This application claims priority from IL Patent Application No. 238839, filed on May 14, 2015. The content of the above document is incorporated by reference as if fully set forth herein.
The invention relates, inter alia, to a delivery device and method for the application of active agents such as plant hormones to a plant.
Different levels of apical dominancy ensure continuous growth and plant design necessary for agriculture. This is true of vegetables, ornamental woody plants and fruit trees. In many of these plants one of the problems is vegetative growth of side branches, termed axillary branches, which require removing constantly. In olives and many other fruit trees, apical dominance is essential in the first stage of growth in the nursery (following rooting and hardening), for the development of one bud only to apical branch. However, in most cases, multiple buds develop simultaneously, thus requiring the investment of manpower for pruning these axillary branches. Other plants such as ornamental plants and fruit trees require defined architecture, which may add value to the determined product. In most of these plants it is necessary to create a section without axillary branching at the base of the stem. Prevention of branching of the plant requires considerable investment and farm work using valuable manpower.
Olive (Olea europaea L.), as a non-limiting example, is one of the most important tree crop species of the Mediterranean area. These are small tree or shrub that grows up to 8-15 meters tall. The fruit of olive trees are used as fruits or for olive oil, which is generally used for cooking, cosmetics and different medicinal applications. In total, about 11,000 tons of olive oil is produced annually. On the tree branches, nodule segments contain two leaves, in each a single bud. Inflorescence and flower differentiation occur in the early spring following a period of winter chilling and dormancy of the vegetative and reproductive buds.
Apart from their benefits, one of the difficulties with olive cultivation is obtaining desired growth architecture. Olive seedlings grow in a bushy structure, not desired at early stages of development. This natural growth architecture requires constant and costly trimming of axillary buds by the farmers.
Plant surfaces play a crucial role in protecting organs against an array of biotic and abiotic stress factors such as the uncontrolled loss of water. On the other hand, plant surface, being hydrophilic, provide permeability to liquids so as to enable absorption of, for example, liquid water, fog and dew. These will be influenced by the wettability and adhesion or repellence of water drops onto plant surfaces.
Natural strigolactones (SLs) are a group of plant hormones shown to act as long-distance branching factors, suppressing the outgrowth of axillary buds in the shoot. Hence, they are prominent effectors of apical dominance in plants. Present in a wide variety of plant species, SLs are derived from carotenoids and are biosynthesized through several steps. Root development of seedlings has been shown to be also regulated by SL activity. They are also involved in plant communication in the rhizosphere, and act as stimulants of parasitic plant (Striga and Orobanche) seed germination and as stimulants of arbuscular mycorrhizal fungi hyphal branching.
Several structural analogs of the naturally occurring strigolactones have been synthesized, and were found to retain the biological activity of the natural hormones. These synthetic compounds are easier to produce in large amounts compared to the natural strigolactones, and are therefore economically more suitable for large scale application to plants.
There is a long-felt need to control the level of branching in plants so as to provide many agricultural benefits, including reduced labor, improved product quality and increased profitability.
The invention relates to a polymeric composition comprising an active agent, a delivery device comprising same, and to a method for the application of active agents such as plant hormones to a plant.
According to one aspect, there is provided a composition comprising at least one polymer characterized by a surface energy having a value that ranges from 20 mJ/m2 to 60 mJ/m2, and an active agent. In some embodiments, the active agent is a plant hormone. In some embodiments, the plant hormone is at a concentration that ranges from 3 mg/L to 30 mg/L.
In some embodiments, the at least one polymer is selected from the group consisting of polyvinyl acetate (PVAc), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), hydroxy ethyl cellulose (HEC), polyvinyl pyrrolidone (PVP), polyethylene, and glycol (PEG), or any combination thereof. In some embodiments, the polymer is HPC. In some embodiments, the polymer is polyvinyl chloride (PVC).
In some embodiments, the composition further comprises a plasticizer. In some embodiments, the plasticizer is in the range of 30% to 50%, by weight of the composition. In some embodiments, the plasticizer is a polymer having a molecular weight (MW) of less than 1000 gr/mole. In some embodiments, the plasticizer is PEG. In some embodiments, the plasticizer is glycerin. In some embodiments, the polymer is HPC and the plasticizer is glycerin. In some embodiments, the polymer is PVC and the plasticizer is PEG.
According to another aspect, there is provided an article comprising the disclosed composition in any embodiment thereof. In some embodiments, the article is a delivery device. In some embodiments, the article is adapted for installation on a part of a plant, thereby enabling the application of the composition to the plant.
In some embodiments, the delivery device is configured to wrap the part of the plant.
In some embodiments, the delivery device has a cylindrical form, a tube form, a ring form or a clamp form.
In some embodiments, the delivery device is made of a rigid, semi-rigid or a flexible material, or any combinations thereof.
In some embodiments, the delivery device is made of a biodegradable polymer, composed of pure or blends of bio-plastics. In some embodiments, the biodegradable polymer comprises or is produced from corn starch, potato starch, agar, gelatin, PLA (polylactic acid) or PLGA (poly(lactic-co-glycolic acid)).
In some embodiments, the part of the plant is the stem, the bud, the root stock, the trunk, the stalk, or any part of the shoot of the plant.
In some embodiments, the active agent is a plant hormone being a compound of formula I:
In some embodiments, the plant hormone is a compound of formula II:
wherein
X is CH2, NH, or NR′;
Y is CH2 or O;
Z is CO or -(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-methylene;
Z′ is CO or -(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-methylene;
R is aryl, heteroaryl, NH2, NHR′, NR′2, or alkyl;
R′ is aromatic or heteroaromatic ring or alkyl;
or pharmaceutically acceptable salts thereof.
In some embodiments, the compound of formula I is selected from Strigol, Strigyl acetate, Sorgolactone, Orobanchol, Orobanchyl acetate, 5-Deoxystrigol, 2′-Epiorobanchol, Sorgomol, 7-Oxoorobanchol, Solanacol, Fabacyl acetate, Alectrol, 7-Hydroxyorobanchol, 7-Oxoorobanchyl acetate, and 7-Hydroxyorobanchyl acetate.
In some embodiments, the plant hormone is a strigolactone analog. In some embodiments, the strigolactone analog is selected from the group consisting of EGO10, GR24, and ST362.
In some embodiments, the plant hormone is EGO10, the medium compound is HPC and the plasticizing agent is glycerin.
According to another aspect, there is provided a method for applying a plant hormone to a plant, the method comprising providing the article or composition described herein to a plant. In one embodiment, the providing the composition is contacting the plant with the delivery device of the invention, the delivery device comprises the composition of the invention.
According to another aspect, there is provided a method for applying a plant hormone to a plant, the method comprising providing a delivery device adapted to contain a plant hormone formulation, and installing the delivery device on a part of the plant, wherein the plant hormone formulation is provided prior to and/or after the installation of the device.
In some embodiments, the plant hormone formulation is provided by filling the device.
In some embodiments, the method comprises sealing the delivery device containing the plant hormone formulation.
According to an aspect of some embodiments of the present invention, there is provided a composition comprising a natural strigolactone or a strigolactone analog, a polymeric compound, and a plasticizer, for use in decreasing or preventing axillary bud growth in a plant.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The invention relates to a composition, a delivery device and a method for the application of active agents, such as plant hormones, to a plant.
Specifically, an embodiment of the invention relates to application of compositions, or delivery devices comprising the compositions (also referred to as: “formulation”) comprising strigolactone and/or strigolactone analogs and at least one additive compound.
The Polymeric Composition
According to some embodiments, the present invention provides a composition comprising at least one polymer.
The terms “composition” and “polymeric composition” are used hereinthroughout interchangeably.
As used herein, the term “polymer” describes an organic substance having polymeric backbone composed of a plurality of repeating structural units (monomeric units) covalently connected to one another.
In some embodiments, the repeating structural units refers to alternating copolymers with regular alternating monomeric units (designated as “A1” and “A2”).
In some embodiments, the polymeric backbone comprises periodic copolymers with A1 and A2 units arranged in a repeating sequence (e.g., A1-A2-A1-A2-A2-A1-A1-A1-A1-A2-A2-A2).
In some embodiments, the polymeric backbone comprises statistical copolymers. As used herein, “statistical copolymers” are copolymers in which the sequence of monomer residues follows a statistical rule. In some embodiments, the polymeric backbone comprises block copolymers. As used herein, block copolymers comprise two or more homopolymer subunits linked by covalent bonds.
In some embodiments, the polymer is water-soluble (also referred to as: “hydrophilic”). In some embodiments, the polymer is water-insoluble (also referred to as: “hydrophobic”).
In some embodiments, the composition is hydrophilic.
Without being bound by any particular mechanism, the hydrophilicity of the composition may assist the mobility of hydrophobic active agent (incorporated within the composition) onto the composition's surface (e.g., in a sustained or controlled release manner).
In some embodiments, the composition comprises a hydrophilic polymer. In some embodiments, the composition comprises a hydrophobic polymer and a hydrophilic plasticizer. Non-limiting exemplary plasticizers are described hereinbelow.
In some embodiments, the term “water-insoluble” is defined to mean that less than e.g., 5 gr, 4 gr, 3 gr, 2 gr, 1 gr, 0.5 gr, 0.4, gr, 0.3 gr, 0.2 gr, or 0.1 gr of the polymeric domain is soluble in 100 gr of water. In some embodiments, the hydrophobicity characteristic is maintained at a defined range of temperature (e.g., 20° C. to 40° C.).
In some embodiments, the polymer is configured to be attachable to a plant surface. In some embodiments, the term “plant surface” is defined as the outermost structuring of part of a plant, formed by the topography of e.g., epidermal cells with its overlying cuticle and/or additional coverings, e.g. wax layers to the surface. In some embodiments, the term “part of a plant” refers to the stem, the bud, the root stock, the trunk, the stalk, or any part of the shoot.
In some embodiments, the water-soluble polymer(s) forms a layer (film) which adheres to the plant surface. The terms “film(s)” and “layer(s)” are used herein interchangeably and refer to a substantially uniform-thickness of a substantially homogeneous substance.
The chemistry and morphological properties of the films are discussed hereinbelow in the Example section. According to some embodiments of the present invention, the layer is homogenized deposited on a plant surface.
In some embodiments, the film of the disclosed polymer(s) is characterized by a thickness of 0.1 micron to 100 micron, e.g., 20 to 50 microns.
In some embodiments, the film of the disclosed polymer(s) is characterized by a thickness of 1 micron, 5 microns, 10 microns, 15 microns, 20 microns, 25 microns, 30 microns, 35 microns, 40 microns, 45 microns, 50 microns, 55 microns, 60 microns, 65 microns, 70 microns, 75 microns, 80 microns, 85 microns, 90 microns, 95 microns, or 100 microns, including any value and range therebetween.
In some embodiments, the composition and/or polymer of the invention is characterized by one or more specific properties. In some embodiments, the term “property”, or any grammatical derivative thereof, refers to properly of the surface. In some embodiments, the term “property” refers to desired permeability. In some embodiments, the term property refers to wettability. In some embodiments, the term “property” refers to adhesive affinity to the plant surface. In some embodiments, the term “property” refers to hydrophobicity. In some embodiments, the term “property” refers to biocompatibility of the composition and/or polymer. By “biocompatibility” it is meant that the composition will sustain the growth of living plant tissue.
In some embodiments, the term “property” refers to dirt and dirt resistance.
In some embodiments, the term “property” refers to mechanical performance of the polymer, e.g., its being film forming polymer.
In some embodiments, the mechanical performance relates to flexibility.
In some embodiments, the term “property” refers the surface energy of the polymeric composition. As used herein and in the art, the term “surface energy” is the energy associated with the intermolecular forces at the interface between two media (e.g., surface and surrounding air). In some embodiments, the surface energy per unit area (also termed “surface free energy”) equals the surface tension.
The surface free energy (SFE) of the pepper fruit plant was found to be 30-36 mJ/m2 (see: Front. Plant Sci. 2015; 6:510) which is a median value between hydrophobic and hydrophilic surfaces.
Without being bound by any particular theory, it is assumed that in order for a plastic film to adhere to a plant stalk, the film's SFE should be approximately (e.g., up to ±30%) equal to that of the stalk. Hydrophobic films with SFE much below this value will be repulsed or rejected by the plant stalk.
In some embodiments, the polymeric composition is characterized by a surface free energy that ranges from 20 to 70 mJ/m2, 20 to 30 mJ/m2, 20 to 40 mJ/m2. For example, the disclosed polymeric composition is characterized by a surface free energy of e.g., 20 mJ/m2, 25 mJ/m2, 30 mJ/m2, 35 mJ/m2, 40 mJ/m2, 45 mJ/m2, 50 mJ/m2, 55 mJ/m2, 60 mJ/m2, 65 mJ/m2, or 70 mJ/m2, including any value and range therebetween.
In some embodiments, the polymeric composition is substantially devoid of cracking defects. As used herein, the term “cracking defects” refers to cracks that are at least observable with the naked eye within and/or at the surface of the polymeric composition.
Therefore, typically, but not exclusively, the composition comprising the polymer and optionally the plasticizer is characterized by a sufficiently flexible so as to be wound around e.g., 1-5 mm, 5-10 mm, 10-20 mm, 20-50 mm, or 50-100 mm, plant structure (e.g., stalk) without fracture or break. Methods for assessing the flexibility at break are known in the art (see e.g., ASTM. F137-08(2013).
In some embodiments, the composition is substantially devoid of water. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 5% of water, by (total) weight. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 4% of water, by (total) weight. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 3% of water, by (total) weight. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 2% of water, by (total) weight. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 1% of water, by (total) weight. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 0.5% of water, by (total) weight. In some embodiments, by “substantially devoid of water” it is meant that the composition comprises less than 0.1% of water, by (total) weight.
In some embodiments, the polymer is selected from polyacryls and polyesters.
Non-limiting examples of polymers useful according to the invention are polyvinyl acetate (PVAc), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), hydroxy ethyl cellulose (HEC), and polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG) and polyvinyl chloride (PVC). Any combinations of the different polymers in a single composition (e.g., formulation) are also encompassed by the invention.
Table 1 below provides the chemical structures of non-limiting exemplary polymeric compounds that may incorporate active agents (e.g., strigolactones as described below), for enhancing and prolonging their biological activity on the plant.
Further non-limiting examples of polymers include both hydrophobic and hydrophilic polymers. Non-limiting examples of hydrophobic polymers include, but are not limited to, ethyl cellulose and other cellulose derivatives, fats such as glycerol palmito-stereate, polymethylmethacrylate, beeswax, glycowax, castorwax, carnaubawax, glycerol monostereate or stearyl alcohol, hydrophobic polyacrylamide derivatives and hydrophobic methacrylic acid derivatives, as well as mixtures of these polymers.
In some embodiments, the polymer (e.g., PEG) is characterized by a weight average molecular weight (Mw; in grams/mole) of at least e.g., 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000, including any range therebetween.
In some embodiments, the term “weight average molecular weight” generally refers to a molecular weight measurement that depends on the contributions of polymer molecules according to their sizes.
Hydrophilic polymers include, but are not limited to, hydrophilic cellulose derivatives such as methyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose and hydroxyethyl methyl-cellulose polyvinyl alcohol, polyethylene, polypropylene, polystyrene, polyacrylamide, ethylene vinyl acetate copolymer, poly-urethane, polyvinylpyrrolidone, polyvinyl acetate, polyacrylamide, polymethacrylic acid, as well as mixtures of these polymers. Furthermore, any mixture of one or more hydrophobic polymer and one or more hydrophilic polymer may optionally be used.
One of the criteria for selection of the various units of the polymer as presented herein, exhibiting suitable functionalities in accordance to the requirement. For example, the disclosed composition may comprise one or more water-sensitive reagents (agents).
Depending on the agent desired to be attached to the polymer, the polymeric composition's properties can be pre-determined so as to allow the formation of the desired interactions between the agent and the polymer.
In some embodiments, these properties can be determined by virtue of the side chains of the polymer by virtue of the polymer backbone, or by virtue of an additive being incorporated within the polymer.
In some embodiments, for attaching an agent via hydrophobic interactions, polymeric composition comprising hydrophobic additive are prepared.
In some embodiments, for attaching a chiral agent, optically active polymers having a complementary stereoselectivity may be used.
The Additive
Accordingly, the composition according to the present invention may further comprise at least one additional component, also referred to herein as “additive”.
The terms “additive”, “plasticizer”, “stabilizer” and “carrier” are used hereinthroughout interchangeably.
According to some embodiments of the invention, the additive is a hydrophobic compound which plasticizes the polymer. In some embodiments, the composition comprises a hydrophilic polymer or a hydrophilic additive. According to some embodiments of the invention, the additive is a hydrophilic compound which plasticizes the polymer.
In some embodiments, the terms “stabilizing compound”, or “additive” as used herein refer to a natural or synthetic material that is combined with the active agent of the invention, e.g., to increase its durability and to increase its penetration to the plant.
According to still an additional aspect of the present invention there is provided a composition comprising a polymer, as described herein, having attached to or incorporated thereto an additive and an active agent.
The additive may allow to achieve higher mobility of active agent(s) to the outer surface of the composition. The mobility, in some embodiments, depends on the morphology of the polymer e.g., presence of defects, chain conformation or one of the properties described hereinabove.
In some embodiments, the term “mobility” refers to the solubility of the active agent(s) in the additive. As used herein, the term “solubility” refers to the ability of the active agents of the present invention to dissolve without substantial aggregation.
In some embodiments, the carrier compound increases the stability of the natural or synthetic hormone, thereby supporting its effective application to the plant.
In some embodiments, the composition imparts long-lasting and stable medium for preserving the active agent.
As used hereinthroughout, the term “stable”, or any grammatical derivative thereof, may refer to chemical stability. In some embodiments, the term “chemical stability” means that an acceptable percentage of degradation of the active agent (e.g., plant hormone) structure disclosed hereinthroughout by chemical pathways such as oxidation or hydrolysis is formed. In particular, the active agent is considered chemically stable if no more than e.g., about 30%, or about 20% breakdown products are formed after e.g., two weeks of storage at the intended storage temperature of the product (e.g., at room temperature, i.e. 15° C. to 40° C.).
In some embodiments, the term “stable” refers to biological stability. “Biological stability” as used herein means that the active ingredients maintain their biological activity.
In a specific embodiment, the strigolactones or strigolactone analogs and the formulations comprising them, are stable for at least 2 to 6 weeks and up to at least one year.
In some embodiments, the stabilizing compound is basically inert, and serves as a medium for maintaining the biological activity of e.g., the plant hormones by extending their half-life and durability.
Further to the active agent, and optionally the carrier, the compositions the invention may also comprise another plasticizing agent which serves as a solidifying substance. The plasticizer compound included in the formulation may be selected depending, inter alia, on the specific properties of the plant hormone presents in the polymeric mixture.
Table 2 below lists non-limiting examples of hydrophilic and hydrophobic plasticizers, useful according to the invention.
In some embodiments, the additive is a low Mw polymeric compound (e.g., PEG).
In some embodiments, low Mw polymeric compound is characterized by a molecular weight (Mw; grams/mole) of less than e.g., 10,000, 5,000, 4,000, 3,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, including any range therebetween.
In some embodiments, the composition comprises PVC and PEG (e.g., PEG 600, i.e. having Mw of 600 gr/mole). In some embodiments, the composition comprises PVC and e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% PEG, by weight, including any value and range therebetween. In some embodiments, the composition comprises PVC and e.g., 40% to 60% PEG, by weight, including any value therebetween.
In some embodiments, the polymer(s), additive(s) and the active agent(s) are attached to each other in a non-covalent manner. By “non-covalent manner” it is meant to refer to binding, by any non-covalent interaction, to another molecule, ion, complex or substance. The non-covalent interactions include, but are not limited to, ionotropic interaction, complexation interaction, electrostatic interactions, hydrogen bonds, receptor-substrate interactions, or any other non-covalent crosslinking and combinations thereof.
According to a specific embodiment of the invention, the plasticizing compound is glycerin (also termed “glycerol”).
In some embodiments, the polymeric composition comprises polysaccharide. In exemplary embodiments, the polysaccharide is selected from amylose, amylopectin, or a mixture thereof. In some embodiments, the composition comprises amylose, amylopectin or a derivative thereof and glycerin. In some embodiments, the composition comprises cellulose or a derivative thereof (e.g., HPC) and glycerin.
In some embodiments, the composition comprises e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% glycerol, by total weight, including any value and range therebetween. In some embodiments, the composition comprises e.g., 30% to 40% glycerol, by total weight. In some embodiments, the composition comprises e.g., 40% to 50%, glycerol, by total weight. According to a specific embodiment, the composition comprises about 40% glycerol, by total weight.
One specific formulation according to another embodiment comprises HPC and glycerin together with an active agent such as plant hormone (e.g., strigolactone) as described hereinbelow. As described hereinbelow, this formulation may have a marked effect on plants grown under greenhouse conditions.
Another specific formulation according to some embodiments of the invention comprises an active agent (e.g., strigolactone such as EGO10) together with PVAc.
The concentration of the active agent to be effective biological control agents may vary depending on the end use, physiological condition of the plant.
In some embodiments, the concentration of the active agent in the composition of the invention (e.g., strigolactone such as EGO10) is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, or 500 μM, including any value and range therebetween. In some embodiments, the concentration of the active agent in the composition of the invention range from 10 μM to 100 μM.
In some embodiments, the concentration of the active agent in the composition is (in ppm or mg/L) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, including any value and range therebetween.
In some embodiments, the present invention provides a composition comprising strigolactone or a derivative thereof wherein a portion of the active agent (e.g., strigolactone or its derivative) is formulated for sustained and/or controlled release and a portion of the active agent (e.g., strigolactone or its derivative) is formulated for immediate release when contacting the plant.
In some embodiments, effective levels of the active agent diffused into the plant are achieved within from about 10 minutes to about 20 or 30 or 40 or 50 or 60, 90 minutes, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h following contacting the composition with the plant.
In some embodiments, effective levels of the active agent diffused into the plant are achieved within from about 1 day, two days, 3 days, 5 days, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, including any value therebetween.
In some embodiments, the compositions used by the invention may be formulated to release up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% of the total active agent (e.g., strigolactone or its derivative) in about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours.
Delivery Device
According to some embodiments, the present invention provides an article comprising the disclosed composition.
In some embodiments there is provided an article for the application of plant hormone formulations to a plant.
According to some embodiments, the article is a delivery device e.g., for the application of plant hormones to a plant. The delivery device may be adapted for installation on a part of a plant, thereby enabling the application of a plant hormone formulation to the plant.
According to one embodiment of the invention, the plant hormone formulation provided by the delivery device is filled after the installation of the device on the part of the plant. According to another embodiment, the delivery device is filled with the plant hormone formulation prior to the fasten of the device on the plant.
The delivery device of the invention, also termed “collar” or “ring”, is, in some embodiment, a container, a case, or a cover, in any suitable form, configured to wrap the part of the plant.
The delivery device may be capable of containing the formulations of the invention, and may enable the long-term application of the plant hormone to a specific locus on the treated plant.
The delivery device may comprise one or more polymers (in some embodiments, in addition to the polymeric composition) used to produce the delivery device. Non-limiting examples of the polymer used to produce the delivery device are corn starch, potato starch, agar, gelatin, PLA (polylactic acid) and PLGA (poly(lactic-co-glycolic acid)).
Non-limiting examples of suitable forms of the delivery device are a cylindrical form, a tube form, a ring form or a clamp form. In some embodiments, the delivery device according to the invention is made of a rigid, semi-rigid or flexible material, or any combinations thereof.
According to one embodiment, the delivery device is made of a biodegradable polymer, composed of pure or blends of bio-plastics with different plasticizers, selected from, but are not limited to, the list in Table 2 above. The term “pure” as used herein refers to one kind of polymer, and the term “blend” refers to a mixture of two or more polymers in different ratios.
According to a one embodiment of the invention, the delivery device is a paraffin film, also referred to herein as “parafilm”, which is used to wrap the composition according to the invention that is placed at a specific locus on the treated plant.
According to a another embodiment of the invention, the delivery device is a flexible plastic tube, that is threaded on the part of the plant, (e.g., on the stem or bark of the plant), and fixed on the desired locus by a transparent adhesive tape. After the installation of the plastic tube on the plant, it is filled with the formulation of the invention, and optionally sealed from both ends by cotton to prevent the leakage of the formulation and to minimize water evaporation.
According to a further embodiment of the invention, the delivery device is a rigid plastic tube, such as an Eppendorf tube. In some embodiments, the tube is cut from top to bottom and placed on the part of the treated plant (i.e., the stem). The tube is then sealed with a small moist cotton at the bottom, filled with the formulation of the invention, and finally covered with a transparent adhesive tape.
In some embodiments, delivery device for an active agent to a plant is made from the disclosed polymeric composition.
In some embodiments, the delivery system used by this invention may be administered in controlled release formulations. For example, in some embodiments, the strigolactone or derivative thereof may be formulated for immediate release upon contacting the plant. In some embodiments, the delivery device may be formulated for sustained and/or controlled release, and may optionally be formulated to have both immediate release and sustained and/or controlled release characteristics upon contacting the plant.
In some embodiments, the active agent (e.g., strigolactone or its derivative) in a composition used by the invention may be formulated to release not less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% of the total active agent (e.g., strigolactone or its derivative) in about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours, including any value therebetween.
In some embodiments, the polymeric composition is used to sustain or control the release of active agent (e.g., strigolactone or its derivative). In certain embodiments, the type of polymeric material and the amount of which is used, has a strong influence on the rate of release of active agent (e.g., strigolactone or its derivative) from the product of the present invention.
The delivery device according to the invention is adapted to be implemented on the part of the treated plant (i.e., the stem or the bark) for any period of time. The application period of the formulations by the delivery device may diverge according to the species and growth stage of treated plant, and with respect to the specific desire of the cultivator.
According to one embodiment of the invention, the delivery device is maintained on the plant for any time between one hour and 100 days. According to another embodiment of the invention, the delivery device is maintained on the plant for between four days and 20 days. According to a specific embodiment of the invention, the delivery device is maintained on the plant for 12 days.
The delivery device and the plant hormone formulation of the invention may be applied at any stage of the plants growth, starting from the first stage of growth in the greenhouse or nursery after the rooting of the seedling, after the hardening of the stem and throughout the entire life time of the mature plant.
Typically, but not exclusively, the delivery device is designed for a single use, and is removed (actively or spontaneity) from the plant after the plant hormone formulation is almost or completely absorbed by the plant. Optionally, once the amount of the formulation comprised in the delivery device decreases, the cultivator can refill the delivery device with an additional amount of the formulation, or remove the empty device and apply another full delivery device onto the plant.
In some embodiments, the delivery device is implemented on any part of the plant, for example, on the stem, on the bud, on root stock, on trunk, stalk or any part of the shoot.
Plant Hormones
In some embodiments, the active agent is a signaling molecule. In some embodiments, the signaling molecule is a plant signaling molecule. In some embodiments, plant signaling molecules refer to molecules that stimulate e.g., cell differentiation, fruit ripening, plant cell elongation, stem elongation and onset of dormancy.
In some embodiments, the active agent is hydrophobic.
In some embodiments, the plant signaling molecule is a plant hormone, e.g., a hydrophobic plant hormone. The term “plant hormone” as used herein refers to plant growth regulators. Non-limiting examples of plant hormones suitable for application according to the invention are auxin (for regulation of root development), cytokinin (for regulation of shoot branching), gibberellins (for regulation of shoot elongation), and any functional derivatives and analogs thereof.
According to a specific aspect of the invention, the plant hormone is strigolactone, a strigolactone analog, or any functional derivative thereof. It is noted that “strigolactones” as used herein, includes, in some embodiments, all forms of natural strigolactones, including, their pre-form, prodrugs, derivatives, recombinants, or any acceptable form thereof.
In some embodiments, by “active agent” it is meant to refer to two or more active agents. In exemplary embodiments, the active agent comprises strigolactone (e.g., EG10) and auxin.
It is noted that “strigolactone analogs” as used herein, includes, in some embodiments, all forms of strigolactones, including, their pre-form, prodrugs, derivatives, recombinants, or any acceptable form thereof which have activity similar to native strigolactones.
In one embodiment, the plant hormone is a compound of formula I:
wherein:
or a pharmaceutically acceptable salt thereof.
Accordingly, the present invention encompasses the use of natural strigolactone hormones.
In a specific embodiment of the invention, the natural strigolactone hormone is selected from the following:
In another embodiment, the plant hormone the plant hormone is a compound of formula II:
wherein
R′ is aromatic or heteroaromatic ring or alkyl;
or individual isomer, pharmaceutically acceptable salt thereof, or a mixture thereof
The examples to follow illustrate the beneficial effects of the compounds of formula II, referred to herein as “strigolactone analogs”.
The compounds of formula II may be present as mixtures of diastereoisomers or as a racemic mixture or as pure isomers, optionally as enantio-pure isomers, that is, individual isomers or mixture of isomers thereof.
Table 3 below lists examples of strigolactone analogs useful according to the present invention, together with their chemical names and given codes.
According to a specific embodiment of the invention, the compound of formula II is selected from EGO10, GR24, and ST362, e.g., having the following formulae:
The plant hormone formulations according to the invention, also referred to herein as the “polymeric composition”, include at least one plant hormone or any synthetic analog thereof (i.e., a natural strigolactone or a strigolactone analog), as the active agent, a compound that serves as the medium, and optionally a plasticizing agent.
As described hereinabove, in some embodiments, the formulations are provided to the plant via a delivery device as described hereinabove.
In some embodiments, the formulations according to the invention further comprises additional solid or liquid materials in accordance with the general formulation techniques acceptable in the field.
In some embodiments, the active strigolactone compound and the medium compound present in the compositions according to the invention are applied to the plant in any ratio between the two.
In some embodiments, compositions comprising a plasticizer, such as a formulation comprising EGO10, HPC and glycerin, are applied to the plant in any ratio between the three.
Method for Applying a Plant Hormone
According to another aspect, there is provided a method for applying an active agent (e.g., a plant hormone) to a plant, the method comprising providing the article or composition described herein to a plant. In one embodiment, the providing the composition is contacting the plant with the delivery device of the invention, the delivery device comprises the composition of the invention.
In another aspect, the present invention provides a method for applying a plant hormone formulation to a plant. In some embodiments, the method comprises
In some embodiments, the method comprises providing a delivery device adapted to contain a plant hormone formulation (i.e. the hereinabove disclosed composition), and installing the delivery device on a part of the plant.
In some embodiments, the plant hormone formulation is provided prior to or after the installation of the device on the plant. According to a specific embodiment, the plant hormone formulation is provided in the delivery device prior to its fastening on the plant, and refilled after a desired period of time. In another embodiment, the method includes the step of sealing the delivery device, containing the plant hormone formulation, optionally by a piece of tape.
The plant hormone applied according to the method of the invention may be any plant growth regulator and/or plant hormone, selected for example, from auxin, cytokinin, gibberellin, strigolactone, and any analog thereof.
In some embodiments, the method is specifically suitable for the application of a strigolactone, a strigolactone derivative or a strigolactone analog to a plant, for decreasing or preventing axillary bud growth.
According to one embodiment of the invention, the formulation provided to the plant by the delivery device is a strigolactone, a strigolactone derivative or a strigolactone analog together with a medium compound. According to another embodiment of the invention, the formulation further comprises a plasticizing agent (plasticizer) as described hereinabove.
According to a specific embodiment, the strigolactone or strigolactone analog is selected from EGO10, GR24, and ST362, the composition comprises a compound is selected from polyvinyl acetate (PVAc), polyvinyl alcohol (PVA), hydroxypropyl cellulose (HPC), hydroxy ethyl cellulose (HEC), polyvinyl pyrrolidone (PVP), or any combinations thereof, and the plasticizing agent is glycerin.
In some embodiments, plants that can be treated with natural or synthetic strigolactone formulations according to the present invention include any plant that can benefit from enhancing its epical dominancy, including woody plants such as fruit trees and ornamental plants, non-woody plants such as vegetables and herbs. Non-limiting examples of plants suitable for the practice of the present invention are olives, Hypericum, roses, tomatoes, pepper, and grapes.
In some embodiments, the formulations according to the present invention comprising a strigolactone or strigolactone analog in a polymeric compound (and optionally at least one plasticizing agent as described herein) may further comprise additional pharmaceutically accepted additives or excipients.
The additives or excipients assist the penetration of the active agent to the plant, preserve the active agent and inhibit its degradation. These activities prolong the biological activity and increase the efficacy of the compounds of the invention. Excipients that can be employed include any excipients known in the art for the preparation of formulations.
In some embodiments, the composition according to the invention is non-toxic and harmless to both the plant and the environment.
According to another aspect, the present invention provides an easy and cost effective method for designing the architecture of a plant by enhancing its apical dominancy, and suppressing axillary bud (e.g., wake-up bud) growth, thereby preventing the development of non-desired axillary branches.
Consequently, the method of the invention dramatically reduces the time and labor required during the entire growth period of the plant, and enhances the yield, growth or vigor of the treated plant, thus increases the profitability of the crop.
In another aspect, the invention provides a method for decreasing or preventing axillary bud growth in a plant, characterized in that a delivery device comprising at least one strigolactone, strigolactone derivative or strigolactone analog compound, optionally in combination with at least one plasticizer agent, is applied to a plant.
Accordingly, in some embodiments, the method of the invention mainly consists of contacting one or more of the formulations of the invention to the plant via a delivery device.
The term “wake-up bud” as used herein refers to axillary buds that are in the process of growing.
As used herein, the term “alkyl” describes an aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 21 to 100 carbon atoms, and more preferably 21-50 carbon atoms. Whenever a numerical range; e.g., “21-100”, is stated herein, it implies that the group, in this case the alkyl group, may contain 21 carbon atom, 22 carbon atoms, 23 carbon atoms, etc., up to and including 100 carbon atoms. In the context of the present invention, a “long alkyl” is an alkyl having at least 20 carbon atoms in its main chain (the longest path of continuous covalently attached atoms). A short alkyl therefore has 20 or less main-chain carbons. The alkyl can be substituted or unsubstituted, as defined herein
The term “alkyl”, as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl.
The term “alkenyl” describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.
The term “alkynyl”, as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.
The term “cycloalkyl” describes an all-carbon monocyclic or fused ring (i.e. rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted, as indicated herein.
The term “aryl” describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted, as indicated herein.
The term “alkoxy” describes both an —O-alkyl and an —O-cycloalkyl group, as defined herein.
The term “aryloxy” describes an —O-aryl, as defined herein.
Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule. Additional substituents are also contemplated
The term “halide”, “halogen” or “halo” describes fluorine, chlorine, bromine or iodine.
The term “haloalkyl” describes an alkyl group as defined herein, further substituted by one or more halide(s).
The term “haloalkoxy” describes an alkoxy group as defined herein, further substituted by one or more halide(s).
The term “hydroxyl” or “hydroxy” describes a —OH group.
The term “thiohydroxy” or “thiol” describes a —SH group.
The term “thioalkoxy” describes both an —S-alkyl group, and a —S-cycloalkyl group, as defined herein.
The term “thioaryloxy” describes both an —S-aryl and a —S-heteroaryl group, as defined herein.
The term “amine” describes a —NR′R″ group, with R′ and R″ as described herein.
The term “heteroaryl” describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
The term “heteroalicyclic” or “heterocyclyl” describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.
The term “carboxy” or “carboxylate” describes a —C(═O)—OR′ group, where R′ is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon) as defined herein.
The term “carbonyl” describes a —C(═O)—R′ group, where R′ is as defined hereinabove.
The above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).
The term “thiocarbonyl” describes a —C(═S)—R′ group, where R′ is as defined hereinabove.
A “thiocarboxy” group describes a —C(═S)—OR′ group, where R′ is as defined herein.
A “sulfinyl” group describes an —S(═O)—R′ group, where R′ is as defined herein.
A “sulfonyl” or “sulfonate” group describes an —S(═O)2—R′ group, where Rx is as defined herein.
A “carbamyl” or “carbamate” group describes an —OC(═O)—NR′R″ group, where R′ is as defined herein and R″ is as defined for R′.
A “nitro” group refers to a —NO2 group.
A “cyano” or “nitrile” group refers to a —C≡N group.
As used herein, the term “azide” refers to a —N3 group. The term “sulfonamide” refers to a —S(═O)2—NR′R″ group, with R′ and R″ as defined herein.
The term “phosphonyl” or “phosphonate” describes an —O—P(═O)(OR′)2 group, with R′ as defined hereinabove.
The term “phosphinyl” describes a —PR′R″ group, with R′ and R″ as defined hereinabove.
The term “alkaryl” describes an alkyl, as defined herein, which substituted by an aryl, as described herein. An exemplary alkaryl is benzyl.
The term “heteroaryl” describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.
As used herein, the terms “halo” and “halide”, which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine or iodine, also referred to herein as fluoride, chloride, bromide and iodide.
The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide(s).
General:
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. The term “consisting of means “including and limited to”. The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.
The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
In those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
The effect of application of various analogues of strigolactone (SL) on olives, given to roots by irrigation was examined. Three different SL analogs; GR24, EGO10 and ST362 were applied at a concentration of 0 (control), 0.03 and 0.3 μM to olive seedlings during vegetative growth. The seedlings were rooted from cuttings and hardened, and were about 6 months old at the time of the experiment. Growth soil mixture was used as in the nursery. Each strigolactone analog was dissolved in acetone to give a 5.6 mM solution. This solution was then further diluted with distilled water to 0.03 and 0.3 μM. The diluted solution was injected to the roots twice a week for 6 weeks, and subsequently all the growing buds were counted. For each treatment (treated and control groups) 10 plants were measured. Experiments were repeated three times.
The results show that application of GR24 lead to a significant reduction in number of lateral branches. The effect was most prominent at a concentration of 0.03 μM (
The implementation of the strigolactone analogs necessitates a degree of material stability, to ensure their activity on the plant under various growth conditions. Accordingly, the stability of the strigolactone analog GR24 was tested under different environmental conditions. GR24 in acetone stock solution of 5.6 mM was diluted in double distilled water (DDW) or HPC pre-made gel to a concentration of 0.56 μM in eppendorf tubes and was subjected to the following treatments:
a. Temperature Range of 4 to 60° C. for Periods of 1 Hour to 7 d:
GR24 solutions of 0.56 μM, diluted either in DDW or in HPC pre-made gel in eppendorf tubes were incubated in a dry block heater at temperatures of 4° C., 20° C., 40° C., 60° C. digitally set for periods of 3 h, 1 d, 2 d, 4 d, 5 d and 7 d under ambient light.
b. UV Radiation:
GR24 solutions of 0.56 μM, diluted either in DDW or in HPC pre-made gel, each in quartz tubes (15 replicates for each treatment) were exposed to UV radiation of λ=312 nm under UV lamp of 600 or 857 μE intensity for periods of 15 min, 30 min or 18 h.
Biological activity was determined by Orobanche Germination Assay: Approximately 30 to 50 seeds of Orobanche aegyptiaca were spread on a glass fiber filter paper disk (9-mm diameter) and put into sterile petri dishes (9-cm diameter) lined with Whatman filter paper wetted with 3 mL of demineralized water. Petri dishes were sealed with parafilm and incubated at 27° C. for preconditioning. After one week of preconditioning period, the glass fiber filter paper disks with Orobanche seeds were removed from the petri dish and dried for 20 min to remove surplus moisture. The disks were transferred to another petri dish within a filter paper ring (outer diameter of 9 cm; inner diameter of 8 cm) wetted with 0.9 mL of water, which maintained a moist environment during the germination bioassay. Forty microliters of the GR24 test solutions were added to the disks. Untreated GR24 at a 0.56 μM concentration was used as a positive control, water or HPC pre-made gel as negative controls. Germination percent was calculated using the counter cell analysis of the IMAGEJ (http://www.//rsbweb.nih.gov/ij/) software. Germinated seeds were distinguished from non-germinated seeds according to the seedling radicles, which are a few millimeters long and visible under a stereomicroscope (Leica MZFLIII, Leica Microsystems, GmbH).
For each of the GR24 treatments fifteen replicates with 30-40 seeds were tested per treatment with 40 μl of 0.56 μM GR24 test solutions pre-treated as described above. The experiment was repeated three times (n=450 seeds).
Mass spectrometry (MS) analysis was performed to GR24 test solutions using the Orbitrap XL (Thermo Fisher Scientific). The samples were injected directly using direct spray injection probe, in 50% acetonitrile solution and at 5 ml/min flow rate. A full scan was acquired to detect the GR24 peaks, at 60,000 resolution, Maximum ion fill time settings were 300 ms for the high resolution full scan in the Orbitrap analyzer. The spectra are sum of 10 sec acquisition. Results observed by Xcalibur© software of Thermo Fisher Scientific Inc. version 2.0.7. were analyzed manually.
The results showed that GR24 loses biological activity and is molecularly degrading following long term exposure (over 5 days) to temperatures above 40° C., under acidic pH solutions and following exposure to UV irradiation.
Next, GR24 was stabilized with Hydroxypropyl Cellulose (HPC), which is a hydrophobic medium. Granular HPC [Sigma-Aldrich Israel Ltd., CAS-No. 9004-64-2] (1 g) was ground to a fine powder using a mortar and pestle, mixed with sterile distilled water to make about 10 mL of gel, and then allowed to stand for 1 day for complete dissolution at room temperature. Stability of GR24 was tested by both biological activity of the strigolactone and by assessing the molecule using LC-MS.
HPC medium stabilized GR24 by increasing its biological activity. The structure of the GR24 molecule remained intact under the examination conditions.
Further to the above, the stabilization effect of HPC on EGO10 at different pH conditions (pH 4, pH 7 and pH 10) and at a temperature of 60° C. was examined on Orobanche germination. The results demonstrate that HPC stabilizes EGO10, as evident by its increased biological activity, under basic and acidic pH conditions (
Development of the Bioassay:
For the purpose of determining whether the active SL analogs may be used as effective repressors of shoot branching by application on the stem itself, a bioassay examining SL analogs effect on shoot branching in vitro was established. Olive seedlings were grown in greenhouse under controlled temperature of 23° C. Partitioned plates containing ½ Murashige and Skoog (MS) solidified with 1.5% w/v agar were prepared. After solidification, the center plastic area (2 cm) of the plate was removed by scalpel. Olive seedlings were cut between the second and fourth node, and the cuttings were surface-sterilized by 70% ethanol and 1% sodium hypochlorite treatment, and then placed on the plates containing the agar media. The SL analog EGO10 was mixed with ½MS in agar, cubes were cut (1.5 cm area) and positioned at the base of the cutting in the plates (
Effect of EGO10 Application at the Base of Plant Cuttings on Bud Outgrowth:
The effect of 5 μM EGO10, supplied in agar cubes at the base of olive seedling cuttings, on bud outgrowth was examined. At day 12, a marked reduction in bud outgrowth was observed compared to the control treatment (
Effect of EGO10 Application at the Bark of Plant Cuttings on Bud Outgrowth:
The ability of EGO10, applied to olive seedling cuttings through the bark of the stem, to reduce bud outgrowth was examined. An agar tube containing EGO10 was placed on the bark of the cutting (
Bud outgrowth following 96, 168 and 264 hours of EGO10 treatment was measured and the length of each bud minus its length at the first day following application was calculated (
As a next step in the development of SL application technology, different substances that can serve as media supporting the diffusion of SLs though the bark of plants were examined. As agar usually contains nutritional compounds, bacteria and fungi can easily contaminate agar cubes. Therefore, other substances were examined, as described hereinbelow. Experiments were performed similarly to those described in Example 3 above, by application of EGO10 in different media on the cutting bark, in plates. Bud outgrowth was monitored as described above.
The use of Hydroxypropyl cellulose (HPC) material instead of agar as the media for EGO10 application on plants was tested. The stabilizing effect of HPC on SL analogs, including EGO10, is described in Example 2 above. For determining which percentage of HPC is suitable for field application, different dilutions of HPC in water were prepared, and their capability to stick to the olive stem was examined.
The results showed that 30% HPC was sufficient for sticking to the stem (
Based on the results above, 30% HPC were mixed with two different concentrations of EGO10; 5 and 10 μM, and applied on the stem of olive seedling (
The results showed that both concentrations of EGO10 in 30% HPC reduced bud outgrowth until day 4 post application. However, after day 4 bud outgrowth resumed, whereas EGO10 application using agar cube reduced bud outgrowth throughout the entire experimental period (
In an attempt to improve HPC suitability to the examined system, HPC was mixed with glycerin in the ratio of 3:1. Glycerin, as a known humectant, has the capability to moist materials. In exemplary procedures, 5 μM EGO10 were added to the HPC and glycerin mixture, and then placed on the bark of olive cuttings. Bud outgrowth was monitored as described above.
The results show that the mixture of HPC, glycerin and EGO10 inhibited bud outgrowth throughout the experimental period, i.e. until day 12 (
A number of additional possible substances suitable as media for EGO10 application were screened, including polyvinyl acetate (PVAc), polyvinyl pyrrolidone (PVP) and hydroxy ethyl cellulose (HEC). The polymers were used at concentrations of 30% in water, however, as 30% PVP was not solid, it could not be tested. Accordingly, HEC and PVAc were examined for their ability to support EGO10 activity. Bud outgrowth was calculated as described above.
The results indicated that PVAc supported EGO10 activity of bud outgrowth inhibition, while HEC did not (
Following, different mixtures of the polymers were examined applied as described above and monitored bud outgrowth. It was found that a mixture of PVA with HEC or PVA and PVP did not sufficiently support EGO10 ability to act to inhibit bud outgrowth (
EGO10 either with PVAc, or HPC+glycerin were applied on olive seedling stem by the use of parafilm cover as the apparatus, under greenhouse conditions (
In attempt to improve the performance of the apparatus, different substances and different cover were used. Four formulations were developed and tested. These formulations are based on polysaccharide-sorbent-humidifier substances while the main difference between them is the viscosity of the final product and the ability to retain and release of the active ingredient (EGO 10). All formulations included preservatives against biological decay and were tested under lab condition and were proven to be less heat susceptible, and less likely to liquefy under greenhouse conditions. The cover consisted of a flexible plastic tube that was cut (
It was concluded that despite their better resistance to greenhouse conditions these substances were not suitable for olive treatments because of their strong retention of the active ingredient and bad release pattern to the plant.
Rigid plastic tubes were tested in order to determine their efficiency as a delivery device for application of the hormone formulations to plants. At first, eppendorf tubes served as prototype. The tubes ware cut from top to bottom and placed on the stem of seedlings. Each tube was sealed with a small moist cotton at the bottom, filled with 1 ml of the examined material and finally covered with transparent plastic tape (
EGO10 (10 μM) or acetone control together with HPC+glycerin or PVAc were applied on the stem of olive cuttings by the use of rigid plastic tubes as described in Example 10 above.
Application of PVAc with the acetone control under the examined conditions led to inhibition of seedling outgrowth (
On the other hand, use of HPC+glycerin (with acetone) under these conditions did not lead to inhibition of outgrowth (
Higher concentrations of 50 μM of EGO10 or acetone control were applied with HPC and glycerin using rigid tubes as described in Example 10. Bud outgrowth of olive cuttings was measured until day 30.
The results indicate that the formulation of 50 μM EGO10+HPC+glycerin significantly inhibits bud outgrowth, compared to control (acetone+HPC+glycerin) (
Experiments were performed in AGROGOLD nurseries under commercial nursery conditions, which include nethouse, 40% shed without temperature control. The tested formulations were applied in rigid tubes. Specifically, each eppendorf tube was cut from top to bottom and placed on the stem of the olive seedlings. The tube was sealed and fixed to the plant by cello tape. The tube was completely filled with ˜1 ml of the experimental formulation and then the top was covered with cotton (
The bud growth effect of EGO10 (50 μM) or acetone control, each supplied together with HPC+glycerin on the stem of the olive cuttings was tested under the nursery conditions.
The results show that the formulation of EGO10+HPC+glycerin inhibits bud outgrowth significantly compared to control (acetone+HPC+glycerin), under commercial nursery conditions (
Application of EGO10-based collar for a variety of agricultural crops. This was done under greenhouse and commercial nursery conditions. Also, an advanced version of collar in terms of its envelop was developed, for promotion of a cost effective and efficient apparatus for agricultural use, for reduction of axillary branches growth.
EGO10-Collar (and Granules) Application to Multiple Crops Roses (Rosa Hybrida):
Three replicates on experiments were done in two commercial plots. EGO10 was applied at the indicated concentrations as a collar in Eppendorf tube (
HPC+glycerin and EGO10 (25 or 50 μM), provided in rigid tubes, were applied as described above (Example 13) to roses (Rosa hybrid golden gate). 20 days following application of the formulation by the delivery device, the length of axillary buds (in centimeters) was measured and the number of lateral buds was counted. In all experiment a clear tendency of reduction of lateral branching was determined (
Application of EGO10 with HPC+Glycerin Via Rigid Plastic Tubes to Hypericum Plants Under Commercial Nursery Conditions:
EGO10 (25 and 50 μM) with HPC+glycerin were applied in rigid tubes, as described above (Example 13) to Hypericum calycinum or Hypericum perforatum (Ivory Spices of Danziger, result of breeding for ornamental).
Two replicates on experiments were done in the greenhouse. EGO10 was applied at the indicated concentrations as a collar. Also, EGO10 was applied in another, new way of application—as granules of slow release of 3×10−8 M—that were applied in the soil, close to the root zone. On Hypericum, this way of application did not result with substantial changes in the no. of axillary branches. In contrast, application of 50 μM EGO10 as collar (in Eppendorf tubes) led to a marked reduction in the no. of axillary branches (
Pomegranate (Punica granatum):
Two replicates on experiments were performed in commercial nursery. Application of 50 μM EGO10 as collar (in Eppendorf tubes) led to a marked and significant reduction in the number of axillary branches (
Almond (Prunus dulcis):
Two replicates on experiments were performed in commercial nursery. Application of 3×10−8 M EGO10 as granules of controlled release (granules are based on sugar polymers), and not in collar, led to a marked and significant reduction in the number of axillary branches (
Olives (Koroneiki olives cultivar):
Since Koroneiki olives are one of the most abundant cultivars worldwide, the effect of application of collar of 50 μM and granules that release 3×10−8 M on development of side branches in this cultivar was examined. The results showed that application of collar was effective in preventing side branches development in Koroneiki olives (
Tomato (Solanum lycopersicum):
Greenhouse tomato (Shirez, Ikram and other cultivars) are one of the crops that necessitate vast investment in the constant trimming of side branches. Hand labor is needed for this trimming, on an everyday basis, to avoid significant yield loses. The constant trimming is a cause for infection at trimming sites. Hence, adapting collar application to tomato growth may allow to reduce the need for the trimming of side branches. For these experiments two cultivars of greenhouse tomato were used, Ikram and Shirez, both vigorously producing side branches that should be trimmed under commercial growth conditions.
Collar was applied at 25 and 50 μM of EGO10, enveloped by parafilm (application of tube is not feasible on tomato, as it leads to scratching of the stem) as demonstrated in
The results (shown as side branch weight) demonstrated that in Shirez, treatments with EGO10 did not change significantly branching (
In Ikram, in the same experiment, yield determined as number of fruit was increased in the EGO10 treated plants (
Following, and to increase the possible effect of EGO10 on tomato branching, additional experiment was performed with tomato plants were treated with collar of 500 μM EGO10. Here, although Shirez did not respond to this treatment in Ikram a significant reduction in side branches was determined (
To summarize, by application of EGO10 as collar, in granules or via irrigation led to reduction of side branches in several agricultural crops.
Tomato (Solanum lycopersicum):
The effect of EGO 10 combined with auxin was tested. For this experiments the Ikram cultivar of greenhouse tomato was used since it showed better response to the EGO 10 treatment in the previous experiment. Collar was applied 50 μM of EGO10 (
The results were consistent throw the experiment and showed a clear inhibition of bud outgrowth above the Implemented EGO10 collar when auxin was applied. A more moderate and not consistent respond was showed as a result of the EGO10 treatment (
Additionally, there was no negative effect of the treatments on the harvest of the tomatoes (
Another aspect of collar development is development of its cover, or envelop. The hormone and its medium (HPC and glycerin) are needed to be covered with some substance to avoid drying or leaking of the compounds once applied on the stem. Moreover, an environmentally friendly collar was developed, which is based on non-hazardous materials. For that purpose several polymers as collar covers was examined. Table 4 below presents polymers that were tested and their assessment.
In another exemplary tomato experiment eight polymers that may act as EGO10 carriers and be placed on the plant with no use of any additions were tested. All polymers were less than 0.2 mm thick.
After one week three polymers showed best reaction to the plants were chosen: PEG MW 100000+EGO10; HPC+40% Glycerol; and PVC+50% PEG 600, (referred to as: “POL1”, “POL2”, “POL3”).
With the chosen polymers a tomato experiment was performed. The polymers were charged with EGO10 with the equivalent amount of EGO10 that the 50 μl gel collars contains. In this experiment the effect of the EGO10 polymers with a daily auxin spray (IAA 5 ppm) was tested in comparison to the EGO10 gel collar (following the same procedures as described above with the auxin treatment and a control treatment).
The results are summarized in Table 5 below
The results showed a clear inhibition of bud outgrowth above the implemented EGO10 collar when POL2 (HPC+40% Glycerol) was applied (
Further, Table 6 summarizes some surface free energy (SFE) values for some typical film forming polymers.
On the basis of these values transparent films were prepared by solvent casting of polyethylene oxide, plasticized PVC, hydroxypropyl cellulose (HPC) and polylactic acid. The films were typically 20-50 microns thick.
As further described hereinabove (e.g., Table 5), to the casting solutions of the high molecular weight polymers, a low molecular weight material (typically <1,000 Daltons) was often added.
Without being bound by any particular mechanism, this low molecular weight material had two functions: 1. To plasticize the polymer film so that it could be wound around a stalk without breaking. 2. To transport the hormone e.g., EGO 10 from the bulk of the polymer to the interface between the film and the stalk.
As noted above, the small molecules tested were glycerol, PEG 600, ethoxy ethanol (all are polar) and dioctyl sebacate (DOS) which is a hydrophobic plasticizer.
The small molecule should meet the requirement of serving as a solvent for the EGO10 and is available in anhydrous forms. Typically, 6 mg of hormone were dissolved in 0.6 ml anhydrous acetone which was then dispersed in the polymer solution.
To summarize, the formulations that adhered to a tomato stalk and those that did not are listed below in Table 7.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
Number | Date | Country | Kind |
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238839 | May 2015 | IL | national |
Filing Document | Filing Date | Country | Kind |
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PCT/IL2016/050504 | 5/11/2016 | WO | 00 |