WO2005077171A1

WO2005077171A1 - Amino acid compositions for seeds and their use in preventing herbicide damage to plants

Info

Publication number: WO2005077171A1
Application number: PCT/US2005/003714
Authority: WO
Inventors: David C. Sands; Alice L. Pilgeram; Daniel R. Cox
Original assignee: Montana State University-Bozeman
Priority date: 2004-02-05
Filing date: 2005-02-07
Publication date: 2005-08-25
Also published as: CA2555271A1

Abstract

The present invention provides methods for protecting plants from herbicide injury and damage. More particularly, this invention relates to coating or priming seeds with one or more amino acids to confer tolerance to herbicides that disrupt production of the amino acids by a plant treated with the herbicide.

Description

AMINO ACID COMPOSITIONS FOR SEEDS AND THEIR USE IN PREVENTING HERBICIDE DAMAGE TO PLANTS

INVENTORS David C. Sands, Alice L. Pilgeram, Daniel R. Cox

RELATED APPLICATIONS This application claims priority to U.S. provisional patent application Serial No. 60/542,109 filed February 05, 2004 which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Seeds are tightly packaged plant organs consisting of a dormant embryo, an endosperm containing complex carbohydrates, proteins, oils and a rigid protective water- permeable seed coat. When planted in moist soil, the seed imbibes water, hormones are activated causing production of hydrolytic enzymes, leading to a breakdown of complex storage carbohydrates, proteins and oils. The seed embryo and more significantly endosperm thus provide the energy and twenty different amino acid building blocks for new cellular protein, oils for membranes, and carbohydrates for cell walls, sufficient for seedling growth until it can develop enough roots and leaves to proceed on its own, typically within about a week. Weed control is often required in field cropping systems because conditions that trigger germination of the crop seed also concomitantly trigger germination of weed seed. Many weed seeds actually are triggered to germinate at lower temperatures and shorter days than most cultivated crops. Herbicides provide an effective means for weed control. But all herbicides have the potential to cause crop injury. The extent of such injury depends largely on the chemical nature of the herbicide, life of the herbicide in the soil, the amount of herbicide taken into the plant, the sensitivity of the plant species to the herbicide and its retention in the active form in the plant. Damage may occur from foliar absorption or uptake from soil by roots of plants near treated areas. The period during germination of the seed, sprouting and initial growth of the plant is particularly critical because the roots and shoots of the growing plant are small and even a small amount of damage can kill the entire plant. Thus, post emergence herbicides can only be used if they do not damage the crop, and it is for this reason that one popular plant breeding strategy is to incorporate herbicide resistant genes into the crop via genetic engineering. The herbicides most often used for such a strategy are those that specifically target a key enzyme in amino acid biosynthesis since an exotic gene encoding an enzyme resistant to the herbicide can be transferred into, and expressed in the crop plant. Several widely used, commercially available synthetic herbicides have been found or designed to inhibit key regulatory enzymes in the biosynthesis of amino acids of plants. These target sites are especially useful because the pathways and specific enzymes targeted do not exist in animals; hence the effects of the herbicides that target them are largely limited to plants. Without amino acid synthesis, in order to make new protein, a plant can only recycle amino acids from its existing proteins. If synthesis of even a single amino acid is inhibited by an herbicide, the plant dies as a result. One such herbicide (glyphosate) inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), a critical enzyme in the biosynthesis of three aromatic amino acids (phenylalanine, tyrosine and tryptophan). A second herbicide family (the sulfonylureas) inhibits acetolactate synthase (ALS), an essential enzyme in the biosynthesis of valine, isoleucine and leucine. A third plant enzyme glutamine synthase in the biosynthetic pathway leading to glutamine is inhibited by the herbicide glufosinate. Another plant enzyme imidazoleglycerol phosphate dehydrase (IGPD) involved in histidine biosynthesis is inhibited by the triazole herbicides. A number of genetically modified EPSP synthase, acetolactate synthase and glutamine synthase resistant crops are now being commercially produced. There are several limitations to this genetically conferred herbicide resistance technology. Some crops are recalcitrant to genetic engineering. Herbicide resistant seeds are expensive and not all plant breeders or producers can afford to use this protected technology. It does not allow the farmer as much flexibility in choosing varieties or herbicides. Similarly, if genetically modified herbicide resistance technology is widely adapted, it may be difficult to prevent an unintended narrowing of genetic diversity in a crop. There is resistance on the part of some consumers to consume foods made from genetically modified plants. There is some fear of herbicide resistance genes escaping into other varieties of the crop, or even into wild species including weeds. There are also legal issues involving the right of farmers to replant their own seed from the previous crop. There is clearly a need for an alternate technology that can protect crop seedlings from some very effective herbicides. There have been suggestions in the art that the toxicity caused by these types of herbicides may be reversed by supplying the appropriate amino acids (Gresshoff 1979, Killmer et al, 1981, Cox et al, 1997, Eason et al, 2000 and Romagni et al, 2000). Although herbicide reversal by the application of exogenous solutions of appropriate amino acids has been documented in the literature, prior to the instant invention there has been no practical use of this knowledge per se, probably because it is not practical to spray plants or irrigate with amino acids in the field. This is at least partially because such non-selective treatment would also help protect the target weeds from the effect of the applied herbicide. The present invention has discovered that coating or priming seeds with appropriate amino acids can reverse damage caused by post-emergent herbicides. When the coated seed germinates, sufficient amino acids are transported into the seedling from the seed coating/priming to permit normal protein synthesis, thereby circumventing the inhibition of the target enzyme by a post-emergence spray of herbicide. The present technology can be applied to natural seeds, seeds generated from plant breeding and genetically modified seeds. Thus, an object of this invention is to protect crop seedlings from post-emergent herbicides by an appropriate amino acid seed treatment of the desired plant species, while allowing herbicide damage to the undesired plant species.

SUMMARY OF THE INVENTION The present invention provides amino acid compositions and methods of using such amino acid compositions for protecting plants against herbicide damage. The present invention involves determining whether the production of one or more amino acids by a desirable plant is reduced or eliminated by the application of a specific herbicide and, if so, providing the one or more of these amino acids to the seed of the desirable plant before, at the time of, or immediately after the application of the specific herbicide to the seed or plant, thereby enabling growth of the plant in the presence of the specific herbicide. The compositions and methods of the present invention are also applicable to protecting transplants. By utilizing the compositions and methods of the present invention, one can enable the growth of the desirable plant that would normally be reduced or prevented by the specific herbicide while allowing the specific herbicide to reduce or prevent the growth of any undesirable plants. The invention is particularly useful for broad spectrum herbicides that would normally inhibit the growth or kill all or almost all species of plants. The invention is also useful for classes of herbicides that would normally inhibit the growth or kill all or almost all species of plants of a certain class of plants (e.g., all or almost all monocots or all or almost all dicots). However, the invention is useful for any type of herbicide regardless of the specific types or classes or plants that it harms or kills. The one or more amino acids required by the desirable plant to maintain growth and health in the presence of the specific herbicide can be provided to the desirable plant by a variety of different methods. In the case of broad spectrum herbicides or herbicides that harm or kill certain classes of plants, it is most useful to provide the one or more amino acids directly to the seed or the seedling of the desirable plant. In one embodiment, the one or more amino acids are provided directly to the seed of the desirable plant by coating or priming the seed with a composition that contains the one or more amino acids. In such a circumstance, the amount of the one or more amino acids are provided to the desirable plant in an amount sufficient to allow or sustain the health and growth of the desirable plant through the time period in which the desirable plant is exposed to the herbicide, thereby reducing damage or preventing death of the desirable plant from the herbicide while reducing the growth of or killing undesirable plants. For example, where an herbicide persists in the soil up to 3 weeks, it would be necessary to provide enough of the one or more amino acids to the desirable plant so that the desirable plant will remain healthy and grow during its up to 3 week exposure to the herbicide. Thus, the amount of the one or more amino acids to provide to the desirable plant depends on the life of the particular herbicide in a particular soil or other growing medium (e.g., vermiculite, peat moss), as well as its retention in the active form in the plant. Other methods of providing the one or more amino acids to the plant are also contemplated by the instant invention, wherein such methods can be used for seeds and/or transplants, such as seedling transplants or for the transplantation of more mature plants. Examples of plants that are often transplanted include but are not limited to petunias, cabbage, pine trees, violas, cucumbers, tomatoes, grapes, peppers, apple trees, tobacco, etc. Such transplanting can be by hand or by machine or by a combination of both. According to the present invention, the one or more amino acids can be provided in a composition that is applied directly or indirectly to the seeds or to the transplants before, during or after planting. For example, the one or more amino acids may be in a solution, slurry or powder that is dribbled, dripped, splashed, or poured directly on the seeds or transplants. Alternatively, the slurry or powder could be applied to the growth medium which is directly in contact with the seeds or transplants. In the case of transplants, it may be desirable to dip the roots of the transplants in a composition that contains the one or more amino acids before or during the time of planting, or alternately to pour such a composition on the plants before, during or after the transplantation. It is important that the one or more amino acids be applied in a localized manner to the seeds and/or transplants so as to avoid or prevent undesirable plants from obtaining the benefits of the one or more amino acids, thereby also avoiding herbicide damage. In another embodiment, a microbe or microbes could be applied to the seed or transplant of the desirable plant, wherein the microbe or microbes produce the one or more amino acids so that the desirable plant obtains the one or more amino acids it needs to be healthy and grow in the presence of the specific herbicide.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effects of three different levels of phenylalanine and tyrosine coatings on damage caused by post germination application of glyphosate in spring wheat. Plants were divided into 4 groups based upon visual reading of herbicide damage. l=healthy. 4=dead. The readings for each treatment were averaged.

DETAILED DESCRIPTION OF THE INVENTION All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

I. Definitions As used herein, the term "amino acid" is used in its broadest sense and refers to any organic compound containing both an amino group and a carboxylic acid group (COOH). Aminocarboxylic acids are the components of proteins and peptides. As used herein, the term "barley" refers to any Hordeum species, including, but not limited to, Hordeum vulgare, H sativum, H. distichum, H. tetastichum, and H. hexastichum. Thus, as used herein, the term "barley" means any type of barley including, but is not limited to, any cultivated barley, any wild barley, any barley species, any intra- and inter-species barley crosses, all barley varieties, all barley genotypes and all barley cultivars. As used herein, the term "sugar beet" refers to any Beta species, including but not limited to Beta vulgaris and Beta maritima. Thus, as used herein, the term "sugar beet" means any type of sugar beet including, but is not limited to, any cultivated sugar beet, any wild sugar beet, any sugar beet species, any intra- and inter-species sugar beet crosses, all sugar beet varieties, all sugar beet genotypes and all sugar beet cultivars. As used herein, the term "chlorosis" refers to yellowing in plant color due to a decline in chlorophyll levels. As used herein, the term "clover" refers to any Trifolium species, including, but not limited to, T. hybridum, T. vesiculosum, T. alexandrinum, T. incarnatum, T. campestre, T. dubium, T. ambiguum, T. arvense, T. pratense, T. fragiferum, T. subterraneum, T. repens, and T. medium. Thus, as used herein, the term "clover" means any type of clover including, but is not limited to, any clover commonly referred to as alsike clover, arrowleaf clover, berseem clover, crimson clover, large hop clover, small hop clover, Kura clover, rabbit's foot clover, red clover, strawberry clover, subterranean clover, white clover, and zigzag clover. As used herein, the term "crop plant" refers to any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food additives, smoking products, pulp production and wood production. As used herein, the term "cultivar" refers to a variety, strain or race of plant which has been produced by horticultural or agronomic techniques and is not normally found in wild populations. As used herein, the term "desiccant" refers to a chemical used to kill or dry plants and, as such, is usually used just prior to harvest. As used herein, the term's "dicotyledon" and "dicot" refer to a flowering plant having an embryo containing two seed halves or cotyledons. Examples include, but not limited to, tobacco; tomato; the legumes, including peas, alfalfa, clover and soybeans; oaks; maples; roses; mints; squashes; daisies; walnuts; cacti; violets and buttercups. As used herein, the term "emergence" refers to an event in seedling or perennial growth when a shoot becomes visible by pushing through the soil surface. As used herein, the term "formulation" refers to a form in which the pesticide is supplied by the manufacturer for use. As used herein, the term "herbicide" refers to any chemical substance used to destroy/kill, delay, reduce, inhibit or otherwise adversely affect the growth of plants, especially weeds. As used herein, the term "herbicide resistance" refers to the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type. In a plant, resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis. As used herein, the term "herbicide tolerance" refers to the ability of a plant to withstand herbicide treatment without marked deviation from normal growth or function. As used herein, the term "hybrid" means any individual plant resulting from a cross between parents that differ in one or more genes. As used herein, the term "maize" refers to any Zea species, including, but not limited to, Z. mays, Z. diploperennis, Z. luxurians, Z. nicaraguensis and Z. perennis. Thus, as used herein, the term "maize" means any type of rice including, but is not limited to, any cultivated maize, any wild maize, any maize species, any intra- and inter-species maize crosses, all maize varieties, all maize genotypes and all maize cultivars. As used herein, the term "onion" refers to any Allium species including, but not limited to, Allium cepa, Allium fistulosum, Allium schoenoprasum, Allium ascalonicum, Allium cernuu, and Allium ampeloprasum. Thus, as used herein, the term "onion" means any type of onion including, but is not limited to, any cultivated onion, any wild onion, any onion species, any intra- and inter-species onion crosses, all onion varieties, all onion genotypes and all onion cultivars. As used herein, the term "pea" refers to any Pisum species including, but not limited to, Pisum sativum, Pisum fulvum and Pisum syriacum. Thus, as used herein, the term "pea" means any type of pea including, but is not limited to, any cultivated pea, any wild pea, any pea species, any intra- and inter-species pea crosses, all pea varieties, all pea genotypes and all pea cultivars. As used herein, the term "pre-emergence application" refers to a time of herbicide application occurring after a crop is planted but before the crop or weeds emerge from the soil. As used herein, the term "post-emergence application" refers to a time of herbicide application occurring after the crop or weeds emerge from the soil. As used herein, the term "residual herbicide" refers to a herbicide that persists in the soil and injures or kills germinating weed seedlings over a relatively short period of time. As used herein, the term "rice" refers to any Oryza species, including, but not limited to, O. sativa, O. glaberrima, O. perennis, O. nivara, and O. breviligulata. Thus, as used herein, the term "rice" means any type of rice including, but is not limited to, any cultivated rice, any wild rice, any rice species, any intra- and inter-species rice crosses, all rice varieties, all rice genotypes and all rice cultivars. As used herein, the term "seedling" refers to a juvenile plant that has developed from a seed. As used herein, the term "seed" refers to a ripened plant ovule containing an embryo. As used herein, the term "seed priming" refers to a physiological method of improving seed performance, wherein the seeds are imbibed to a water content below that required for radicle emergence, but sufficient to allow geπninative metabolism to proceed. As used herein, the term "seed coating" refers to a procedure in which the seed is treated with one or more adhering coating layers. As used herein, the term "soybean" refers to any Glycine species, including, but not limited to, G. max, G. gracillis, G hispida, Soja hispida, and S. max. Thus, as used herein, the term "soybean" means any type of soybean including, but is not limited to, any cultivated soybean, any wild soybean, any soybean species, any intra- and inter-species soybean crosses, all soybean varieties, all soybean genotypes and all soybean cultivars. As used herein, the term "synthetic" refers to a set of progenies derived by intercrossing a specific set of clones or seed-propagated lines. A synthetic may contain mixtures of seed resulting from cross-, self-, and sib-fertilization. As used herein, the term "systemic herbicides" refers to a class of herbicides that are able to move away from the site of absorption to other parts of the plant. As used herein, the term "translocation" refers to the movement of water, plant sugars and nutrients, herbicides and other soluble materials from one plant part to another. As used herein, the term "transplant" refers to any plant that is uprooted and replanted. As used herein, the term "variety" refers to a subdivision of a species, consisting of a group of individuals within the species which are distinct in form or function from other similar arrays of individuals. As used herein, the term "weed" refers to any plant considered undesirable, unattractive, or troublesome, especially one growing where it is not wanted or any plant that is objectionable or interferes with the activities or welfare of man. As used herein, the term "alfalfa" refers to any Medicago species, including, but not limited to, M. sativa, M. murex, M.falcata, and prostrata. Thus, as used herein, the term "alfalfa" means any type of alfalfa including, but is not limited to, any alfalfa commonly referred to as cultivated alfalfa, diploid alfalfa, glanded alfalfa, purple- flowered alfalfa, sickle alfalfa, variegated alfalfa, wild alfalfa, or yellow-flowered alfalfa. As used herein, the term "wheat" refers to any Triticum species, including, but not limited to, T. aestivum, T. monococcum, T. tauschii and T. turgidum. Thus, as used herein, the term "wheat" means any type of wheat including, but is not limited to, any cultivated wheat, any wild wheat, any wheat species, any intra- and inter-species wheat crosses, all wheat varieties, all wheat genotypes and all wheat cultivars. Cultivated wheats include, but are not limited to, einkom, durum and common wheats. II. Amino Acids Amino acids play central roles both as building blocks of proteins and as intermediates in metabolism. There are 20 naturally occurring amino acids. They exist as either of two enantiomorphs. But only L-amino acids are found in proteins. An alpha- amino acid consists of an amino group(-NH₂), a carboxyl group(-COOH), a hydrogen atom, and a distinctive R group bonded to a carbon atom, which is called the alpha- carbon because it is adjacent to the carboxyl (acidic) group. An R group is referred to as a side chain. Each of the 20 α-amino acids can be distinguished by the R-group substitution on the α-carbon atom. The side chains (R groups) of the amino acids can be divided into two major classes, those with non-polar side chains and those with polar side chains. The nonpolar amino acids are characterized by having only carbon and hydrogen in their side chains. The non-polar amino acids include: alanine (Ala, A), cysteine (Cys, C), glycine (Gly, G), isoleucine (He, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V). The polar amino acids include: arginine (Arg, R), asparagine (Asn, N), aspartic acid or aspartate (Asp, D), glutamine (Gin, Q), glutamic acid or glutamate(Glu, E), histidine (His, H), lysine (Lys, K), serine (Ser, S), and threonine (Thr, T). Amino acids have the ability to rotate the plane of polarized light. They are optically active and exist in two isomeric forms known as enantiomers. The isomer that rotates the plane of polarized light to the left (counterclockwise) is called levorotatory (L). The other isomer that rotates the light to the right (clockwise) is called dextrorotatory (D). The optical isomers are mirror images of each other. The isomers result from the tetrahedral geometry around the chiral carbon center. A chiral carbon is one that has four different "groups" attached to it. The groups can be anything from a single H to functional groups to one or more other carbons. Except for glycine, all amino acids exist as D and L enantiomers. L amino acids represent the vast majority of amino acids found in proteins. D amino acids are found in some proteins produced by exotic sea-dwelling organisms, such as cone snails. They are also abundant components of the cell walls of bacteria. When amino acids are synthesized in vitro, they form as a racemic mixture of the D and L enantiomers. A racemic mixture is an equimolar mixture of the two enantiomeric isomers of a amino acid. As a consequence of the equal numbers of laevo- and dextro-rotatory molecules present in a racemate, there is no net rotation of plane polarized light. Amino acid compositions of the seeds of some plant species namely wheat (Triticum aestivum), common vetch (Vicia sativa), chickpeas (Cicer arientum), Cyprus vetch (Lathyrus ochrus), field beans (Viciafaba), narbon vetch (Vicia narbonensis), peas (Pisum sativum) and on imported solvent extracted soybean meal (Glycine max) are shown in Table 1, Table 2 and Table 3.

Table 1. Distribution of protein in the wheat kernel

* based on Hinton (1953) and MacMasters et al. (1971)

Table 2. Amino acid composition of wheat grain, germ and aleurone proteins (g/100 g protein)

a From FAQ (1970) b From Bushuk and Wrigley (1974) c From Pomeranz et al. (197) (g/100 g amino acids) d From Fulcher et al (1972)

Data from M Hadjipanayiotou and S Economides (2001)

Plants can synthesize all 20 amino acids used in protein synthesis from simple organic precursors and metabolic intermediates. The pathways for the biosynthesis of amino acids are diverse. However, they have an important common feature: their carbon skeletons come from intermediates of glycolysis, the pentose phosphate pathway, or the citric acid cycle. On the basis of these starting materials, amino acids can be grouped into six biosynthetic families 1. Glutamate family (α-ketoglutarate); 2. Aspartate family (Oxalo acetate); 3. Alanine-valine-leucine (Pyruvate); 4. Serine₇glycine (3- Phosphoglycerate); 5. Aromatic amino acids family (Phosphoenolpyruvate and Erythrose 4-phosphate) and 6. Histidine (Ribose 5-phosphate). Nitrogen-fixing microorganisms (such as Rhizobium in root nodules of leguminous plants) use ATP and reduced ferredoxin to reduce atmospheric N₂ to NH₃. Plants take up nitrogen as nitrate, and in smaller amounts also as ammonium ions, h a first step, nitrate (NO ^") is reduced to nitrite (NO₂ ^") by the enzyme nitrate reductase. The reducing agent is NADH + H+ that is gained during glycolysis. In the second step, nitrite is reduced to ammonium by nitrite reductase, an enzyme that is located in plastids. Free ammonium ions are toxic to plant cells and are rapidly incorporated into organic compounds. NH₄ is incorporated into amino acids via glutamate and glutamine.

A. Glutamate Family The amino acids glutamate, glutamine, proline and arginine are members of this family. Glutamate is synthesized from NH₄ ⁺ and α-ketoglutarate, a citric acid cycle intermediate, by the action of NADP⁺-dependent glutamate dehydrogenase. Glutamate is accordingly the amino acid generated first. Most amino acids obtain their α-amino group from glutamate by transamination. Glutamate itself can bind a further ammonium ion to form glutamine, a second amino acid. This reaction takes place in chloroplasts, catalyzed by enzyme glutamine synthase. Both reactions are ATP-dependent. Glutamine contributes its side-chain nitrogen atom in the biosynthesis of a wide range of important compounds, including the amino acids tryptophan and histidine. Glutamate is the precursor of two other amino acids: proline and arginine. First, the γ-carboxyl group of glutamate reacts with ATP to form an acyl phosphate. This mixed anhydride is then reduced by NADPH to an aldehyde. Glutamic γ-semialdehyde cyclizes with a loss of H₂O in a nonenzymatic process to give Δ^pyrroline-S-carboxylate, which is reduced by NADPH to proline. Alternatively, the semialdehyde can be transaminated to ornithine, which is converted in several steps into arginine B. Aspartate Family The amino acids aspartate, asparagine, threonine, isoleucine, methionine and lysine are counted among this group. Aspartate is synthesized by the transfer of a ammonia group from glutamate to oxaloacetate. Asparagine is made either by transamination from glutamine or by adding ammonia directly to aspartate. Threonine, methionine, isoleucine and lysine are all made starting from aspartate. The pathways for these 4 amino acids all start the same way. Aspartate is phosphorylated and reduced to form the aldehyde. This requires an ATP and NADPH. At this point lysine biosynthesis branches off, through an 8 step pathway involving the addition of pyruvate to aspartate semialdehyde, the use of a Co A intermediate (either acetyl CoA or succinyl-CoA) and the addition of an amino group from glutamate. The group added from CoA (either succinyl or acetyl) serves as a blocking group, protecting the amino group from attack during transamination by glutamate. NADPH + H⁺ is required for reduction in the second step of the pathway. One carbon is removed in a decarboxylation reaction at the end of the pathway to form lysine. The three remaining amino acids have one more common step before they branch off. This is the reduction of the aspartyl semialdehyde to an alcohol by NADPH. The alcohol of aspartic acid is called L-homoserine. The biosynthesis of threonine has again several intermediates and consumes two molecules of ATP, one NADPH + ET^1" and one NADH + H⁺. First a second reduction with NADPH + H⁺, yields homoserine. This is phosphorylated to homoserine phosphate by ATP and finally converted into threonine. Threonine biosynthesis is completed in three steps. Isoleucine is made from threonine after it is oxidatively deaminated to form alpha ketobutyrate. This compound is similar to pyruvate, except it has one more methylene group. Synthesis of methionine requires sulphur. Sulphur is taken up by plants as sulphate ions and is subsequently converted to its reduced form in a two step reaction. The intermediate is sulphide. The reduced sulphur is finally converted to the -SH group of cysteine (assimilatory sulphate reduction). Homoserine is also the precursor for methionine. Again the hydroxyl is activated, but this time it is by succinyl CoA that transfers a succinyl group and releases CoA. Methionine has a sulfur, and this is donated from cysteine. The whole cysteine molecule is covalently attached to the activated carbon through the sulfur. This is cleaved off as pyruvate and ammonia to leave the SH group on the product homocysteine. Finally methionine is formed by the addition of a methyl group donated by N5 methyl tetrahydrofolate.

C. Alanine-Valine-Leucine Group Alanine is derived from pyruvate, the end product of glycolysis, through a transamination reaction with glutamate as the amino group donor. Valine and leucine are synthesized independently of each other by elongation of the pyruvate chain, interconversion and subsequent transamination. Valine is made by the same pathway as isoleucine starting from the alpha keto acids pyruvate and alpha ketobutyrate. In the first common step, pyruvate donates an hydroxyethyl group to the alpha carbon of the keto acid. In the next step, the newly added acetyl group inserts itself between the alpha and beta carbons, forming a dihydroxy acid. This is then dehydrated to make the alpha keto acids that are directly transaminated by glutamate to make valine and isoleucine. Leucine biosynthesis branches from alpha ketoisovalerate, the immediate precursor to valine. In the first step Acetyl-CoA is used to add an acetyl group to the molecule. Electrons are transferred to NAD (note these can be used for other cellular processes) and one carbon is lost in the form of CO at the fourth step of the pathway, hi the final step, the amine from glutamate is added to a-ketoisocaproate to form leucine.

D. Serine-Glycine-Cysteine Group Serine family amino acids include 3 amino acids: serine, glycine and cysteine. Synthesis of serine and glycine starts with oxidation of 3-phosphoglycerate forming 3- phosphohydroxy pyruvate and NADH. A transamination reaction with glutamate forms 3-phosphoserine and removal of the phosphate yields serine. Serine is the precursor of glycine and cysteine. Glycine is generated by removal of the methyl group from serine, catalyzed by enzyme serine hydroxy methyl transferase. Energy is not required for this pathway, actually it yields energy in the form of reduced NADH. The conversion of serine into cysteine requires the substitution of a sulfur atom derived from methionine for the side-chain oxygen atom. Synthesis of cysteine is a two step reaction. Serine and acetyl-CoA combine to form O-acetylserine. Sulfide from sulfur assimilation is then added to O-acetylserine to form cysteine.

E. Aromatic Amino Acids Aromatic amino acids phenylalanine, tyrosine, and tryptophan are synthesized by a common pathway. The initial step is the condensation of phosphoenolpyruvate (a glycolytic intermediate) with erythrose 4-phosphate (a pentose phosphate pathway intermediate). The resulting seven-carbon open-chain sugar is oxidized, loses its phosphoryl group, and cyclizes to 3-dehydroquinate. Dehydration then yields 3- dehydroshikimate, which is reduced by NADPH to shikimate. Phosphorylation of shikimate by ATP gives shikimate 3-phosphate, which condenses with a second molecule of phosphoenolpyruvate. This 5-enolpyruvyl intermediate loses its phosphoryl group, yielding chorismate, the common precursor of all three aromatic amino acids. The importance of this pathway is revealed by the effectiveness of glyphosate, a broad- spectrum herbicide. This compound inhibits the enzyme that produces 5- enolpyruvylshikimate 3-phosphate and, hence, blocks aromatic amino acid biosynthesis in plants. Because animals lack this enzyme, the herbicide is fairly nontoxic. The pathway bifurcates at chorismate. A mutase converts chorismate into prephenate, the immediate precursor of the aromatic ring of phenylalanine and tyrosine. Dehydration and decarboxylation yield phenylpyruvate. Alternatively, prephenate can be oxidatively decarboxylated to p-hydroxyphenylpyruvate. These α-ketoacids are then transaminated to form phenylalanine and tyrosine. The branch starting with anthranilate leads to the synthesis of tryptophan.

Chorismate acquires an amino group derived from the hydrolysis of the side chain of glutamine and releases pyruvate to form anthranilate. Then anthranilate condenses with 5-phosphoribosyl-l-pyrophosphate (PRPP), an activated form of ribose phosphate. PRPP is also an important intermediate in the synthesis of histidine, purine nucleotides, and pyrimidine nucleotides. The C-1 atom of ribose 5-phosphate becomes bonded to the nitrogen atom of anthranilate in a reaction that is driven by the release and hydrolysis of pyrophosphate. The ribose moiety of phosphoribosylanthranilate undergoes reanangement to yield l-(o-carboxyphenylamino)-l-deoxyribulose 5-phosphate. This intennediate is dehydrated and then decarboxylated to indole-3-glycerol phosphate, which is cleaved to indole. Then indole reacts with serine to form tryptophan. In these final steps, which are catalyzed by tryptophan synthetase, the side chain of indole-3 -glycerol phosphate is removed as glyceraldehyde 3-phosphate and replaced by the carbon skeleton of serine. F. Histidine Biosynthesis The synthesis of histidine is long and complex and its pathway is intertwined with nucleic acid biosynthesis (specifically purine). In the first step of histidine synthesis, 5- phosphoribosyl-α-pyrophosphate (PRPP) condenses with ATP to form a purine, N -5 - phosphoribosyl ATP, in a reaction that is driven by the subsequent hydrolysis of the pyrophosphate that condenses out. Glutamine plays a role as an amino group donor, resulting in the formation of 5-aminoamidazole-4-carboximide ribonucleotide (ACAIR), which is an intennediate in purine biosynthesis. This product is converted to imidazole glycerol phosphate by the enzyme glutamine amido transferase. Imidazole glycerol- phosphate dehydratase catalyzes the dehydration of imidazole glycerol phosphate to imidazole acetol phosphate, which is transmainated by glutamate to form histidinol phosphate. Histidinol phosphate is dephosphorylated and oxidized twice by histidinol dehydrogenase to form histidine.

III. Weed Management Weeds are considered as "plants growing in places where they are not wanted"

(Zoschke and Quadranti, 2002). Weeds are troublesome in many ways. Primarily, they reduce crop yield by competing for water, light, soil nutrients, space and CO . They reduce the crop quality by contaminating the commodity and interfering with harvest. They also serve as hosts for crop diseases or provide shelter for insects and animals (rodents, box turtles, snakes, etc.). Weeds produce chemical substances that can be allergins or toxins to humans, animals, or crop plants (allelopathy). They are usually plants that are very prolific, unattractive, invasive, competitive, harmful, destructive, or difficult to control.

A. Classification of Weeds Weeds may be classified as grasses, sedges, and broadleaf weeds. They may be further classified by the length of their life cycle. The three basic life cycles of weed plants are annuals, biennials, and perennials (Vandiver and Teem, 2002). Grass Weeds: True grasses have hollow, rounded stems and nodes (joints) that are closed and hard. The leaf blades are alternate on each side of the stem, have parallel veins, and are much longer than they are wide. Some examples are crabgrass, goosegrass, crowfootgrass, sandbur, annual bluegrass, torpedograss, and vaseygrass. Sedges: Sedges are an important group of "grass-like" weeds; however, they are not true grasses and are characterized by a solid, triangular-shaped stem with leaves extending in three directions. There are annual sedges (some are often called water grass), and the predominant and difficult to control perennial sedges. Of the latter group, yellow nutsedge is yellow-green in color and reproduces by seed, rhizomes (underground stems), and tubers. The rhizomes radiate from the plant, with a single bulb or tuber at the end, which may produce a new plant. Purple nutsedge is usually smaller in growth habit than yellow nutsedge, has reddish purple seed heads and produces a series of bulbs on radiating rhizomes called "tuber chains". Broadleaf Weeds: Broadleaf weeds are a highly variable group of plants, but most have showy flowers and net-like veins in their leaves. They are easy to separate from grasses due to their generally different leaf structure and habits of growth. Some examples of broadleaf weeds are cudweed, creeping charlie, henbit, spurges, burning nettle, pennywort, creeping beggarweed, cocklebur, sicklepod, and Florida beggarweed. Annual Weeds: Annual weeds, as the name implies, complete their life cycle within one year. They germinate from seed, grow, mature, produce seed and die in 12 months or less. They may be annual grasses, sedges, or broadleaf weeds. In addition, their life cycle may begin at different seasons of the year. Thus, summer annuals emerge in the spring, mature, produce seed, and die before winter of each year. Weeds such as crabgrass and cocklebur are typical of summer annuals. Similarly, winter annual weeds sprout from seed in the fall, and complete their life cycle before summer of the next calendar year. Sowthistle, henbit, annual bluegrass, and chickweed are examples of winter annual weeds. Biennial Weeds: Compared to annual weeds, biennial weeds are few in number. These weeds have a 2-year life cycle. They germinate from seed in the fall, and develop large root systems and a compact cluster of leaves during the first year. The second year they mature, produce seed and die. Examples of biennial weeds are cudweed, Carolina falsedandelion, wild canot, and bull thistle. Perennial Weeds: Weeds that live more than two years are perennials. They reproduce by vegetative parts such as tubers, bulbs, rhizomes or stolons (above-ground stems). Some also produce seed in addition to vegetative reproduction. During the winter season most live-over in a donnant state and many lose their above-ground foliage and stems. With the beginning of spring they regenerate from food reserves in their root systems. Torpedograss, nutsedge, johnsongrass, bermudagrass, guineagrass, vaseygrass and various vines are members of this group of weeds.

B. Methods of Weed Control Weeds are hard to control because they grow rapidly, produce vast numbers of seeds, and spread aggressively by vegetative structures and/or seeds. There are several methods of weed control, including mechanical, cultural, , biological, and chemical. Best weed control is usually achieved by a combination of two or more of these methods. Many times this combination of weed control methods is called an integrated weed control program. Cultural Control: Cultural control involves methods or management practices which favor the growth of desirable plants over noxious weeds, including maintaining optimum fertility and plant moisture in an area, planting at optimum density and spatial anangement in an area, and planting species that are most suited to a particular area. Crop rotation, reseeding, fertilization, irrigation are all cultural practices that can enhance weed control. Reseeding to reintroduce desirable plants once weeds have been removed is important. Mechanical Control: Mechanical weed control refers to the physical removal of undesirable plants. Mowing eliminates a wide variety of plants that have upright growth habits, such as velvetleaf and lambsquarter. One type of mechanical control is burial. This method is most effective on annual weeds in which all the growing points can be buried. Burial is usually less effective on perennial weeds which have underground stems and roots and are capable of regrowth from these underground storage organs. Manually removing plants by pulling or digging is an efficient means of control in small lawns, or when only a few weeds are present. Perennial weeds also may be controlled in time by continually removing the top-growth of the weed, which depletes food reserves in the root system. Another method of mechanical control is cultivation. The main objective in cultivation is to cut the root system of the weeds; deep cultivation should usually be avoided due to damage to the crop roots. Deep cultivation may also bring more weed seed to the surface where they will germinate. Burning is an old method of weed control, and in certain instances can be used to favor selectively certain species over others. Controlled burning can be useful to remove weeds from ditch banks, roadsides and other waste areas. Fire has been used for many years to favor the growth of pine seedlings over hardwoods. Special equipment for flaming is available. Fire is usually more effective on annual weeds than on perennial weeds and usually does not kill weed seed in the soil. Biological Control: Biological control is the use of animals, fungi, or other microbes to feed upon, parasitize or otherwise interfere with a targeted pest species. Biological weed control is therefore an approach using living organisms to control or reduce the population of a selected, undesirable, weed species, whilst leaving the crop unharmed (TeBeest, 1991). Biological control is often viewed as a progressive and environmentally friendly way to control pest organisms because it leaves behind no chemical residues that might have harmful impacts on humans or other organisms, and when successful, it can provide essentially permanent, widespread control with a very favorable cost-benefit ratio. The most commonly used natural enemies are insects (herbivores such as leaf-eaters, seed-eaters, or root-eaters) and fungi (rob nutrients from plants, cause diseases in plants, which often results in necrosis of leaves or complete leaf loss). The fungi can also be turned into a specific bioherbicide which can be applied (e. g. sprayed) onto weeds to control them. Agents can attack different parts of the weed. For example, seed-eating agents can eat the seeds and stop new weed seedlings taking over when the leaf-eating agents attack and kill the mature weeds. Chemical Control: Chemicals are the most modern and efficient means for controlling unwanted plant species. Chemicals can be applied to relatively large areas quickly with relatively little labor. A wide variety of weeds can be effectively controlled by the use of herbicides. They kill plants by working on plant anatomy or physiology. The success of chemical weed control depends on the type of herbicide used, where it is placed, when it is applied, and what environmental conditions prevail at the time of application. Prevention: Once effective weed control has been achieved, further steps should be taken to prevent weeds from re-infesting the area. Weed seed may be distributed in crop seed, hay, straw, by wind, water, animals, machinery and other ways. Certified, registered, and foundation seed, or clean planting material should be used to prevent weeds from infesting fields. It is also important to clean equipment before entering fields or when moving from one field to another. Soil on tractor tires or other areas of equipment may contain large numbers of weed seed. Cultivators and mowers should be cleaned to prevent the movement of vegetative plant parts such as rhizomes and stolons from different areas in the fields or from field to field.

IV. Herbicides Herbicides belong to a group of chemicals known as pesticides, which prevent, destroy, repel, or mitigate any pest. Herbicides are any chemical substance used to destroy/kill, delay, reduce, inhibit or otherwise adversely affect the growth of plants, especially weeds. Herbicides are an important tool for managing weeds. They provide an effective means for weed control. Herbicides may be classified as selective or non-selective depending on how and when they are used. In addition to classification based in selectivity herbicides may also be classified based on time of application, area covered, mode of action, and chemical structure (Fenell et al, 2004). Selective herbicides are chemicals which can remove certain plant species without seriously affecting the growth of other plant species. The majority of herbicides used are selective herbicides. Non-selective herbicides are chemicals which are toxic to all plants. They may be used to remove a wide range of vegetation from an area. When no selectivity is intended, these chemicals can be used for vegetation control along fence rows, around pipe lines, traffic signs, storage areas, parking lots, and other areas where total vegetation control is desired. Glyphosate, glufosinate and diquat are a few examples of nonselective herbicides.

A. Time of Application: There are several methods of herbicide application based on when they are applied. These include pre-plant incorporated, pre-plant, pre-emergence, post-emergence, and lay-by (Ferrell et al, 2004). Pre-plant refers to applications made before the crop is planted. Currently, in most cases, these materials are incorporated into the soil and are called pre-plant incorporated treatments. The great advantage of these incorporated treatments is that the herbicide is placed in the zone where weed seed germinate and is not dependent on rainfall to move the herbicide into this zone. This type of treatment adds to the cost of incorporation and requires that the crop be tolerant of the herbicide, as the crop seed and the herbicide will be in contact. Examples of such herbicides are trifluralin, profluralin, benefm, and vernolate. Pre-emergence treatments usually refer to applications made after the crop is planted but prior to crop and/or weed emergence. These pre-emergence applications are usually applied to the soil surface and require rainfall or irrigation to move the herbicide into the soil. If the herbicide is not moved into the soil where the weed seed are located it will not be effective. If left on the soil surface, these herbicides are often lost due to photodecomposition and vaporization. A pre-emergence treatment is generally the best method for control of annual weeds. Post-emergence treatments are applied following crop or weed emergence. If the crop has emerged but no weeds are present then the application is post-emergence to the crop but pre-emergence to the weeds and would be applied to the soil surface. If the crop has emerged and the weeds have emerged, then the application is post-emergence to both weed and crop and would be applied to the foliage of the weeds. Lay-by treatments are applications of herbicides after the last cultivation.

B. Types of Herbicide Treatments Foliage Treatments: Foliage treatments are herbicide applications to the leaves, stems or shoots of growing plants usually as sprays, mists, or dusts. They are applied directly to the weeds and kill them primarily by being absorbed into the foliage. Types of foliage treatments include contact and translocated or systemic herbicides. Contact herbicides affect only the portion of green plant tissue that is contacted by the herbicide spray. These herbicides are not translocated or moved in the vascular system of plants. Therefore, these will not kill underground plant parts, such as rhizomes or tubers. Repeat applications are often needed with contact herbicides to kill regrowth from these underground plant parts. They are most effective against annuals. Complete coverage is essential in weed control with contact materials. Examples of contact herbicides include the organic arsenicals (MSMA, DSMA), bentazon (Basagran), glufosinate and diquat. Systemic herbicides enter the plant through the roots and leaves and move throughout the inside of the plant. The vascular system transports the nutrients and water necessary for normal growth and development. Systemic herbicides generally are slower acting and kill plants over a period of days. Systemic herbicides may be effective against all weed types; however, their greatest advantage is in the control of established perennials, those weeds that continue their growth from year to year. Uniform application is needed for the translocated materials, whereas complete coverage is not required. Examples of systemic herbicides include glyphosate, 2,4-D, dicamba (Banvel), imazaquin (Image), and sethoxydim (Vantage). Soil Treatments: Soil applied herbicides are applied to the soil, although some require incorporation into the soil. They may also be contact or translocated herbicides. They are carried into the soil by water from irrigation or rainfall and absorbed by the root system of the weed. These herbicides effectively control weeds for a few weeks to several months, with the exact time depending on the particular herbicide, rate and time of application, weather and type of soil. Because of this long-term residual effectiveness, soil-applied herbicides are sometimes refened to as residual herbicides.

C. Herbicide Formulations A herbicide formulation is the total marketed product, and is typically available in fonns that can be sprayed on as liquids or applied as dry solids. It includes the active ingredient(s), any additives that enhance herbicide effectiveness, stability, or ease of application such as surfactants and other adjuvants, and any other ingredients including solvents, carriers, or dyes. The application method and species to be treated will determine which formulation is best to use. In most cases, manufacturers produce formulations that make applications and handling simpler and safer. Some herbicides are available in fonns that can reduce risk of exposure during mixing, such as pre-measured packets that dissolve in water, or as a liquid form already mixed with surfactant and dye. Sprayable/liquid formulations include: Water-soluble formulations: soluble liquids (SL), soluble powders or packets (SP), and soluble granules (SG). Only a few herbicidal active ingredients readily dissolve in water. These products will not settle out or separate when mixed with water. Emulsifiable formulations (oily liquids): emulsifiable concentrates (E or EC) and gels (GL). These products tend to be easy to handle and store, require little agitation, and will not settle out of solution. Disadvantages of these products are that most can be easily absorbed through the skin and the solvents they contain can cause the rubber and plastic parts of application equipment to deteriorate. Liquid suspensions (L for liquid or F for flowable) that are dispersed in water include: suspension concentrates (SC), aqueous suspensions (AS), emulsions of water- dissolved herbicide in oil (EO), emulsions of an oil-dissolved herbicide in water (EW), micro-encapsulated formulations (ME), and capsule suspensions (CS). All these products consist of a particulate or liquid droplet active ingredient suspended in a liquid. They are easy to handle and apply, and rarely clog nozzles. However, they can require agitation to keep the active ingredients from separating out. Dry solids that are suspended in water: wettable powders (W or WP), water- dispersible granules (WDG, WG, DG), or dry flowables (DF). These formulations are some of the most widely used. The active ingredient is mixed with a fine particulate carrier, such as clay, to maintain suspension in water. These products tend to be inexpensive, easy to store, and are not as readily absorbed through the skin and eyes as ECs or other liquid formulations. These products, however, can be inhalation hazards during pouring and mixing. In addition, they require constant agitation to maintain suspension and they may be abrasive to application pumps and nozzles.

Dry formulations include: Granules (G) - Granules consist of the active ingredient absorbed onto coarse particles of clay or other substance, and are most often used in soil applications. These formulations can persist for some time and may need to be incorporated into the soil. Pellets (P) or tablets (TB) - Pellets are similar to granules but tend to be more uniform in size and shape. Dusts (D) - A dust is a finely ground pesticide combined with an inert or inactive dry carrier. They can pose a drift or inhalation hazard.

D. Method of Application The way in which herbicides are applied can reduce the risk of haiming native plants nearby. Herbicides may be applied as band, broadcast, spot treatments, and directed spraying (Fenell et al. 2004). Band applications usually refer to treating a nanow strip directly over the row. This reduces the amount of chemical required and the cost per acre; however, with this type application the area between the rows is not treated and usually will require cultivation or chemical treatment later in the season. Broadcast applications cover the entire area. These treatments, while requiring the largest amount of chemical and highest cost per acre, usually result in the best weed control. Spot treatments are used for weeds which are localized in certain areas but are not widespread over the entire area. When only isolated areas of weeds are present, this is the most economical and best method to control and prevent their spread to other areas. Directed applications are applied to a particular area or part of the plant. These applications are usually directed to the base of the crop plant and away from the leaves. The ability to use directed sprays usually depends on a height differential between the crop and the weed. If the crop is taller than the weeds then drop nozzles can be used to direct the spray treatment over the weeds but below the leaves of the crop. Directed sprays are very useful in late season control of weeds and usually follow a preplant or pre-emergence application. In many cases preplant or pre-emergence applications do not persist long enough to control late germinating weed seed or may not be used on certain soil types, hi such cases directed sprays are used to obtain effective weed control and improve harvest efficiency.

E. Mode of Action An herbicide's mode of action is the mechanism (biochemical or physical) by which it kills or suppresses plants. The mode of action is generally dictated by its chemical structure, and therefore, herbicides in the same family, tend to have the same mode of action. The seven major modes of action are: growth regulation, lipid synthesis inhibition, seedling growth inhibition, photosynthesis inhibition, cell membrane disruption, pigment inhibition, and amino acid synthesis inhibition. 1. Growth Regulators Growth regulator herbicides closely mimic functions of auxin plant hormones, most notably IAA (indoleacetic acid). Auxins are known for their roles in cell elongation, controlling lateral growth, and cell wall formation. Susceptible plants have abnormal cell wall development when treated. Characteristic symptomology of bent and twisted stems results from increased cell wall plasticity due to induced proton movement out of the cell. This significant movement of protons changes the pH around the cell, increasing the activity of certain enzymes around the cell wall, which causes the cell to abnonnally elongate. Growth regulator herbicides also increase the production of RNA polymerase, which stimulates the production of RNA, and many proteins. This in turn leads to increased cell division. Together, uncontrollable cell division and cell elongation leads to destruction of the vascular tissues of the plant. Decreased ability to translocate through these tissues leads to starvation for nutrients and water and the inability to move sugars away from the source of production. The growth regulators include the following herbicide families: phenoxy acetic acids [e.g. 2,4-D, 2,4-DB (Buctyrac®, Butoxone®), 2,4-DP, MCPA, MCPB (Thistrol), MCPP]; benzoic acids [e.g. dicamba (Banvel®, Clarity®)]; and the pyridines [e.g. clopyralid (Stinger®), picloram (Tordon®), triclopyr (Garlon, Crossbow)]. Growth regulator herbicides selectively kill broadleaf weeds; however, they are capable of injuring grass crops. Herbicides in this group can move in both the xylem and the phloem to areas of new plant growth. As a result, many herbicides in this group are effective on perennial and annual broadleaf weeds. Herbicide uptake is primarily through the foliage but root uptake is possible. Injury symptoms are most obvious on newly developing leaves.

2. Lipid Synthesis Inhibitors Herbicides in this family inhibit the production of fatty acids within plants. Fatty acid synthesis in plants is an essential process in the development of plant lipids that are required for cell membrane integrity and normal plant growth. The lipid synthesis inhibitor herbicides prevent the activity of acetyl-CoA carboxylase (ACCase) enzyme, which is involved in fatty acid biosynthesis. The lipid synthesis inhibitors include the following herbicide families: aryloxyphenoxypropionates [e.g. diclofop (Hoelon®), fluazifop (Fusilade DX®), quizalofop (Assure II®), fenoxaprop (Option II®)] and cyclohexanediones [e.g. sethoxydim (Poast®, Poast Plus®), clethodim (Select®)]. Broadleaf plants are tolerant to these herbicides; however, almost all perennial and annual grasses are susceptible. Herbicides in this family are taken up by foliage and are translocated via the phloem to areas of new growth. Injury symptoms are slow to occur (7 to 14 days) and first appear on new leaves emerging from the whorl of the plant. The plants will gradually turn purple, brown, and die, but older leaves may stay green for a long time.

3. Seedling Growth Inhibitors Seedling growth inhibitors interfere with new plant growth, thereby reducing the ability of seedlings to develop nonnally in the soil. Seedling growth inhibitors are active at two main sites, the developing shoot and the root. The root inhibitors stop plant cells from dividing, which in turn inhibits shoot elongation and lateral root formation. Uptake of these herbicides is through developing shoots and roots. Translocation of the herbicide is limited within the plant, and therefore the injury is mainly confined to the uptake areas. Shoot-inhibiting herbicides are absorbed by developing shoots and roots and are transported via the xylem and phloem to areas of new growth. The seedling growth inhibitors include the following three herbicide families: Carbamothioate family (Shoot Inhibitors) e.g. EPTC (Eradicane, Eptam), butylate (Sutan+), triallate (Far-Go) ;Acetamide family (Shoot Inhibitors) e.g. alachlor (Lasso®, Partner, Micro-Tech®), metolachlor (Pennant/Dual II® MAGNUM), propachlor (Ramrod®), dimethenamid (Outlook), acetochlor (Surpass®, Harness®, Degree, Topnotch®), flufenacet (Axiom, Define); Dinitroanaline family (Root Inhibitors) e.g. ethalfluralin (Sonalan®), pendimethalin (Pendimax, Prowl®), oryzalin (Surflan®), prodiamine (Baracade), benefin (Balan®), trifluralin (Treflan®), oryzalin + isoxaben (Snapshot), etc. Herbicides in these families must be soil-applied. Plants can take up these herbicides after germinating until the seedling emerges from the soil. Therefore, these herbicides are only effective on seedling annual or perennial weeds. Plants that have emerged from the soil uninjured are likely to remain unaffected.

4. Photosynthesis Inhibitors Photosynthesis is the process by which chlorophyll-containing cells in green plants use the energy of light to synthesize carbohydrates from carbon dioxide and water. Photosynthesis inhibitors shut down the photosynthetic (food producing) process in susceptible plants by binding to specific sites within the plant's chloroplasts. This group of compounds inhibit photosynthesis in photosystem II (PS II) by blocking the transfer of electrons from plastiquinone and cytochromes. Since the electrons are not converted to stored chemical energy (ATP and NADPH) in PS II, they fonn free radicals that result in cell membrane destruction leading to plant death. Injury symptoms include yellowing (chlorosis) of leaf tissue followed by death (necrosis) of the tissue. The photosynthesis inhibitors include the following herbicide families: triazines [e.g atrazine (various), cyanazine (Bladex®), simazine (Princep®), propazine (Milogard), ametryn (Evik), metribuzin (Sencor®, Lexone®), prometon (Pramitol), hexazinone (Velpar®)], phenylureas [e.g. inuron (Lorox®), tebuthiuron (Spike®)], uracils [e.g. terbacil (Sinbar®), bromacil (Hyvar®)], benzothiadiazoles [e.g. bentazon (Basagran®)], and nitriles [e.g. bromoxynil (Buctril®)] Three of the herbicide families (triazines, phenylureas, and uracils) are taken up into the plant via the roots or foliage and move in the xylem to plant leaves. As a result, injury symptoms first appear on the older leaves, along the leaf margin. After foliar application, triazine, phenylurea, and uracil herbicides are less mobile and do not move out of the leaf tissue. The nitrile and benzothiadiazole herbicide families are not mobile in plants and are classified as post-emergence contact herbicides. These herbicides have no soil activity. Contact herbicides must thoroughly cover a susceptible plant's foliage if complete control is to be achieved. Photosynthetic inhibitors may control annual or perennial grass or broadleaf weeds.

5. Cell Membrane Disrupters These herbicides are post-emergence contact herbicides that are activated by exposure to sunlight to form oxygen compounds such as hydrogen peroxide. These oxygen compounds destroy plant tissue by rupturing plant cell membranes. Destruction of cell membranes results in a rapid browning (necrosis) of plant tissue. On a bright and sunny day, herbicide injury symptoms can occur in 1 to 2 hours. There are two types of cell membrane disrupters. One inhibits protoporphyrinogen oxidase (PPO) enzyme and the other inhibits photosystem I. The chemical families that inhibit PPO-enzyme are the diphenyl ethers [e.g. acifluorfen (Blazer®), lactofen (Cobra®), fomesafen (Reflex®), oxyfluorfen (Goal®)], N- phenylphthalimides [e.g. flumioxazin (valor), flumiclorac pentyl ester (Resource)] and the triazolinones [e.g. sulfentrazone (Authority/Spartan), carfentrazone methyl (Aim)]. The chemical family that inhibits photo-system I is the bipyridilium family of herbicides [e.g. paraquat (Gramoxone Extra®), difenzoquat (Avenge®), diquat (Diquat®, Reward®)] Because these are contact herbicides, they are excellent for bumdown of existing foliage and post-emergence control of annual weeds. Because of the limited translocation of this chemical within plants, underground root systems are not destroyed and perennial weeds usually regrow after their top growth has been destroyed. These herbicides have little soil activity.

6. Pigment Inhibitors: Pigment inhibitors prevent plants from forming photosynthetic pigments. They inhibit the formation of carotenoid pigments or carotenoid and chlorophyll pigments. Carotenoids are yellow/orange pigments that are almost always associated with chlorophyll. These pigments protect chlorophyll by dissipating the oxidative energy of singlet oxygen produced during photosynthesis. Loss of carotenoid pigment results in the formation of massive amounts of singlet oxygen that leads to photo-destruction of chlorophyll and bleaching (whitening) of plant tissue. The pigment inhibitors include the following herbicide families: isoxazol [e.g clomazone (Command®), isoxaflutole (Balance)], pyridazinone [e.g. norflurazon (Zorial, Evital®, Solicam®)], triazole [e.g. amitrole (Amitrol®)], Isoxazolidinones [e.g clomazone (Command®)], and Pyridazinones [norflurazon (Zorial®)]. The most striking symptom resulting from treating plants that inhibit carotenoid biosynthesis is the totally white foliage produced following treatment. These herbicides are absorbed by roots and translocate to the shoot tissue where they inhibit the production of carotenoids. These herbicides do not cause a destruction of carotenoids already fonned. Rather, they prevent the production of new carotenoids after exposure. Thus, plant tissues fonned before treatment do not show typical albino symptoms; only new growth in treated plants is white. Growth does continue for some time, but without production of green photosynthetic tissue, growth ceases and necrosis begins to occur.

7. Amino Acid Synthesis Inhibitors Herbicides with this mode of action reduce or block the production of amino acids, the essential building blocks of proteins. They generally inhibit a key enzyme necessary for production of the particular amino acid(s). The amino acid synthesis inhibitors initially block amino acid biosynthesis at the rapidly growing regions of the plant. This is followed by inhibition in other tissues. Death of the plant occurs slowly and may take several days. However, since most cells of the plant are killed, these herbicides are systemic. In general, lower doses of AA herbicides are sufficient to inhibit plant growth (Kishore and Shah, 1988). Inhibitors of amino acid synthesis are divided into three classes - inhibitors of EPSP synthase (5-enolpyruvyl-shil imate-3 phosphate synthase), the ALS or AHAS (acetolactate synthase, also known as acetohydroxyacid synthase) inhibitors, glutamine synthetase inhibitors and the inhibitors of histidine biosynthesis. The ALS inhibitors are composed of four families of herbicide chemistry, the imidazolinones (Pursuit®, Scepte®r, others); the sulfonylureas (Accent®, Beacon®, Classic®, Harmony Extra, others) the sulfonamides (FirstRate® and Python®); and the thiopyrimidines (Staple®). Members of these chemical families have the same mode of action, which is inhibition of ALS. ALS catalyzes the condensation of 2 mol of pyruvate to produce acetolactate or 1 mol of pyruvate and 1 mol of 2-ketobutyrate to produce acetohydroxybutyrate in the pathways leading to biosynthesis of amino acids valine, leucine and isoleucine (Shaner et al, 1984). These herbicides, in general, are absorbed readily and move throughout the plant. EPSPs inhibitors inhibit the EPSP synthase enzyme, which is involved in the synthesis of the aromatic amino acids (tyrosine, tryptophan, and phenylalanine). The EPSP inhibitor herbicides are readily absorbed through plant foliage and translocated in the phloem to the growing points. The effect of this group of compounds is noted on the entire plant. They affect grasses and dicots alike, e.g. Glyphosate (Roundup®, Glyfos, Glyphomax®, Acquire, Credit™, Touchdown®, ClearOut, etc. ) The Glutamine synthetase inhibitors inhibit the conversion of glutamic acid and ammonia to glutamine. Ammonia accumulates and glutamine, glutamate and aspartate decrease. This interruption of important nitrogen metabolism and indirect inhibition of electron flow in photosynthesis causes a disruption of membranes, e.g. Glufosinate (Liberty®, Rely®, etc.). Histidine biosynthesis inhibitors inhibit the enzyme imidazoleglycerol-phosphate dehydratase, which is involved in the biosynthesis of amino acid histidine. e.g. Amitrole (Amitrol®, Amino Triazole, etc.)

V. Glyphosate Glyphosate [N-(phosphonomethyl)glycine C₃H₈NO₅P] is a broad spectrum, post- emergence herbicide (Eason et al, 2000). In pure chemical terms glyphosate is an organophosphate in that it contains carbon and phosphorous. The herbicidal activity of glyphosate and its salts was first described in 1971 and since then glyphosate has become the world's most popular herbicide (Baird et al, 1971). Glyphosate is a post-emergent herbicide active by plant translocation. Plants treated with glyphosate translocate the systemic herbicide to their roots, shoot regions and fruit, where it interferes with the plant's ability to form aromatic amino acids necessary for protein synthesis. It is absorbed mainly through the leaves and rapidly moves throughout the plant, killing all parts of it. It acts by inhibiting a biochemical pathway, the shikimic acid pathway, which leads to the biosynthesis of aromatic compounds including amino acids (phenylalanine, tyrosine and tryptophan), plant hormones and vitamins. This pathway exists in higher plants and microorganisms but not in animals. Specifically, glyphosate inhibits the conversion of phosphoenolpyruvic acid (PEP) and 3- phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) (Franz, J.E., M.K. Mao, and J.A. Sikorski. 1997. Glyphosate: A unique global herbicide. ACS Monograph 189. Washington D.C.: American Chemical Society, U.S. Patent No. 5,804,425 ). Glyphosate can affect plant enzymes not connected with the shikimic acid pathway. In sugar cane, it reduces the activity of one of the enzymes involved in sugar metabolism. It also inhibits a major detoxification enzyme in plants. Glyphosate is metabolized or broken down by some plants, while other plants do not break it down. Aminomethylphosphonic acid is the main break-down product of glyphosate in plants. At low levels of application glyphosate acts as a growth regulator. Pure glyphosate is a colorless, odorless, crystalline solid with a melting point of 185 °C and decomposes at 187 °C producing toxic fumes including nitrogen oxides and phosphorus oxides. Pure glyphosate is slightly soluble in water (12 g/liter at 25 °C), and is practically insoluble in most organic solvents. U.S. Patent No. 3,969,398, U.S. Pat. No. 3,799,758, U.S. Pat. No. 3,927,080, U.S. Pat. No. 4,237,065, and U.S. Patent No. 4,065,491 describe different processes for preparing glyphosate. The parent acid of glyphosate has a negative charge, and salts with a positive charge are formulated with glyphosate to produce a finished product. Commercial formulations of glyphosate are available as the isopropylamine salt, the diammonium salt, and the trimethylsulfonium salt. Concentrations of the formulated products range from 3.57 lb./gal of the trimethylsulfonium salt to 5 lb./gal of the isopropylamine salt. Most glyphosate-based herbicides are also formulated with one or more surfactants. The surfactant spreads the solution across the leaf, penetrates the leaf and enhances the uptake of glyphosate by the plant(13). A class of surfactants known as polyoxyethylene tallowamines (POEA) are used most frequently. Glyphosate formulations are stable for extended periods below 60 °C. Glyphosate is one of the most toxic herbicides, with many species of wild plants being damaged or killed by applications of less than 10 micrograms per plant. About 0.3 to 4.0 pounds of active ingredient is used per acre. Method of application is usually via aerial spraying, spraying from a truck, backpack or hand-held sprayer, wipe application, frill treatment and cut stump treatment. Glyphosate is used to control a great variety of annual, biennial, and perennial grasses, sedges, broad-leafed weeds and woody shrubs. It is used in fruit orchards, vineyards, conifer plantations and many plantation crops (e.g. coffee, tea, bananas); in pre-crop, post-weed emergence in a wide range of crops (including cereals, vegetables, soybean, citrus, stone fruits, pine-apple, asparagus and cotton); on non-crop areas (e.g. turf, road shoulders and rights of way); in cereal stubble; forestry; gardening and horticulture. Other uses of salts of glyphosate are in growth regulation in peanuts and in sugarcane to regulate growth and speed fruit ripening (World Health Organisation, Food and Agriculture Organisation of the United Nations, Glyphosate, WHO/FAO Data Sheets on Pesticides No. 91, WHO/PCS/DS/96.91, July 1997). Glyphosate is sold around the world and is formulated into dozens of products by many pesticide companies. It is generally distributed as water-soluble concentrates and powders. There are three forms of glyphosate used as weed killers; glyphosate- isopropylammonium and glyphosate-sesquiodium patented by Monsanto and glyphosate- trimesium, patented by Zeneca. Trade names for products containing glyphosate include Accord®, Annada, ClearOut, Dardo, Fuste, Gallup, Kleenup®, Landmaster, MON-0573, Poledo® (sesquisodium), Pondmaster, Quotemeter, Ranger, Rodeo®, Roundup®

(isopropyl ammonium), Spasor®, Squadron, Sting®, Stirrup, Touchdown® (trimesium), Tumbleweed®, Vision and Wellop. Concentrations of glyphosate in products are usually 360g/liter or 450 g/liter in products approved for agricultural use and lOOg/liter or 3.6g/liter (ready to use) in products approved for home garden use. Roundup is the major formulation and contains 480 g mono (isopropylammonium) glyphosate/liter (equivalent to 360 g glyphosate/liter).

VI. Glufosinate Glufosinate is a short name for the ammonium salt, glufosinate-ammonium

[ammonium 4-[hydroxy(methyl)phosphinoyl]-DL-homoalaninate] (U.S. Patent No. 4,168,963). It was first reported as a herbicide in 1981. Glufosinate is non-selective herbicide and affects all actively growing green plants. The herbicidal property of glufosinate is due to L-phosphinothricin (PPT), an analogue of glutamate (Bayer et al, 1972; Lea et al, 1984). Glufosinate is the most potent known inhibitor of the enzyme glutamine synthetase (GS) (Devine et al., 1993). GS is critical to the assimilation of nitrogen by plants, and its inhibition leads to several immediate metabolic dysfunctions. GS converts glutamate into glutamine and is also involved in the pathways for detoxification of ammonia released by nitrate reduction, amino acid degradation or photorespiration. The application of glufosinate leads to a deficiency in glutamine (Tachibana et al., 1986a), rapid accumulation of ammonia in the plant tissues (Tachibana et al., 1986b) and also glyoxylate accumulation, which inhibits Rubisco and carbon fixation (Wendler et al., 1992). This causes photosynthesis to stop (Sauer et al., 1987) resulting in death of the plant cells within a few days (Bayer et al, 1972; Tachibana et al., 1986a, 1986b). Glufosinate is marketed under a number of trade names including Rely®, Finale®, Basta®, Ignite®, Liberty® and Challenge®. It is applied in spring or early summer when young weeds are actively growing. Method of application is by broadcast spray (0.25 to 0.75 lb. ai/acre; 2 to 6 qtper acre) or spot/directed spray (11.7 to 46.9 ml per liter water). It is absorbed through foliage with minimal translocation through xylem and phloem - depending on the application rate and species treated. Chlorosis and wilting usually occur within 3 to 5 days after application, followed by necrosis in 1 to 2 weeks. Rapidity of symptom development is increased by bright sunlight, high humidity, and moist soil. Glufosinate is a broad-spectrum contact herbicide and is used to control a wide range of weeds after the crop emerges or for total vegetation control on land not used for cultivation. Glufosinate herbicides are also used to desiccate (dry off) crops before harvest (e.g. at harvest of oilseed rape or potatoes or to desiccate grassland before sowing or resowing). It is used primarily to control annual and perennial weeds in crops like corn, canola, apples, grapes, peaches, pears, plums, strawberry, walnut, citruses, string bean, asparagus, tomato, cabbage, turnip, sugar beet, carrots, lettuce and onions.

VII. Sulfonylurea Herbicides Sulfonylureas are a family of environmentally compatible herbicides that were discovered in 1975 and first commercialized for wheat and barley crops in 1982. Members of this herbicide class are known for high toxicity toward plant growth at low application levels, short half-lives and low toxicities to animal species. Minute amounts of these herbicides are capable of disrupting plant reproduction processes, even without visible damage to the plant or its vegetative organs. All sulfonylureas share the same basic structure, containing an aryl ring linked to a heterocycle (triazine or pyrimidine) through the herbicidally active sulfonylurea (SU) 'bridge'. To increase herbicidal activity, the aryl ring is usually substituted 'ortho' to the sulfonylurea bridge, with the substituent typically a carboxylic acid ester (e.g. metsulfuron- methyl) or halogen (e.g. chlorsulfuron). Substituents on the heterocycle are typically alkyl or alkoxy, and can be partially halogenated. Certain sulfonylureas possess a thienyl (e.g. thifensulfuron-methyl) or pyridinyl (e.g. nicosulfuron and rimsulfuron) moiety rather than the analogous aryl ring. The basic SU molecular structure can be easily altered to produce many derivatives such that designer herbicides can be synthesized to target specific weeds in particular crops. Sulfonylureas work by inhibiting the plant-specific enzyme acetolactate synthase (ALS), which is required for the biosynthesis of branched-chain amino acids in bacteria, fungi and plants (Chaleff and Mauvias 1984). Branched-chain amino acids, like valine, leucine and isoleucine are required components of the growth processes of cell division. By blocking ALS and preventing branched-chain amino acid production, sulfonylurea herbicides rapidly inhibit cell division at the root and shoot tips. Sulfonylureas can be applied both 'pre' and 'post' emergence of the crop and are very effective in the control of a wide range of annual and perennial grasses, as well as broad leaf weeds. The high selectivity for the ALS enzyme, allows application rates of the g/ha level compared to the kg/ha level for conventional herbicides. Sulfonylureas are absorbed by foliage and roots. Once absorbed into the plant, they rapidly translocate acropetally from the root to the shoot, and basipetally from the shoot to the root , and inhibit growth at both locations. Studies detected cell division inhibition as quickly as one to two hours after application (Brown, 1990). However, whole-plant symptoms, such as vein reddening, leaf chlorosis and terminal bud death are not evident for several days. Usually one to three weeks are required from the time of application to complete control of the target weed (McCarty, 1997). Sulfonylurea family has been extensively developed and is cunently the most populous group of related herbicides with at least 35 of them cunently on the market. Few examples of the sulfonylureas include, but not limited to, chlorimuron (Classic) for soybean, primisulfuron (Beacon) for corn, thifensulfuron (Harmony Extra) for small grains, (Pinnacle) for soybean, triasulfuron (Amber) for small grains, nicosulfuron (Accent) for corn, metsulfuron (Ally) for small grains, grass pastures, and CRP, tribenuron (Express, Harmony Extra) for small grains, rimsulfuron (Matrix) for potato, and triflusulfuron (Upbeet) for sugar beet.

VIII. Amitrole Amitrole [3, Amino- 1,2,4-triazole] is a nonselective systemic triazole herbicide. Amitrole interferes with histidine biosynthesis inhibiting the enzyme imidazoleglycerol-phosphate dehydratase (IGPD) which leads to the accumulation of imidazoleglycerol (IG). It is available as soluble powders, soluble concentrates, suspension concentrates, water dispersible granules, liquid solutions, and wettable powders. Trade names include Amerol, Amino Triazole, Amitol, Amitrol, Amizine, A izol, Azaplant, Azolan, Azole, Campaprim, Cytrol, Diurol, Domatol, Emisol, Herbizole, Vorox, Weedazin and Weedazol. The chemical is absorbed by the foliage and roots and readily translocated to the meristematic zones. The most obvious response of plants treated with amitrole is albinism of the emerging leaves. However, amitrole causes cessation of growth before these leaves emerge. Amitrole has also been shown to decrease the levels of soluble protein and increase the free amino acid levels. In addition this herbicide causes a rapid decrease in the growth of roots and disrupts the development of chloroplasts. It is an effective herbicide on annual and perennial monocots and dicots. It is used on non- cropland for control of annual grasses and perennial and annual broadleaf weeds, for poison ivy control, and for control of aquatic weeds in marshes and drainage ditches.

IX. Seed Priming Presence of water is known to be the most important physiological factor needed to bring about seed germination. The purpose of seed priming is to reduce the germination time, make germination occur over a short period and improve stand and percentage germination. Seed priming involves allowing seeds to absorb sufficient water to initiate metabolic processes, but insufficient water to allow completion of germination. Different priming methods are known, such as osmo-priming (using liquid carriers of water e.g. Polyethylene glycol, glycerol mannitol, and Agro Lig), matrixpriming (using solid water carriers e.g. vermiculite compounds, Celite and Micro Cel) or hydropriming (using pure water). The principle of all priming methods is the same: pre-treatment of seeds in order to provide water in a controlled manner. Examples of priming procedures are known in the art and include drum priming and steep priming. After the priming treatment, the seeds can be dried, packaged, distributed and planted in the same way as untreated seeds. When the seeds are imbibed again, the lag period before radicle emergence occurs is considerably reduced, improving the rate and imiformity of germination. In addition, priming can overcome some types of environmental stresses, such as high temperature inhibition of lettuce seed germination. There may be little or no differences between primed and non- primed seed if the field conditions are closer to ideal. A disadvantage of priming is that in most cases, the storage life of the seeds is shortened after the treatment and the seeds are more sensitive to poor storage conditions.

X. Seed Coating Seed coating is a process designed to create a nutritious environment in the immediate vicinity of the germinating seed. The purpose of seed coating in particular is to improve the characteristics of germination, to provide various additives capable of playing a part at any time during the establishment and growth of plants, to provide protection against mechanical or environmental damage, to give the seed a shape or size that is suitable for automatic sowing, and to use such coatings as a carrier for various materials such as, for example, fertilizer, fungicides, herbicides, insecticides, etc. (U.S. Patent No. 5,876,739). Coated seed benefits the plant in its critical seedling stage thus ensuring early vigor and maximum establishment. Usually the procedure for seed coating comprises a) mixing one or more binders with one or more active ingredients, wherein the binder serves as a matrix for the active ingredients; b) applying the mixture to a seed; c) allowing the mixture to dry on said seed and/or d) applying a film overcoating to the seed. Active ingredients may include, but not limited to, fertilizers, micronutrients, plant growth regulators, fungicides, insecticides, herbicides and inoculants like nitrogen fixing Rhizobia or Myconhizal fungi. Amino acids, the active ingredients of the present invention may be used solely or in combination with other active ingredients. In view of the fact that the active ingredients generally will not adhere directly to the seed surface, which can vary in surface texture and the degree of hydrophobic or hydrophilic character, the active ingredients are usually dispersed or dissolved in a liquid coating composition which is applied to the seed. The active ingredient may be one or more types of microbes that produce amino acids which can be utilized by a plant. The binder component of the coating is composed preferably of an adhesive polymer that may be natural or synthetic and is without phytotoxic effect on the seed to be coated, eg. Vinamul. The binder may be selected from polyvinyl acetates, polyvinyl acetate copolymers (-ethylene), polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses, polyvinylpyrolidones, dextrins, maltodextrins, polysaccharides, fats, oils, proteins, gum arabics, shellacs, vinylidene chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, starches, polyvinylacrylates, zeins, gelatin, carboxymethylcellulose, chitosan, polyethylene oxide, acrylimide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylimide monomers, alginate, polychloroprene and syrups or mixtures thereof. Use of fillers in the coating is particularly effective for protecting the seed during stress conditions. Fillers for such formulations are known in the art and may include woodflours, clays, activated carbon, sugars, diatomaceous earth, cereal flours, fine-grain inorganic solids, calcium carbonate and the like. Clays and inorganic solids which may be used include calcium bentonite, kaolin, china clay, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite and mixtures thereof. Sugars which may be used include dextrin and maltodextrin. Cereal flours include: wheat flour, oat flour and barley flour. Prefened fillers include diatomaceous earth, perlite, silica and calcium carbonates and mixtures thereof. For example, a product containing diatomaceous earth and amorphous silica such as that manufactured by Celite Corporation (Celite™) is most prefened. Other recognized filler materials may be used depending on the seed to be coated and the materials used in the coating (U.S. Patent No. 5,876,739). Examples of seed coating or treatment techniques are found in the prior art. For example, U.S. Patent No. 4,735,015 discloses a seed protective coating using a film forming composition comprising polyoxyethylene-polyoxybutylene block co-polymers for controlling water uptake by the seed. U.S. Patent No. 3,911,183 discloses a seed coating process in which a seed is coated with polymer-pesticide film. Halogenated vinyl resin is used as a film former and pesticide carrier. U.S. Patent No. 3,698,133 discloses a plant seed with a multiple coating. The coating is in two layers, an inner porous coating permeable to water and an outer coating of a polymer with a controlled permeability to water. This patent discloses the use of additives to enhance particular functions of the plant. U.S. Patent No. 4,735,017 discloses a coated seed having inorganic additives in the coating. U.S. Patent No. 5,044,116 discloses a method for polymer coating of seed, which coating may include additives. Conventional means of coating may be used for canying out the coating of the invention. Additionally, various coating machines are available to one skilled in the art. Three well known techniques include the use of drum coaters, and fluidized bed techniques. Other methods, such as spouted beds may also be useful. The seeds may be presized prior to coating. After coating the seeds are dried and then optionally sized by transfer to a sizing machine. These machines are known in the art for example, a typical machine used when sizing seed corn in the industry. Film-forming compositions for enveloping coated seeds are well known in the art, and a film overcoating can be optionally applied to the coated seeds of the present invention. The film overcoat protects the coating layers and optionally allows for easy identification of the treated seeds. In general, additives are dissolved or dispersed in a liquid adhesive, usually a polymer into or with which seeds are dipped or sprayed before drying. Alternatively a powder adhesive can be used. Various materials are suitable for overcoating including but not limited to, methyl cellulose, hydroxypropylmethylcellulose, dextrin, gums, waxes, vegetable or paraffin oils; water soluble or water disperse polysaccharides and their derivatives such as alginates, starch, and cellulose; and synthetic polymers such as polyethylene oxide, polyvinyl alcohol and polyvinylpynolidone and their copolymers and related polymers and mixtures of these (U.S. Patent No. 5,876,739). Further materials may be added to the seed coat, such as a dye so that an observer can immediately determine that the seeds are treated. The dye is also useful to indicate to the user the degree of uniformity of the coating applied. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the prefened embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. XI. EXAMPLES

Example 1. General Procedure for Coating Seeds with Herbicide Reversing Amino Acids Healthy seeds are selected and approximate protein content of the seed is determined, using combustion method, as percentage of total dry weight. Other methods known in the art, for example the kjeldahal method, can be used to determine the protein content. Seed storage protein is purified from the seed and the percentage of each amino acid present in the seed storage protein is determined by measuring relative amounts of each amino acid type using HPLC or gas chromatography or other methods known in the art (D'Aniello et al, 1993). Alternatively, one could rely upon the known amino acid amounts of a particular plant species. For example, such information is provided in Tables 1, 2 and 3 for some plant species namely wheat, common vetch, chickpeas, Cyprus vetch, field beans, narbon vetch, peas and soybean meal. Seeds are planted in the soil and allowed to germinate. Wet weight, dry weight and protein content of the seedlings is determined at 1 week, 2 weeks, 3 weeks and 4 weeks after emergence. The amount of each amino acid required by the plant, to make adequate cellular protein for growth, up to these weekly stages is calculated by subtracting the amino acid content present in the unplanted seed. To recognize the amino acids that are not synthesized after herbicide treatment, seedlings are treated with a herbicide. Free amino acid pools are determined before and after the herbicide treatment, and over a specific period of time, such as a 2-week period after spraying. From the data obtained, it is determined when the pool of any amino acid approaches zero. This is the point at which the herbicide halts the protein synthesis. The ratio of amino acids needed for seed coating is calculated based on the ratio of amino acids used in protein synthesis during, for example, the 2-week protective period. The seeds are then coated with at least enough of those amino acids that are not synthesized during that period to provide a protective window of, for example, 2 weeks after herbicide treatment. For example, in the case of ALS, this reversal of inhibition by adequate supplies of valine, isoleucine, and leucine, depends on the life of the particular sulfonourea herbicide in a particular soil, as well as its retention in the active form in the plant. In addition to the active ingredients (i.e., one, two, three, etc. amino acids), the seed coating materials usually also consist of at least an inert powder and a binder. Alternatively or in addition to the amino acids coated on the seed, microbes can be applied to the seed wherein the microbes produce the required amount of the one or more amino acids. Coated seeds are sown in the soil and the amount of amino acid released from the coating into the spermosphere and seedling root zone is determined. According to the data obtained the seed coating content of the amino acids is re-adjusted to compensate for incomplete solubility. Coated seed are planted into soils with different moisture contents to test the effectiveness of the coatings under field conditions.

Example 2. Effects of Phenylalanine, Tyrosine, and Tryptophan on Glyphosate Reversal in Spring Wheat Spring wheat (Triticum aestivum) seeds were surface sterilized and washed with sterile water and dried. The seeds were then treated with seed coatings containing single aromatic amino acids or equal mixtures of two or all three aromatic amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) as indicated in Table 4. To a mixture of 2.3 ml of a liquid polymer-based film forming agent (Sentry Protective Seed Coating Polymer), 4.7 ml of water and 3.5 grams of seed coating powder (Seed Systems) was added 1 gram of each amino acid and this coating mixture was rolled with 14 grams of seed. The seed was dried and immediately planted into pots with premoistened and well-drained soil. Controls consisted of seeds without any seed coatings ('No Coat') and seeds with the same seed coating but without any added aromatic amino acids ('Seed Coating - No Amino Acid'). Two week-old seedlings (after planting) were sprayed with recommended rates of glyphosate (0.75% to 1.5%) on a calibrated spray table.. Two weeks later, the seedlings were evaluated for herbicide damage. The results are provided in Table 4.

Table 4. Effect of phenylalanine (F), tyrosine (Y) and tryptophan (W) on glyphosate reversal in spring wheat.

• Herbicide tolerance was rated on a scale of 0 to 3 where 0=Healthy (visible reversal of herbicide effect and 3=Dead (no visible reversal of herbicide effect). • These treatments are described in the text.

As shown in Table 4, seedlings supplied with exogenous amino acids through the applied seed coating showed reversal of herbicide damage. Seeds coated with phenylalanine and tyrosine showed near normal growth. Thus, the seed coatings with these amino acids reversed the inhibitory effect of glyphosate on the spring wheat seedlings.

Example 3. Effect of Three Different Levels of Phenylalanine and Tyrosine Coatings on Glyphosate Damage in Spring Wheat Spring wheat (Triticum aestivum) seeds were surface sterilized and washed with sterile water. The seeds were then treated with a single (IX), double (2X) or triple (3X) seed coating containing mixture of phenylalanine (Phe) and tyrosine (Tyr). The same controls were used as in Example 2 as presented in Table 5.

Table 5. Spring Wheat Seed Coatings

* FY = Phenylalanine plus Tyrosine (1:1 ratio)

The seeds were then planted in the soil and allowed to germinate. Two weeks after their emergence from the soil, the seedlings were sprayed with glyphosate (0.75% to 1.5%) and evaluated two weeks later for herbicide damage. The results for spring wheat are presented in Table 6 and Figure 1.

Table 6. Effect of three different levels of phenylalanine and tyrosine coatings on damage caused by post germination application of glyphosate in spring wheat.

* Herbicide damage was rated on a scale of 1 to 4 where l=Healthy and 4=Dead. Seeds without a seed coating or seeds with a seed coating without amino acids showed inhibition of plant growth when sprayed with post-emergent herbicide glyphosate. h contrast, seeds with a seed coating containing phenylalanine and tyrosine showed reversal of herbicide damage. It is also be noted that amount of herbicide damage decreases with increased level of amino acid coating. Seeds treated with triple coating of amino acids showed high percentage of healthy plants (Figure 1.) when compared to single or double seed coating. These results indicate that when significant enough quantities of amino acids are added to a seed coating, they are supplied to the plant when the seed germinates. This permits the plants to undergo normal protein synthesis, thereby circumventing the inhibition of the target enzyme by a post-emergence spray of herbicide.

Example 4. Reversal of Damage Caused by Herbicides of Sulfonylurea Family Sugar beet (Beta vulgaris) seeds can be coated with the amino acids valine, isoleucine and/or leucine by following the methods discussed elsewhere in this application. Sugar beet seedlings can then be sprayed with the herbicide, triflusulfuron (Upbeet). Two weeks later the seedlings can be observed for any damage caused by the herbicide. The results will show that sugar beet seeds coated with amino acids valine, isoleucine and/or leucine demonstrate reversal of herbicide injury whereas seedlings genninated from uncoated seeds would be injured or die. As discussed above, triflusulfuron belongs to a family of herbicide which kills plants by inhibiting the enzyme acetolactate synthase (ALS), an essential enzyme in the biosynthesis of valine, isoleucine and leucine. By supplying adequate amounts of these amino acids to sugar beet seed coating as determined by the methods disclosed herein, the damage caused by this herbicide can be reversed.

Example 5. Effects of Glutamine on Glufosinate Reversal Onion (Allium cepa) seeds can be coated with glutamine as discussed herein and allowed to germinate. Two weeks after emergence from the soil, the seedlings can be sprayed with recommended rates of glufosinate and observed for damage caused by the herbicide. The results will demonstrate that onion seeds coated with glutamine would show reversal of herbicide injury whereas seedlings germinated from uncoated seeds would show injury or death as caused by the herbicide. The germinating onion seeds and seedlings will uptake the glutamine present in the seed coating enabling them to perform normal protein synthesis and overcome the damage caused by glufosinate spray.

Example 6. Effect of Three Different Levels of Phenylalanine and Tyrosine Coatings on Glyphosate Damage in Soybean Soybean (Glycine max) seeds can be surface sterilized and washed with sterile water and dried. The seeds can then be treated with a single (IX), double (2X) or triple (3X) seed coating containing mixture of phenylalanine (Phe) and tyrosine (Tyr) as shown in Table 7 and allowed to germinate. Controls would consist of seeds without any seed coatings ('No Coat') and seeds with the same seed coating but without any added aromatic amino acids ('Seed Coating -No Amino Acid'). Two week-old seedlings can be sprayed with recommended rates of glyphosate (0.75% to 1.5%) on a calibrated spray table. Two weeks later the seedlings can be observed for any damage caused by the herbicide. The results will indicate that soybean seeds supplied with exogenous amino acids from the seed coating would show reversal of herbicide damage. Seeds treated with triple coating of amino acids will show high percentage of healthy plants when compared to single or double seed coating.

Table 7. Soybean Seed Coatings (See Table 5 for Details)

Example 7. Effect of Three Different Levels of Phenylalanine and Tyrosine Coatings on Glyphosate Damage in Pea

Pea (Pisum sativum cv A-14) seeds can be surface sterilized and washed with sterile water and dried. The seeds can then be treated with a single (IX), double (2X) or triple (3X) seed coating containing mixture of phenylalanine (Phe) and tyrosine (Tyr) as shown in Table 8 and allowed to germinate. Controls would consist of seeds without any seed coatings ('No Coat') and seeds with the same seed coating but without any added aromatic amino acids ('Seed Coating - No Amino Acid'). Two week-old seedlings can be sprayed with recommended rates of glyphosate (0.75% to 1.5%) on a calibrated spray table. Two weeks later the seedlings can be observed for any damage caused by the herbicide. The results will indicate that soybean seeds supplied with exogenous amino acids from the seed coating would show reversal of herbicide damage. Seeds treated with triple coating of amino acids will show high percentage of healthy plants when compared to single or double seed coating.

Table 8. A14 Pea Seed Coatings (See Table 5 for Details)

The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. All publications cited herein are incorporated herein by reference for the purpose of disclosing and describing specific aspects of the invention for which the publication is cited.

REFERENCES

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14. Forlani, G., Kafarski, P., Lejczak, B. and Wieczorek, P. 1997. Mode of action of herbicidal derivatives of aminomethylene bisphosphonic acid. II. Reversal of herbicidal action by aromatic amino acids. Journal of plant growth regulation 16 (3):147-151.

15. Forlani, G., Leiczalc, B. and Kafarski, P. 1999. The herbicidally active compound Ν- 2-(6-methyl-pyridyl)-aminomethylane bisphosphonic acid inhibits in vivo aromatic biosynthesis. Journal of plant growth regulation 18 (2):73-79.

16. Fulcher, R.G., O'Brien, T.P. and Simmonds, O.H. (1972). Localization of arginine- rich proteins in mature seeds of some members of gramineae. Aust.J.Biol.Sci. 25:487.

17. Gresshoff, P.M. 1979. Growth inhibition by glyphosate and reversal of its action by phenylalanine and tyrosine. Australian journal of plant physiology 6:177-187.

18. Gresshoff, P.M. 1979. Growth inhibition oi Arabidopsis thaliana by glyphosate and its reversal by aromatic amino acids. Arabidopsis infonnation service 16: 73-75.

19. Hadjipanayiotou M. and Economides S (2001) Chemical Composition, in situ Degradability and Amino Acid Composition of Protein Supplements Fed to Livestock and Poultry in Cyprus Livestock Research for Rural Development Volume 13, Number 6, December 2001 20. Hinton, J.J.C. (1953). The distribution of protein in the maize kernel in comparison with that in wheat. Cereal Chem. 30:441 21. Killmer, J., Widholm, J. and Slife, F. 1981. Reversal of glyphosate inhibition of canot cell culture growth by glycolytic intermediates and organic and amino acids. Plant physiology 68 (6):1299-1302. 22. Kishore, Ganesh M. and Shah, Dilip M. 1988. Amino acid biosynthesis inhibitors as herbicides. Annual Review of Biochemistry 57:627-663.

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24. MacMasters, M.M., Hinton, J.J.C. and Bradbury, D..(1971). Microscopic structure and composition of the wheat kernel. In 'Wheat: Chemistry and Utilization', ed. Y. Pomeranz, p.51. American Association of Cereal Chemists, St Paul, Mimi.

25. Miflin, B.J. 1971. Cooperative Feedback Control of Barley acetohydroxyacid synthetase by leucine, isoleucine, and valine. Archives of Biochemistry and Biophysics 146:542-550.

26. Pomeranz, Y., Carvajal, M.I., Hoseney, R.C. and Ward, A.B. (1970). Wheat germ in bread making. I. Composition of germ lipids and germ protein fractions. Cereal Chem. 47:373. 27. Romagni, J.G., Duke, S.O., and Dayan, F.E. 2000. Inhibition of plant aspargine synetase by monoterpene cineoles. Plant Physiology 123(2):725-732. 28. Sauer H, Wild A, Riihle W (1987) The effect of phosphinothricin (glufosinate) on photosynthesis. 11. The causes of inhibition of photosynthesis. Z Naturforsch 42c: 270-287 29. Shaner, C.L., Anderson, P. and Stidham, M.A. 1984. hnidazolines. Potent inhibitors of acetohydroxyacid synthase. Plant Physiology 76:545-546. 30. Tachibana K, Watanabe T, Seikizawa Y, Takematsu T (1986a) Inhibition of the glutamine synthetase and quantitatwe changes of free amino acids in shoots of bialaphos treated Japanese barnyard millet. :I Pestic Sci 11 : 27-31 31. Tachibana K, Watanaba T, Seikizawa Y, Takematsu T (1986b) Accumulation of ammonia in plants treated with bialaphos. J Pestic Sci 11 : 33-37 32. Vandiver N.N, and Teem D.H. (2002) General principles of weed management SSAGR 123, Dept. of Agronomy, Florida Cooperative extension Service, institute of Food and Agricultural Sciences, Univ. of Florida.

Claims

WHAT IS CLAIMED IS:

1. A method of protecting a plant from herbicide damage, said method comprising applying at least one amino acid to a seed of the plant, germinating the seed to produce a plant, and applying an herbicide to the plant, wherein the applied amino acid is an amino acid whose production by the plant is reduced or eliminated by action of the herbicide and wherein the amount of the applied amino acid is sufficient to sustain growth of the plant, thereby protecting the plant from herbicide damage.

2. The method of claim 1 , wherein the herbicide is selected from the group consisting of glyphosate, sulfonylureas, glufosinate and amitrole.

3. The method of claim 1, wherein the herbicide inhibits an enzyme selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), acetolactate synthase (ALS) , glutamine synthase and imidazoleglycerol-phosphate dehydratase.

4. The method of claim 1, wherein the amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan, valine, isoleucine, leucine, glutamine and histidine.

5. The method of claim 1, wherein the plant is a dicot or a monocot.

6. The method of claim 1, wherein two or more amino acids are applied to the seed.

7. A seed priming or coating for the seed of a plant to be treated with an herbicide, said seed coating prepared by a method comprising: (a), identifying what amino acid produced by the plant will be reduced or eliminated due to the action of the herbicide on the plant; (b). identifying the amount of the amino acid the plant will need in order to maintain growth over a time period equal to that during which the plant is exposed to the herbicide; and, (c). adding the amount of the amino acid identified in step (b) to the seed priming or coating for the plant, thereby preparing a seed coating for the plant to be treated with the herbicide.

8. The seed priming or coating of claim 7, wherein the herbicide is selected from the group consisting of glyphosate, sulfonylureas, glufosinate and amitrole.

9. The seed priming or coating of claim 7, wherein the herbicide inhibits an enzyme selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), acetolactate synthase (ALS), glutamine synthase and imidazoleglycerol- phosphate dehydratase.

10. The seed priming or coating of claim 7, wherein the amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan, valine, isoleucine, leucine, glutamine and histidine.

11. The seed priming or coating of claim 7, wherein the plant is a dicot or a monocot.

12. The seed priming or coating of claim 7, wherein the seed coating comprises two or more amino acids.

13. A method of protecting a plant from damage caused by an herbicide that disrupts amino acid production by the plant, said method comprising applying to the seed a priming or coating prepared according to claim 2 for the herbicide, germinating the seed, and applying the herbicide to the resultant seedling, thereby protecting the plant from herbicide damage.

14. The method of claim 13, wherein the herbicide is selected from the group consisting of glyphosate, sulfonylureas, glufosinate and amitrole.

15. The method of claim 13 , wherein the herbicide inhibits an enzyme selected from the group consisting of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), acetolactate synthase (ALS), glutamine synthase and imidazoleglycerol-phosphate dehydratase.

16. The method of claim 13, wherein the amino acid is selected from the group consisting of phenylalanine, tyrosine, tryptophan, valine, isoleucine, leucine, glutamine and histidine.

17. The method of claim 13, wherein the plant is a dicot or a monocot.

18. The method of claim 13, wherein the seed priming or coating comprises two or more amino acids.

19. A method of protecting a transplant from herbicide damage, said method consisting of applying at least one amino acid to a plant to be transplanted, transplanting the plant in a growth medium, and applying an herbicide to the transplant and/or growth medium, wherein the applied amino acid is an amino acid whose production by the transplant is reduced or eliminated by action of the herbicide and wherein the amount of the applied amino acid is sufficient to sustain growth of the transplant, thereby protecting the transplant from herbicide damage.

20. The method of claim 19, wherein the one or more amino acids are applied to the transplant by dipping roots of the plant in a solution containing the one or more amino acids.

21. A method of protecting a plant from herbicide damage, said method consisting of applying a microbe to seed of the plant, germinating the seed to produce a plant, and applying an herbicide to the plant, wherein the applied microbe produces an amino acid whose production by the plant is reduced or eliminated by action of the herbicide and wherein the amount of the amino acid produced by the microbe is sufficient to sustain growth of the plant, thereby protecting the plant from herbicide damage.