CHEMICAL ABSTRACTS, Volume 82 No. 25, issued 23 June, 1975, (Columbus, Ohio, U.S.A.) RONALD A. STANLEY, "Interaction of Calcium and 2,4-D on Eurasian Watermilfoil" see page 120, column 1, the Abstract no. 165806 Y. Weed Sci. 1975 23 (3) 182-4 (Eng.)
CHEMICAL ABSTRACTS, Volume 84, No. 25, issued 21 June, 1976, (Columbus, Ohio, U.S.A.) JERRY D. COHEN et al, "Calcium Requirement for Indoleacetic Acid-Induced Acidification by Avena Coleoptiles." see page 314 column 1, the Abstract no. 176868 n. Plant Physiol. 1976 57 (3) 347-50 (Eng.)
CHEMICAL ABSTRACTS, Volume 87 No. 19, issued 07 November, 1977, (Columbus, Ohio, U.S.A.) A.C. LEOPOLD et al, "Regulation of Growth with Inorganic Solutes." see page 306 column 2, the Abstract no. 148785 B. Plant Growth Subst., Proc. Int. Conf., 8th 1973 (Pub. 1974) 780-8 (Eng.)
|1.||A plant growth stimulator formulation, compris¬ ing: an effective plant growth stimulating amount of 1 triacontanol; a polar organic solvent in which 1 triacontanol is soluble; metal ions selected from the group consisting of Ca +2, La+3, Sr+2, Ba+2, Cd+?",Pb+2, Mn +2, Ce+4 and Mg+2 said metal ions being present in an amount effective to increase the plant growth stimulating effects of said formulation; and water.|
|2.||The formulation according to claim 1, and fur¬ ther including an effective plant growth affecting amount of at least one plant growth substance selected from the group consisting of auxins, gibberellins, cytokinins, kinins, brassins, brassinosteroids and anticytokinins .|
|3.||The formulation according to claim 1, wherein said metal ions are selected from the group consisting of.|
|4.||The formulation according to claim 2, wherein said metal ions are selected from the group consisting of Ca+2 and La+3.|
|5.||The formulation according to claim 3 or 4, wherein the concentration of said metal ions is about 10"1 to 10'5 molar.|
|6.||The formulation according to claim 3 or 4, wherein the concentration of said metal ions is about 10"2 to 10"4 molar.|
|7.||The formulation according to claim 3 or 4, wherein said polar organic solvent is selected from the group consisting of alcohols, ketones, water soluble ethers, glycols, sulfoxides and organic carboxylic acids.|
|8.||The formulation according to claim 3 or 4, wherein said polar organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, methanol, ethanol, isopropanol, diethylene glycol, nbutanol, pro pylene glycol, dioxane, acetic acid, and dimethyl sulfoxide.|
|9.||The formulation according to claim 8, wherein said polar organic solvent is acetone.|
|10.||The formulation according to claim 4, wherein said plant growth substance is an auxin, gibberellin or cytokinin.|
|11.||The formulation according to claim 4, wherein said plant growth substance is indole3acetic acid.|
|12.||The formulation according to claim 4, 10 or 11, wherein the concentration of said plant growth substance is about 10" to 5 molar.|
|13.||The formulation according to claim 4, 10 or 11, wherein the concentration of said plant growth substance is about 10" to 10" molar.|
|14.||The formulation according to claim 1, wherein said metal ion is Ca +2.|
|15.||The formulation according to claim 1, wherein said metal ion is La +3.|
|16.||The formulation according to claim 3 or 4, hav ing a pH of greater than 7.|
|17.||A method for stimulating plant growth which comprises applying an effective plant growth stimulating amount of the formulation according to claim 1 to the area where plants are growing.|
|18.||A method for stimulating plant growth which comprises applying an effective plant growth stimulating amount of the formulation according to claim 2, to the area where plants are growing.|
|19.||A method for stimulating plant growth which comprises applying an effective plant growth stimulating amount of the formulation according to claim 3 to the area where plants are growing.|
|20.||A method for stimulating plant growth which comprises applying an effective plant growth stimulating amount of the formulation according to claim 4 to the area where plants are growing.|
|21.||The method according to claim 19, wherein the formulation is applied to the leaves of the plants by spraying.|
|22.||The method according to claim 20, wherein the formulation is applied to the leaves of the plants by spraying.|
|23.||The method according to claim 21, wherein the formulation is sprayed onto the leaves of the plant when the plant has 2 to 5 true leaves.|
|24.||The method according to claim 21, wherein the concenitration of said metal ions is about 10 to 10 molar,.|
|25.||The method according to claim 21 or 22, where _2 in the concentration of said metal ions is about 10 to 10 4 molar.|
|26.||The method according to claim 21 or 22, where¬ in said polar organic solvent is acetone.|
|27.||The method according to claim 20, wherein said plant growth substance is an auxin, gibberellin or cytokinin.|
|28.||The method according to claim 20, wherein said plant growth substance is indole3acetic acid.|
|29.||The method according to claim 22, wherein the concentration of said plant growth substance is about 10"10 to 5 molar.|
|30.||The method according to claim 21, wherein said metal ion is Ca +2.|
|31.||The method according to claim 21, wherein said metal ion is La +.3.|
|32.||The method according to claim 21, wherein the triacontanol is sprayed in an amount of at least 1 mg per acre. Q FI .|
|33.||The method according to claim 21, wherein said plants are selected from the group consisting of field corn, sweet corn, sugar cane, cucumbers, beans, tomatoes, soybeans, radishes, carrots, asparagus, peas, beets, watermelons, cantelopes, strawberries, potatoes, and sugar beets.|
This application is a continuation-in-part (CIP) of U.S. Serial No. 202,705 filed October 30, 1980, which is a CIP of U.S. Serial No. 146,005 filed May 2, 1980, which is a CIP of U.S. Serial No. 47,696 filed June 12, 1979.
The present invention relates to a chemical composi- ' tion which, when applied to growing plants, is effective in stimulating plant growth. More particularly, the present invention is directed to a chemical formulation of 1- triacontanol in combination with a polar organic solvent, metal ions, other plant growth substances, and water. BACKGROUND ART
Recently, 1-triacontanol, CH,(CH 2 ) has been under investigation as a naturally-occurring plant growth stimulant (see Ries, et al., Science, 195: 1339 (1977)).
In fact, field trials are presently being conducted in an attempt to optimize the conditions at which a chemical formulation of this compound can be applied effectively to plants.
In the research that is presently being conducted utilizing 1-triacontanol as a plant growth stimulant, use is being made of a relatively large amount of surfactants in the chemical formulation in an attempt to render the
1-triacontanol soluble in water. Of course, the use of
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a large amount of water is imperative in order to economically and effectively apply the chemical formula¬ tion to large areas of growing plants. Accordingly, it is imperative to render the 1-triacontanol water-soluble so that it can be properly dispersed in a large quantity of water which is to be subsequently applied to plants. However, the organic solvents which are presently being utilized to make the 1-triacontanol soluble in water, for example, the use of certain chemicals such as chloroform and chemical surfactants and also the use of other water- insoluble solvents, have been found to be detrimental to both plant life and the environment.. Thus, it has been found, for example, that the use of surfactants may prevent entry of the 1-triacontanol into the plant and, consequently, the plant growth-stimulating properties of the triacontanol are rendered less effective.
It is known that calcium can alter the effects of plant hormones including indole-3-acetic acid (IAA) . Other cations have also been found to affect plant growth and to affect the effects of plant growth regulators (see Poovaiah and Leopold, Plant Physiol., 58: 783 (1976)). The following cations have the ability to increase auxin binding to the cell membrane and inhibit IAA-stimulated growth in the order La ^> Ba , Ca , Sr ->Mg , Mn > Li , Na , K (Poovaiah and Leopold, Plant Physiol., 58: 182 (1976)). IAA is known to rapidly stimulate cell elongation and enlargement, a process that involves loosening of the cell wall. IAA occurs primarily in esterified form, the myo-inositol ester comprising about fifty percent in
Zea mays . Only about .one to ten percent of the relatively large amount of IAA present compared to other plants occurs as free IAA. Auxin binding to cell membranes is a reversible process with a Km between 10 M, and there are apparently two binding sites. Site 1 binds both active and inactive auxin analogs while site 2 appears to be auxin-specific.
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The disclosure of H. H. Ashmead in U.S. Patent No. 4,169,716 dated October 2, 1979, teaches that 1-triacontanol may have a synergistic effect when incorporated into a formulation containing plant growth substances in the presence of metal proteinates. In accordance with the present invention, however, only metal ions and not metals complexed with proteins or surfactants are suitable. Furthermore, the prior art does not clearly show a syner¬ gistic effect with metal proteinates and triacontanol since triacontanol was not sprayed in solution without additives .
DISCLOSURE OF THE ' INVENTION
Accordingly, an object of the present invention is to provide an inexpensive and effective means of formulating 1-triacontanol without the use of surfactants of large quantities of organic solvents which have been found to adversely affect plant growth. Also, since it has been determined that the action of 1-triacontanol appears to require calcium for optimum activity, as described herein, the use of surfactants must be avoided to prevent com- plexation of the calcium ion.
Another object of the present invention is to provide a chemical formulation which contains a polar organic solvent which renders 1-triacontanol soluble in water and at the same time poses no threat to plant life or the environment.
Pursuant to the present invention, the above problems have been eliminated by providing a chemical formulation which can be used with water for application to plant life. According to the present invention, 1-triacontanol is dissolved in a polar organic solvent in an amount sufficient to form a water-soluble concentrate. Typically, a con¬ centrate can be formed by mixing together one part by weight (grams) of 1-triacontanol with up to about 5,000,000 parts by volume (ml) of the polar organic solvent, preferably between 10,000 and 500,000 ml. of polar organic solvent to one gram of 1-triacontanol, more preferably one
part by weight of 1-triacontanol to up to about 35,000 parts by volume of the polar organic solvent and most preferably about one part of 1-triacontanol to about 17,000 to 35,000 parts of solvent. The polar organic solvent can be any water-soluble solvent or solvent mix¬ ture containing one or more functional groups which renders the 1-triacontanol solution soluble in water. This solu¬ tion is then dissolved in a large quantity of water which contains metal salts and other plant growth substances with stirring and/or shaking. Alternately, other plant growth substances may be dissolved in the concentrate, the purpose of which will become clear in the following discussion.
The polar organic solvents which are utilized in the present invention to aid in the solubility of the 1- triacontanol in water include alcohols, ketones , water- soluble ethers, glycols, sulfoxides, and organic carboxylic acids and any other solvent or solvent mixture containing one or more functional groups contained in any one class or classes of said solvents. Typical polar organic sol- vents include acetone, methyl ethyl ketone, methanol, ethanol, isopropanol, n-butanol, diethylene glycol, propylene glycol, dioxane, acetic acid, and dimethyl sulfoxide (DMSO) .
Typical ratios of the 1-triacontanol-organic solvent solution to water may vary from 1:10,000 to 1:1 parts by volume, preferably 1:1000 to 2:100 parts by volume, depending upon the desired concentration of the 1-tria¬ contanol in the final solution.
In accordance with another aspect of the present invention, it has been discovered that indole-3-acetic acid, which normally stimulates plant growth, inhibits the plant growth-stimulating effects of 1-triacontanol in plants when the two are sprayed simultaneously as a binary mixture. Various plant growth substances were added to triacontanol formulations in order to improve the effectiveness of the plant growth-stimulating properties
of the 1-triacontanol formulations. The following plant growth substances were tested: indole-3-acetic acid (IAA), gibberellic acid (GA 3 ) , kinetin, 2 ,4-dichloro- phenoxyacetic acid (2,4,-D), 2,3,5-triiodobenzoic acid (TIBA) , and maleic hydrazide (MH) . Various metal salts were also tested for their effectiveness in improving the plant growth-stimulating properties of the 1-triacontanol formulations. The salts tested included CaCl , LaCl, " , Ce(S0 4 ) 2 , MgCl 2 , MnCl , and mixtures thereof. It has been discovered that the presence of both 1-triacontanol and metal salts in the formulation creates a synergistic effect in increasing the plant growth-stimulating properties of the formulations . Although not wishing to be bound to any specific theory as to the precise mechanism by which the invention achieves its results, the plant growth- stimulating properties may be related to the fact that the metal ions (Ca , etc.) increase the binding of IAA to the cell membrane, and possibly make it unavailable for interaction with 1-triacontanol. Other possible mechanisms will become clear in the following discussion. It has been found that the metal ions of the metal salts are most effective when applied to the leaves by spraying at concentrations of about 10 molar to 10 molar, preferably at concentrations of 10 -2 molar to 10-4 molar and most preferably at concentrations of between 10 -2 and 10-3 molar. Spraying should be done no earlier than about the fifth day (for corn) after germination or when the plant has 2 to 5 true leaves for best results, preferably 3 to 4 true leaves . The metal ions should be sprayed on the area where the plants are grown in an amount of between 10 -4 and 10-1 moles per acre, preferably in an amount of between 10 -2 and 10-1 moles per acre using from 10 liters/acre of the formulation or a pro¬ portional amount using higher volumes per acre. The preferred volumes of solution per acre are 10 to 100 liters per acre.
Typically, the 1-triacontanol-polar organic solvent concentrate-water mixture is applied to the growing plants in an amount sufficient to achieve a distribution of at least 1 mg of 1-triacontanol per acre, advantageously, 5 to 20 mg per acre.
The chemical formulation, according to the present invention, can be applied to plant life in any desired manner although the spraying of the growing plant life has been found to be particularly effective when performed as described above.
The present invention can also be carried out by applying the metal salts or aqueous solutions of the metal salts to the soil where the seeds are planted and at a time up to several hours before the plants are treated with 1-triacontanol. For example, 10 -1 to 10-3 molar aqueous solutions of CaCl 2 can be applied to the soil at a rate of about 200 ml per plant. It is preferable that each plant receive at least 10 -4 moles of th * e metal ion. After several hours, in which time the plants have absorbed some of the metal ions, the plants can be sprayed with the
The metal salts which are useful are any salts or compounds which release metal ions in water. Inorganic salts are preferred. The size of the ionic radii of the metal ions appears to be related to the effectiveness of the ions radii of about between 0.60 and 1.5 angstroms are useful, metal ions with radii of between 0.85 and
1.5 angstroms are preferred, and metals ions with radii between 0.95 and 1.3 angstroms are most preferred. The average ionic radii of the preferred metal ions are listed below.
Ion Avg. radius Ton Avg. radius
Sr ++ 1.24 La* 3 1.10
Ba ++ 1.42 Ce +4 0.94
Cd ++ 1.05 M S ++ 0.77
Pb ++ * 1.19
The ionic radii of other metal ions which may be used in accordance with the present invention are listed below.
Ion Avg. radius Ion Avg. radius
Ce +'3 " 1.034 Pr +4 0.90
Cr +2 0.89 Sm +3 0.964
Cu +2 0.96 Sm 0.93
Er +3 0.881 Tb +3 " 0.923 E _u +3 0.950 Tb +4 0.84
Eu +2 0.950 Ti ++ 0.94
M +3 0.938 τi ÷3 0.95
In * " 3 0.81 Tm +3 0.87
Lu +3 0.93 V +2 0.88
Nd +3 0.995 γ +3 0.893
Pa +S 0.89 Yb +2 0.93
Pa* 4 0.84 Yb +3 0.858
Pm +3 0.979
Pr +3 1.013
Metal ions which have a positive valence of at least +2 are most effective. Salts of the alkaline earth metals such as calcium (Ca ) , barium (Ba ) , and strontium (Sr ) may be used with Ca being preferred from this group because of its effectiveness , availability as a water- soluble salt, and its non-toxicity and exemption from tolerance by the U.S. Environmental Protection Agency (EPA).
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Other metals of Group II of the Periodic Table are also useful such as beryllium (Be ) and magnesium (Mg ) . Metal ions from the lanthanide series are also very effective with metal ions from the lower end of the series such as lanthanum (La ) and cerium (Ce ) being preferred and lanthanum being most preferred from this series. Various metal ions from the transition metals are also useful. Of the transition metals, the lanthanide series metals are useful as discussed above as well as other
_l * -f- transition metals such as manganese (Mn ) . Other useful metals include cadmium (Cd + 2), lead (Pb+2), and iron salts, along with other similar metals as described above. In summary, many different metal ions and mixtures thereof
■ are useful in increasing plant growth when used in combina- tion with triacontanol. The effectiveness of the metal ions is dependent upon the concentration at which they are used, the particular plant species to which they are applied, and various other f ctors, including pH, a pH of about 8 or greater being preferred but not required for positive results. Therefore, it is possible that other metal ions than those discussed above might be useful in stimulating plant growth in accordance with the present invention.
The metal ions that appear to be most effective are LTa + 3, iC.a++, S _ .r++, -B-,a++, „Cd, ++, ,P,-b, ++, Mn++, C r.e+4, and-, Mg++, with La +3 and Ca ++ showing the highest increase in crop
++ yield and Ca being most preferred because of its low toxicity and abundance in nature.
The most reactive metal ions are contained in the Hofmeister series, listed below, and are the most membrane- destabilizing ions:
La +3 , Ca +2 >Mg +2 , Mn +2 , Sr +2 , Ba ÷2 >Na + , K + »NH 4 + The synergistic effect of the metal ions when combined with triacontanol is proportional to their position in this series.
The chemical formulations of the present invention, witho
metal ions, when applied to field and sweet corn, sugar cane, tomatoes, cucumbers, beans and the like, have been found to increase growth in a greenhouse-controlled environment in an amount up to about 36% based upon the dry weight of the plants. Similar tests under field conditions on about 1,000 acres have resulted in an increase in crop yield of field corn of 6 to 16% measured in terms of bushels per acre.
The formulations of the present invention containing metal salts increased the dry weight yield of sweet corn up to 72% in a greenhouse-controlled environment as com¬ pared to up to 36% increased observed with formulations having no metal salts. The formulations having metals salts have also shown increases in the field ranging from about 50 to 90% for beans, sweet corn, tomatoes, cucumbers, and peas, and 16 to 32% for soybeans, and may be expected to increase the yield of crops such as radishes, field corn, carrots, asparagus, sugar cane, beets, watermelons, cante- lopes, strawberries, potatoes, sugar beets, and a variety of other crops. Other plants, such as those used for ornamental purposes, may be expected to respond equally well to the formulations with and without metal salts, as well as tree seedlings.
In accordance with another aspect of the present invention, the effects seen using the formulations con¬ taining various metal ions are also affected by other plant growth substances when they are incorporated into the solutions for application to plant life. Plant growth substances and growth-stimulating agents such as gibberellins, cytokinins, and auxins are especially effective. Examples of such substances are: gibberellic acid (GA,, a gibberellin) , kinetin and benzyladenine (cytokinins and kinins) , indole-3-acetic acid and its salts and analogs (IAA, an auxin) , other auxins such as naphthaleneacetic acid (NAA) , indole-3-butyric acid (IBA) , indole-3-acetonitrile, 2,4-dichlorophenoxyacetic acid (2,4-D), and brassins and brassinosteroids , such as brassinolide and its analogs. Certain anticytokinins are
It has been discovered that certain concentrations of metal ions in the formulations produce an optimum effect. Also, concentrations up to about 10-fold less than the optimum concentrations of the metal salts in the formulations containing 1-triacontanol produce signi¬ ficant increases in both dry weight and water uptake in plant life. Higher-than-optimum concentrations, however, tend to reverse the growth-stimulating effects of the 1-triacontanol formulations. While not wishing to be bound by the following mechanism whereby the invention may achieve its results, it may be hypothesized that the action of 1-triacontanol requires the presence of calcium or similar metal ions for its growth-stimulating action. Calcium, being basically a plant growth inhibitor at certain concentrations, is known to exert its inhibi¬ tory effect through the inhibition of endogenous auxin. The binding of auxins such as indole-3-acetic acid and naphthalene acetic acid is known to be promoted by the metal salts of the present invention, thereby causing the naturally-occurring growth promoters to be unavailable for normal growth stimulation. Therefore, high concen¬ trations of calcium or the other metal ions, concentrations above the optimum concentrations used in the formulations containing 1-triacontanol, appear to be detrimental to plant growth through the inhibition of auxin. Because of this, the increases observed in dry weight and water uptake in plant life are lowered above this optimum concentration of the-me al salts even in the presence of 1-triacontanol.
It has been discovered during the course of research leading to the present invention that the inhibition of growth observed using high concentrations of metal ions is reversed when amounts of auxins and other plant growth- stimulating agents and hormones are added to the formula¬ tion. Therefore, in accordance with the present invention,
other plant growth stimulating agents were added to the formulations -of 1-triacontanol and metal ions.
It has further been discovered that, through the addition of other plant growth substances , much higher concentrations of metal ions than the optimum concentra¬ tions show favorable results. Increases in dry weight and water uptake were observed, for example, when the
_2 calcium concentration was raised from 10 molar to
_2 5 x 10 molar in the formulation of 1-triacontanol sprayed on sweet corn under a greenhouse-controlled environment. It was further found that the increases were comparable to those seen at the optimum calcium
_2 concentration of 10 molar, above which no increases in growth were observed without added auxin. An advantage of this procedure is that costly determinations of calcium contained in the water used for spraying in order to obtain a formulation having the most effective Ca concentration are effectively circumvented, since an excess of calcium or other metal ions may be used in the presence of the plant growth stimulants or hormones.
The preferred class of plant growth stimulants which show this effect when combined with 1-triacontanol and metal ions in solution for spraying plant life are auxins , examples of which are given above. While the plant growth stimulants may exert their effect at many concentrations , they are especially effective at concentrations between
10 molar and 10 molar, with concentrations between
5 x 10 and 5 x 10 being preferred. It is interesting to note that the preferred range of concentrations is the range of endogenous concentrations present in plant life.
The formulations of the present invention may include one or more metal ions in solution and, advantageously, one or more plant growth substances or stimulants in addition to an effective amount of 1-triacontanol. The formulations are effective in stimulating plant growth
using all plants and crops and the conditions described previously. The plant growth substance added to the formulation may be dissolved previously ' in the concen¬ trate containing 1-triacontanol and any of the polar organic solvents described herein, or may be dissolved in the water used to dissolve the concentrate prior to spraying. In addition to the plant growth stimulants used, their salts are likewise effective and show in¬ creased water solubility. In addition to the above, higher concentrations of the metal ions may be added in the presence of the plant growth substances and 1-triacontanol. Concentrations of 10 molar to about 5 molar may be used, preferably concentrations of 10 -4 to 1 molar, and most preferably concentrations of between 10 -3 molar to 10-1 molar are effective .
Other objects and further scope of applicability of the present invention will become apparent from the detaile description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graph showing the reproducibility in stimulating growth of sweet corn as determined by dry weight and water uptake increases and the formulation used which contained 100 μg/1 of 1-triacontanol without the addition of metal ions ; and
Fig. 2 is a graph showing the relationship between the concentration of CaCl 2 and LaCl^ and the increase in the plant growth-stimulating effects of the formulation for sweet corn with a concentration of 1-triacontanol of
Fig. 3 is a graph showing the relationship between the concentration of CaCl 2 and the plant growth-stimulating effects of the formulation for sweet corn with a concen¬ tration of 1-triacontanol of 500 μg/1, with and without the addition of indole-3-acetic acid (10 M) ; and
Fig. 4 is a graph showing the relationship between the concentration of indole-3-acetic acid and the plant growth-stimulating effects of the formulation for sweet corn with a concentration of 1-triacontanol of 500 μg/1
_2 and concentration of CaCl 2 of 5 x 10 molar; and
Fig. 5 is a graph showing the relationship between the concentration of CaCl and the plant growth-stimulating effects of the formulation for field corn with a concen¬ tration of 1-triacontanol of 500 μg/1, with and without the addition of indole-3-acetic acid (10 M) .
The following examples are presented herein as being exemplary of the present invention and, accordingly, should not be considered, in any way, as being limitative of the applicant's inventive contribution.
A 1 mg quantity of 1-triacontanol was dissolved in 20 ml of boiling acetone (or methyl ethyl ketone) and the solution was cooled to room temperature. This was added to 980 ml of water having a pH of 8 or more with vigorous stirring. This may be applied to plant life aerially at a rate of 10 mg per acre using 10 to 100 liters of solution per acre. The solution may further be diluted with ten parts of water resulting in a rate of 1 mg per acre. Other dilutions or higher concentrations of 1-triacontanol may also be used.
A 10 mg quantity of 1-triacontanol was dissolved in 100 ml of methanol (or 50 ml of ethanol) at the boiling point. The mixture was diluted to one liter with water at room temperature with vigorous stirring. The concen¬ trate may be diluted resulting in a solution containing 1 mg of 1-triacontanol per liter. This solution may be applied as described under Example 1.
One mg of 1-triacontanol was dissolved in 25 ml of hot isopropanol and the hot solution was poured into 975 ml of water with vigorous stirring over a one-minute period. This solution may be applied as described under Example 1.
One mg of 1-triacontanol was dissolved in 25 ml of hot diethylene glycol and added to 975 ml of rapidly stirring water. The resulting solution may be used as described above.
One mg of 1-triacontanol was dissoved in 10 ml of hot n-butanol and the mixture was added with stirring to 990 ml of water at 60 degrees. The solution was cooled to room temperature before use.
Ten mg of 1-triacontanol was dissolved in 100 ml of warm dioxane and added over 60 seconds to 950 ml of warm water. The solution may be used as described under Example 1.
* EXAMPLE 7
One mg of 1-triacontanol was dissolved in 50 ml of hot propylene glycol and added to 950 ml of water with stirring. The solution may be used as described under Example 1.
One mg of 1-triacontanol was dissolved in 20 ml of hot dimethyl sulfoxide (DMSO) and cooled to room tempera¬ ture. The resultant solution was added to 980 ml of water with stirring prior to use. This solution may be used as described under Example 1.
1-Triacontanol was prepared in accordance with U.S. Patent 4,167,641 and had a melting point of 87°C. Indole- 3-acetic acid, gibberellic acid (potassium salt) , and kinetin were obtained from Calbiochem-Behring Corporation, LaJolla, California. 2,4-Dichlorophenoxyacetic acid, 2,3,5-triiodo- benzoic acid, and maleic hydrazide were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. CaCl 2 and LaCl-T were from Fisher Scientific Co. and were of reagent grade. Hybrid sweet corn seed (Zea mays, cv. Silver Queen) was the product of Wetzel Seed Co., Harrisonburg, Virginia. Tomato seed (Lycopersicon esculentum Mill., cv. Ponderosa Red Beefsteak) was from Ferry Morse Seed Co., Inc., Fulton, Kentucky, and soybean seed (Glycine max) was from the Better Food Foundation, Greencastle, Pennsylvania. Field corn seed (Zea mays, cv. Pfizer 95-day singlecross hybrid) was generously supplied by Pfizer Genetics, Olivia, Minnesota, and pea seed (Pisum sativa) was suppled by the Western Chemical Corp.
Randomized complete block designs were used with four replications per treatment. Corn and tomato seed were sown eight per pot and soybeans twelve per pot in verniculite:peat
(1.1, v/v) , and each received an average of 100 ml of
2 water per day. Plants received 200 to 300 w/m artificial light. Sixteen hour days and eight hour nights were maintained at temperatures of 25°C and 20°C, respectively. After shoots emerged, plants were fertilized with 200 ml of a soluble 15N-30P-15K fertilizer at a concentration of 2.5 g/1. Prior to spraying, corn and tomato plants were thinned to 6 plants per pot on the tenth day after germination. Triacontanol concentrate was formulated by dis¬ solving 5 mg of triacontanol in 1 liter of acetone at the boiling point. Portions of 20 ml were further diluted to 1 liter prior to foliar application with distilled water at a pH of 8 to 10 (adjusted with NaOH) in which inorganic salts were optionally dissolved. IAA was dis¬ solved in the triacontanol concentrate when included in the formulation, however, an indole-3-aceta.te salt may alternately be dissolved in water prior to- the addition of the triacontanol concentrate. Plants were sprayed to the drip point with the variou formulations at mid-day and harvested six to nine days after treatment. Control groups were sprayed with solvent only. Entire plants were dried in an oven in groups of four (or six) below 100°C until constant weight was achieved. The dry weights were subtracted from fresh weights to give water uptake increases.
A comparison study was made between the formulation of the Ries et al patent, U.S. Patent 4,150,970, and the surfactant-free formulation of the present invention. The following formulations were used in all experiments.
Pots of similar sized plants were assigned to the same blocks with treatments being assigned randomly within blocks. A randomized complete block design was used and
analysis of variance performed for each test. Means were compared using Duncan's Multiple Range Test (2) to deter¬ mine the level of significance of growth difference, measured in dry weight, of each treatment compared to the control mean.
A 100 μg quantity of 1-triacontanol was dissolved in 1 ml of chloroform and shaken prior to use with one liter of water containing 1 g of Tween 20 (Example II of the Ries, et al., patent 4,150,970).
A 100 μg quantity of 1-triacontanol was dissolved in 20 ml of acetone at 50°C and the resultant solut * - , which had an indefinite shelf life, was dissolved in 980 ml of water at room temperature.
A 100 μg quantity of 1-triacontanol was dissolved in 50 ml of hot ethanol and the resultant solution was dissolved in 950 ml of water.
A 100 μg quantity of 1-triacontanol was dissolved in 20 ml of acetone.at 50°C and the resultant solution was dissolved in an additional 20 ml of water to form a concentrate. Any additional plant growth substance to be added was then dissolved in the concentrate. The solution was diluted to one liter with water prior to use, with or without a specified amount of a metal salt.
A 100 μg quantity of 1-triacontanol was dissolved
in 20 ml of acetone at 50°C and the resulting solution was dissolved in 980 ml of water containing a specified amount of a waters-soluble plant growth substance, with or without the addition of a specified quantity of a metal salt.
A 500 ' μ"g quantity of 1-triacontanol was dissolved in 20 ml of diozane at 60°C and the resultant solution was dissolved in 980 ml of water prior to use.
A 500 μg quantity of 1-triacontanol was dissolved in 25 ml of propylene glycol at 90°C and the solution was diluted to one liter for use.
A 500 μg quantity of 1-triacontanol was dissolved in 20 ml of hot dimethyl sulfoxide (DMSO) and cooled to room temperature. The resulting solution was added to 980 ml of water prior to use.
A 500 μg quantity of 1-triacontanol was dissolved in 20 ml of acetone at 50°C and a specified quantity of a plant growth substance was added. The resultant solu¬ tion was diluted to "one liter in water containing a specified amount of a metal salt.
A 500 μg quantity of 1-triacontanol was dissolved in 20 ml of acetone at 50°C and the resulting solution was added to 980 ml of water containing a specified amount of metal salts with or without the addition of a specified amount of a water-soluble plant growth substance.
A 100 μg quantity of 1-triacontanol was dissolved in 1 ml of chloroform and the solution was shaken with one liter of water containing one gram of Tween 20 and a specified amount of metal salts.
TABLE 1 Increases in Dry Weight and Water Uptake of 14-Day Old Hybrid Sweet Corn (var. Silver Queen) Sprayed with Various Formulations of 1<-Triacontanol without the Addition of Metal Salts or Other Plant Growth Substances.
FORMULA¬ PERCENT INCREASES LEVEL OF SIGNIFICANCE
DRY WT. H 2 0 UPTAKE DRY WT. H 2 0 UPTAKE
1 -19% - 8% 0,005 0.05
1 +10 -10 0.07 0.07
2 +21 +16 0.005 0.005
2 +19 +29 0.06 0.02
2 +36 +19 0.01 0.08
3 +19 + 6 0.06 N.S.
6 ' +17 + 8 0.07 N.S..
7 +33 +20 0.002 0.03
8 +18 - 9 0.02 N.S.
TABLE 2 Increases in Crop Yields Observed in the Field Spraying Two Different Formulations of 1-Triacontanol (1 mg/1) without the Addition of Metal Salts or Other Plant Growth Substances
CROP FORMULATION INCREASE PER ACRE
Hybrid Sweet Corn' 'Silver Queen' 1 -24% 2 +21
Beans 'Blue Lakes Stringless |-C 1 +29 2 +60
Cucumbers 'Straight Eight' 0 1 -10 2 +39
Increases were characterized by the increases in the fresh weight of marketable ears of corn.
Results over +_ 20% were significant at better than the 0 .05 level .
' Increases characterized by a greater number of beans or cucumbers , which resulted in an increase in total fresh weight .
TABLE 5 Increases in Dry Weight and Water Uptake of 14-Day Old Hybrid Sweet Corn Seedlings (var. Silver Queen) Sprayed with a Variety of Plant Growth Substances with and without the Addition of 1-Triacontanol .
PLANT CONCEN¬ TRIACON¬ FORMU¬ INCREASES a
GROWTH TRATION TANOL LATION SUBSTANCE (M) (100 μg/1) DRY WT. H 2 0 UPTAKE
IAA lo "5 NO 4 + 4% - 7%
IAA 10 "5 YES 4 + 2 - 5%
GA 3 10 "5 NO 5 -24 - 6
GA 3 10 "5 YES 5 -15 - 3
Kinetin 10 "5 NO 4 -24 - 3
Kinetin 10 "5 YES 4 -21 +14
2,4-D 10 "5 NO 4 +10 - 9
2,4-D 10 "5 YES 4 +17 -13
TIBA 10 "4 NO 4 +10 -12
TIBA lO "4 YES 4 +19 + 1
MH 10 "4 NO 4 + 7 - 1
MH 10 "4 YES 4 +12 - 7
———— YES 2 +20 +16
Least Significant Differences: 0.05 Level, 7
0 . 01 Level , 12
TABLE 4 Increases in Dry Weight and Water Uptake of 14 -Day Old Hybrid Sweet Corn Seedlings (var . Silver Queen) Sprayed with a Variety of Metal Salts and Formulations , with and without the Addition of 1 -Triacontanol
METAL CONCEN¬ TRIACON¬ FORMU¬ INCREASES a SALT TRATION TANOL LATION (M) (100 μg/1) DRY WT. H 2 0 UPTAKE
CaCl 2 10 "2 NO 4 + 4% - 2%
CaCl 2 10 "2 YES 4 +65 +11
CaCl 2 5 x 10 "3 YES 4 +50 +21
CaCl 2 10 "3 YES 4 +42 +35
CaCl 2 10 "2 YES 11 - 5 + 9
CaCl 7 5 x 10 "3 YES 11 + 5 + 6
LaCl 3 10 "2 NO 4 + 5 - 2
LaCl 3 10 "2 YES 4 +72 +18
Ce(S0 4 ) 2 10 "3 YES 4 +21 +26
MgCl 2 10 "3 YES 4 +21 +20
MnCl 2 10 "3 YES 4 + 9 +29
MgCl ? + .10 "3 ea. YES 4 +39 +30
YES +20 +16
Least Significant Differences : 0.05 Level, 11% ;
0.01 Level, 20%
TABLE 5 Increases in Dry Weight and Water Uptake of 14-Day Old Field Corn Seedlings (var. Pfizer 95-Day Singlecross Hybrid) Sprayed With a Variety of Metal Salts and Formula¬ tions, with and without the Addition of 1-Triacontanol .
METAL CONCEN¬ TRIACON¬ FORMU¬ INCREASES a SALT TRATION TANOL LATION
CM) (100 μg/1) DRY WT. H 2 0 UPTAKE
CaCl 2 10 ° YES 4 +20* -30% +21%
CaCl 2 10 "3 YES 11 - 8 -26 CaCl 2 5 x 10 "4 YES 4 .-22 + 8
CaCl 2 10 "3 YES 11 -10 -19
LaCl 3 10 "3 YES 4 +25* -32 +25 CaCl 2 + 10 "3 ea. YES 4 +20--30 +6-18
YES 1 - 3- - 4
Least Significant Differences: 0.05 level, 9%
0.01 level, 14%
TABLE 6 Increases in Dry Weight and Water Uptake of 45-Day Old Tomatoes (var. Ponderosa Red Beefsteak) Sprayed on Day 36 with Calcium Chloride and 1-Triacontanol (100 μg/1) Using Different Formulations .
CONCENTRATION FORMULATION INCREASES OF CaCl DRY WEIGHT H 2 0 UPTAKE
5 x 10 M 4 +38%' +34% a -2
10 4 +43 a +10 b
4 - 3 - 2
11 + 6 - 4
, Significant at the 0.05 level Significant at the 0.01 level
TABLE 7 Differences in Lengths between the Stems Cto the First leaf, Length "a") and Total Plant Length (Length "b") over Controls of Hybrid Sweet Corn Seedlings (var. Silver Queen) Sprayed on Day 7 with a Variety of Plant Growth Substances, with and without l÷Triacontanol (T) .
PLANT LENGTH INCREASE LENGTH INCREASE
GROWTH "a" "b"
IAA 11.6 cm 0% 24.4 cm - 2%
IAA + T 12.6 +10 29.8 +19
GA 3 11.2 - 3 26.6 + 6
GA 3 ÷ T 13.0 +13 32.5 +30
Kinetin 12.1 + 8 26.8 + 7
Kinetin ÷ T 11.5 0 32.5 +30 Control, 11.5 0 25.0 0
T 9.3 +15 41.6 +15
CaCl 2 + T 9.4 +16 39.9 +10
Control 2 8.1 0 36.3 0
Note: These data are presented to indicate changes in geometry of seedling growth when sprayed with various substances and do not reflect actual increases in tissue growth which would be indicated by increases in weight. Controls were sprayed with.2% acetone in water only.
TABLE ' 8 Results of Field Trials Spraying 1 mg/1 of 1-Triacontanol (2% acetone) Containing Various Concentrations of Calcium Chloride at Alkaline pH.
CROP pH CALCIUM INCREASE'
Tomatoes ('Ponderosa Red Beefsteak' )
Early 7.8 5 x 10 -3 72%
Late 7.8 5 x 10 67
Sweet Corn ('Silver Queen')
Trial 1 8.2 10 "2 51
Trial 2 . 8.2 10 "2 53
Trial 3 8.2 10 "2 54
('Blue Lakes Stringless ' ) 8.7 4, .5 x ,-3 90
Cucumbers ('Straight Eight') 8.7 4.5 x 10 -3 101
Increase in fresh weight of marketable yield.
TABLE 9 Results of Spraying Second-Generation Hybrid Sweet Corn Seedlings (var. Silver Queen) with 1-Triacontanol (500 μg/1) in the presence of Calcium Chloride at a pH of 8.1 Using Acetone (2%) as the Polar Organic Solvent.
GENERATION CONCENTRATION INCREASES
TEST SEED CONTROL SEED CaCl„ DRY WT. H 2 0 UPTAKE
SECOND SECOND 1x10 ' M +44-1 +40%
(First was (First and
Sprayed Second not
SECOND SECOND 1 x 10 "2 +80 +78
(First and (First and (First)
Second Second not
SECOND SECOND 1 x 10 "2 +41 ■■ 46
(First and (Second was (First)
FIRST FIRST 1.1 x 10 +44 +41
(Sprayed) (Not Sprayed)
TABLE 10 Increases in Dry Weight and Water Uptake of Hybrid Sweet Corn Seedlings (var. Silver Queen) Sprayed with Formulations of 1-Triacontanol (500 μg/1, 2% Acetone) Containing Various Concentrations of CaCl 2 and Various Plant Growth Substances (pH 8) .
PLANT GROWTH CONCEN¬ CONCEN¬ INCREASES
SUBSTANCE TRATION TRATION CM) CaCl 2 DRY WT. H 2 0 UPTAKE
IAA l.OxlO '*6 0.05 •• -37% +18%
IAA 1.0x10 " ° 0.05 +56 +25 IAA l.OxlO "4 0.05 +36 . +14
GA 3 4.0xlθ "5 0.01 +16 +15
GA 3 4.0xlθ "5 0.05 +22 +13
Kinetin l.OxlO "5 0.01 + 3 +13
Kinetin l.OxlO "5 0.05 + 1 - 2
Note: Results over 15% significant at 0.01 level
TABLE 11 Increases in Dry Weight and Water Uptake of Field Corn Seedlings (Pfizer 95-Day Single- cross Hybrid) Sprayed with a Brassinosteroid with and without a Formulation of 1-Triacontanol (500 μg/1, 2% Acetone) Containing 0.01 M CaCl 2 .
FORMULATION DRY WEIGHT WATER UPTAKE INCREASE INCREASE
Brassinosteroid (4 x 10 "7 M) without 1-Triacontanol +11% ÷ 8-
Brassinosteroid (4 x 10 "7 M) with- 1-Triacontanol +19' +10
The compound used, 2α,3α,22 ,23α-tetrahydroxy-24 -methyl* B-homo-5 -cholestan-6-one, a brassinolide analog, was dissolved in the acetone concentrate containing 1-tria¬ contanol according to formulation 9.
Significant at 0.01 level
TABLE 12 Increases in Dry Weight and Water Uptake of Soybean Seedlings Sprayed with and without various Concentration of CaCl 2 and 1-Triacontanol at a pH of 10.4.
TRIAL TRIACONTANOL CaCl 2 INCREASES NO. (μg/D ( M) DRY WT, H 2 0 UPTAKE
1 0 0 2 100 0 + 2 % - 12 b %
3 100 1 +ιo a + 2 4 100 3 + 19 b + 17 b 5 100 5 - 1 ÷ 3
Result Significant at 0.05 level
Result Significant at 0.01 level
TABLE 13 Increases in Dry Weight and Water Uptake of Peas Sprayed with 1-Triacontanol (100 μg/1) and Various Formulations con¬ taining CaCl 2 and IAA (2% acetone)
CaCl 2 IAA DRY OUT WATER
CONCEN¬ CONCEN¬ INCREASE UPTAKE INCREASE
TRATION TRATION .OVER CONTROL OVER CONTROL
0 mM 0 - 4% „. 0%
1 mM 0 +13 - 8 a
5 mM 0 + 7 + 7
7 mM 0 + 4 - 7
10 mM 0 +18 b + 5
50 mM 10 "5 M +46 b +.
Significant at 0.05 level Significant at 0,01 level
V " •ii
As shown in Table 1, the formulations of 1- triacontanol, containing 1-triacontanol and polar organic solvents in water only, significantly increased the dry weight and water uptake of sweet corn seedlings while the formulation of U.S. Patent No. 4,150,970 gave inconsistent results (see Figure 1 and Tables 1 and 2). The dry weight increases were seen to correlate with the fresh weight (marketable yield) of crops when sprayed in the field. Table 3 shows that no improvement in the formula- tions containing polar organic solvents, triacontanol, and water were found when other plant growth substances were added to the formulation without metal ions. However, when metal ions of the present invention were added to the formulation, significant increases in dry weight and water uptake were observed over these found using the formulations without metal ions (Tables 4, 5, and 6 and Figure 2) . The addition of surfactant additives to aid in the solubilization of nonpolar solvents, such as those used in U.S. Patent application 4,150,970, was found to negate this effect and cause decreases in dry weight and water uptake. Since the present invention teaches that metal ions may be necessary for the growth-stimulating action of 1-triacontanol, complexing the ions would result in little or no growth stimulation. U.S. Patent No. 4,169,716 discloses formulations containing 1-triacontanol, plant growth substances, and polyvalent metals co plexed to proteins, the combination of which shows increases in plant weight when applied to seedlings. The significant increases disclosed therein, however, are observed only when metal proteinates are applied without the addition of plant growth sub¬ stances or 1-triacontanol, since the percent increase observed when.triacontanol was added to the metal pro¬ teinates or when triacontanol was added to a mixture of metal proteinates and one or more plant growth substances was only about 1 to 4%.
Table 7 shows the change in the geometry of plants at harvest when sprayed with various formulations of the present invention. These data should not be interpreted as indicating increases in actual tissue growth. The dry weight increases found with the seedlings, a parameter indicating actual growth increases, are shown to correlate with those increases in marketable yield found in the field when the formulations containing metal ions and 1-triacontanol were sprayed (Table 8) . When the metal ion concentration exceeds its optimum, a reversal of the plant-growth stimulating effect becomes apparent. This effect was altered or reversed by the addition of plant growth substances described in the present invention (see Tables 10 and 11 ^ and ' Fig ' . 3).* Indole- acetic acid is seen to reach an optimum effect at 10 " M, approximately the endogenous IAA concentration, when the concentration of CaCl 2 was raised to five times its optimum level. Figure 4 shows the effect of IAA when combined with a formulation- containing 1-triacontanol and 0.05 M CaCl 2 (five times the optimum concentration).
The concentration range for the metal ion is shovm to be wider when IAA is added, and the Ca +2 concentration is no longer critical since a range of concentrations all produce an optimum effect. This observation becomes important when considering the case for field corn (Figure 5) . Field corn seedlings show increases in dry weight and water uptake at a very
_3 narrow range which is optimum at only 10 M CaCl-. Since the Ca +2 concentration of hard water used to economically formulate 1-triacontanol for spraying in the field varies to a high degree, and may be in excess of
-3 10 M m some cases, analysis of the water used must be undertaken in order to provide a formulation which will assure positive results when sprayed on crop seedlings Since this adds considerable expense to the operation, the addition of IAA (or another plant growth substance that
allows for increases in dry weight when triacontanol is sprayed with higher-than-optimum concentrations of metal ions) is desirable. The addition of IAA at 10 ' M gives a formulation that allows for significant increases in dry weight even at ten times the optimum level of
CaCl , i.e., 10 -2M. At elevated levels of Ca+2, the Ca +2 present in hard water becomes negligible, since optimum results are seen not only at concentrations about
3 x 10 M, but a concentrations twice that. Therefore, the use of the formulations of the present invention * . that contain 1-triacontanol, metal ions, and plant growth substances does not require the added expense of water analysis to successfully increase crop yields of field corn. It has also been found that different cultivars of field corn require slightly different formulations for optimum growth stimulation, e.g. WF9 x Bear 38 does not respond well at 10 " M Ca , but does with higher Ca concentrations with the addition of 10 M IAA. Also, Pioneer 3780 responds to the former and not the latter. Table 9 shows that the effect of spraying the 1-triacontanol formulatios of the present invention does not cause subsequent generations of the same plant to become unaffected by triacontanol spraying. Also, the second generation (the plants grown from the seed of sprayed plants) give increases in yield without being sprayed with triacontanol. Spraying these seedlings gives an additional increase equal to the increase found when the first generation seedlings are sprayed. This effect may be due to the fact that ears of sweet corn from triacontanol-sprayed plants contain the same number of kernels as those ears which were not sprayed, however, the kernels are larger in size. Planting the larger seed produces larger seedlings. Table 11 shows the effective range of Ca con¬ centrations for soybeans.
Significant increases in the yield of peas may be expected using relatively high concentrations of CaCl 2 or preferably ,CaCl 2 and IAA, as shown in Table 13. From these examples it is clear that metal ions significantly increase the growth-stimulating effect of 1-triacontanol, and also that auxins and other plant growth substances significantly increase the tolerance of the plant toward large concentrations of the metal ions. The invention being thus described, it will be obvious that the same may be varied in many ways . Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.