were determined to examine the effectiveness of different concentrations of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid in conferring drought resistance.

[0071] Sets of wheat (Var. VitPro) seedlings were given foliar treatments of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid at concentrations of 1 mM or 0.1 mM in aqueous solutions. Two treatments were made; one at Zaddok GS 12(FIG. 1) and a second at Zadoks GS 14 (FIG. 1) and the plants grown until the first flag leaf was fully developed on the control plants. The treated plants and one set of controls were then drought stressed and the relative water content was determined for each set of plants. There were two sets of control plants; one was maintained with normal watering and a second control was drought stressed along with the treated plants. When each of the plants had at least two wilted leaves the water content of the drought stressed plants was determined relative to that of the normal, well-watered control plants. The flag leaves were harvested and used to determine the relative water content. The untreated, drought stressed control plants retained only 35.8%,the drought stress plants treated with 0.1 mM 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid retained 67.7% and the plants treated with 1 mM 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid retained 68.3% of the water content of the normally watered plants.

Example 18

[0072] Sets of wheat seedlings were given two foliar treatments with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid to examine seedling recovery from drought stress.

[0073] Sets of wheat seedlings were given foliar treatments of 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid at concentration of 0.1 mM. Seedlings were given two foliar treatments: the first at Zadoks GS 12 and at Zadoks GS 14 (FIG. 1). The plants were grown in a greenhouse and fed a standard plant nutrient solution. The plants were drought stressed for 10 days after which water and nutrients were resumed and the plants were allowed to grow for 16 days.

TABLE-US-00013 TABLE 7 6-oxo-1,4,5,6-tetrahydropyridizin-3- carboxylic acid concentration in treatment solution and water available 0.0 0.0 0.01 mM Normal Drought Drought Plant Parameter water stressed stressed Total foliar fresh wt., avg., g 32.3 28.2 33.4 Number of seed heads, avg. 5.4 7.1 8.3

Example 19

[0074] Soybean seed were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid to examine the effect on root nodulation by a nitrogen fixing bacterium.

[0075] Soybean seeds, cv. Viking, were treated with 6-oxo-1,4,5,6-tetrahydropyridizin-3-carboxylic acid (0.1 mM) and the resulting seedlings were grown in a greenhouse at 75° F. under natural light during a winter season. The extent of root nodulation by Bradyrhizobium japonicum, a nitrogen fixing bacterium was determined 4 weeks after planting. Plants from the untreated seeds averaged 655.8 mg of nodules per plant versus 763.7 mg for the plants from treated seeds.