Patent Application
20190289849
This disclosure relates to methods for regulating growth in a plant. Specifically, the methods of the disclosure regulate growth of the plant by administration of a composition comprising an effective amount of one or more apocarotenoids to the plant, its part, or the seed.
The rapidly increasing world population, coinciding with changes in climate, creates a need for new ways to stabilize and improve plant growth under harsh environmental conditions. One such way is to regulate root branching and root growth, which are essential for nutrient and water uptake. Two promising methods of optimizing root architecture for increased plant performance are to regulate root system depth and branching (Rogers, E. D. and P. N. Benfey, Current Opinion in Biotechnology, 2015. 32: p. 93-98; Manschadi, A. M., et al., Functional Plant Biology, 2006. 33(9): p. 823-837; Champoux, M. C., et al., Theoretical and Applied Genetics, 1995. 90(7-8): p. 969-981). In developing Arabidopsis plants, branching is initiated by the formation of de novo lateral root primordia. Positioning of lateral root primordia is controlled by a “lateral root clock”—an oscillation cycle of gene expression changes that establishes sites competent to form primordia (Moreno-Risueno et al., Science, 2010. 329 (5997): p. 1306-1311).
The carotenoid pathway is a rich source of metabolites, called apocarotenoids, several of which are important regulators of plant development and root system architecture. Strigolactones, hormones derived from β-carotene, reduce root growth and lateral root and shoot branching (Gomez-Roldan, V., et al., Nature, 2008. 455(7210): p. 189-U22; Ruyter-Spira, C., et al., Plant Physiology, 2011. 155(2): p. 721-734). Abscisic acid (ABA), a stress-responsive hormone metabolite of zeaxanthin, has many roles throughout plant development, including in germination, abscission, drought response and root growth (De Smet, I., et al., Trends in Plant Science, 2006. 11(9): p. 434-439). Previously, an inhibitor of carotenoid metabolism, called D15, was found to decrease lateral root branching through an ABA and strigolactone independent-mechanism in Arabidopsis thaliana (Van Norman, J. M., et al., Proceedings of the National Academy of Sciences of the United States of America, 2014. 111(13): p. E1300-E1309.)
Natural signaling molecules capable of increasing root depth and branching are highly desirable tools for enhancing stress tolerance in crops. The inventors have found novel regulators of root architecture in plants when applied exogenously to the plants.
Thus, one aspect of the disclosure provides methods for regulating growth in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
Another aspect of the disclosure provides methods for improving drought tolerance in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
Another aspect of the disclosure provides methods for fertilizing plant soil, the method comprising providing a composition comprising an effective amount of one or more apocarotenoids to the soil.
Yet another aspect of the disclosure provides compositions comprising, consisting of, or consisting essentially of one or more an effective amount of one or more apocarotenoids present in the composition in a concentration of about 0.01 μM to about 100 mM and an agriculturally acceptable carrier, excipient, and/or diluent.
The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.
Before the disclosed processes and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, apparati, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.
Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
As used herein, the term “contacting” includes the physical contact of at least one substance to another substance.
As used herein, the term “root architecture” means the spatial arrangement of a plant's root tissue within the soil.
As used herein, the term “improving growth” refers to promoting, increasing or improving the rate of growth of the plant or increasing or promoting an increase in the size of the plant.
As used herein, the term “reducing growth” refers to decreasing or slowing the rate of growth of the plant or decreasing or promoting a decrease in the size of the plant.
All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included (e.g., on the total amount of the catalyst material).
In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need. In general, the disclosed materials, methods, and apparati provide apocarotenoids that can modify root architecture when applied exogenously to plants. Root architecture plays a major role in plant fitness, crop performance, and/or grain yield. The inventors have found novel regulators of root architecture in plants when applied exogenously to the plants. For example, nanomolar concentrations of apocarotenoids of the disclosure (e.g., β-cyclocitral) were found to fully rescue lateral root branching in the presence of an apocarotenoid synthesis inhibitor. In addition, apocarotenoids of the disclosure (e.g., β-cyclocitral) increased primary and lateral root growth, even in the absence of inhibitors. Lateral roots constitute the majority of a mature plant's root system and are vital for plant anchorage, absorption of water and nutrients, and survival in the presence of diverse environmental stresses such as drought. One of the apocarotenoids of the disclosure, β-cyclocitral, was also found to significantly increase primary root growth, as well as lateral and crown root growth, in tomatoes and rice, respectively.
Thus, one aspect of the disclosure provides methods for regulating growth in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed.
In one embodiment, the plants may be regulated to improve growth of the plant. For example, the growth may be improved by modifying the root architecture of the plant. The growth also may be improved by altering (e.g., improving) lateral root formation in the plant. The lateral roots of a plant, which constitute the majority of a mature plant's root system, are vital for plant anchorage, absorption of water and nutrients, and survival in the presence of diverse environmental stresses such as drought. In certain embodiments, the growth of the plant is improved by at least about 10% compared to the plant not contacted with the composition of the disclosure. For example, in certain embodiments of the methods as otherwise described herein, the growth of the plant is improved by at least about 12%, or by at least about 15%, by at least about 17%, or by at least about 20%, or by at least about 25%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 75%, or even by at least 100% or more as compared to the plant not contacted with the composition.
In one embodiment, the plants may be regulated to reduce growth of the plant. This method may be particularly useful in controlling unwanted plants, such as weeds, that grow among plant crops (i.e., act as herbicides). For example, the growth may be reduced by modifying the root architecture of the plant. In certain embodiments, the growth of the plant is reduced by at least about 20% compared to the plant not contacted with the composition of the disclosure. For example, in certain embodiments of the methods as otherwise described herein, the growth of the plant is reduced by at least about 25%, or by at least about 30%, or by at least about 40%, or by at least about 50%, or by at least about 75%, or even by at least 100% or more as compared to the plant not contacted with the composition.
One aspect of the disclosure provides methods for improving drought tolerance in a plant, the method comprising exogenously contacting a composition comprising an effective amount of one or more apocarotenoids to the plant, a plant part, or a plant seed. The improvement of drought tolerance is essential for stable and adequate crop production in drought-prone areas. Recent studies have determined that alteration of root system architecture affects (e.g., improves) drought tolerance (Uga et al., Nature Genetics, 2013, 45:1097-1102; Comas et al., Front. Plant Sci., 2013, 4:442; Rogers and Benfey, Curr Opin Biotechnol. 2015, 32:93-8; Manschadi et al., Functional Plant Biology, 2006, 33:823-837). The composition comprising an effective amount of one or more apocarotenoids, such as β-cyclocitral, improves plant's root systems by, for example, making them deeper and more compact, which enhances drought tolerance.
One aspect of the disclosure provides methods for fertilizing plant soil, the method comprising providing a composition comprising an effective amount of one or more apocarotenoids to the soil.
The methods of the disclosure require a composition comprising, consisting of, or consisting essentially of one or more apocarotenoids. As used herein, the term “apocarotenoid” means a cleavage product derived from one or more carotenoids. Most apocarotenoids are normally present at very low levels in the roots of plants. Apocarotenoids are formed by the action of nine different carotenoid cleavage dioxygenases (CCDs). The specific products generated by each individual CCD are difficult to characterize because CCDs seem to have functional redundancy. For instance, although it is known that D15 (N-(4-fluorobenzyl)-methoxycinnamic hydroxamic acid) can inhibit CCD8, ccd8 mutants do not have reduced lateral root capacity. In fact, even quadruple ccd8/ccd1/ccd4/ccd7 mutants (which are the most closely related of the CCDs), do not have a lateral root phenotype, suggesting that CCDs are highly functionally redundant. This makes it very difficult to specifically reduce the levels of a particular apocarotenoid, such as β-cyclocitral, using genetic approaches. In addition, overexpression or mutagenesis of one or more CCDs that normally produce a particular apocarotenoid would likely affect other, less desired, apocarotenoids. This makes it extremely challenging to change endogenous levels of a particular apocarotenoid in plants using genetic approaches. The inventors have found that exogenous application of a composition comprising one or more apocarotenoids can profoundly alter root architecture.
In one embodiment, the apocarotenoid of the disclosure is selected from the group consisting of β-ionone, β-cyclocitral, safranal, dihydro-β-Ionone, dimethyl-β-cyclocitral, dihydroactinidiolide (DHAD), α-ionone, pseudoionone, and combinations thereof. The chemical structures of these compounds are provided below:
In one embodiment, the apocarotenoid of the disclosure is selected from the group consisting of β-ionone, β-cyclocitral, safranal, dihydro-β-Ionone, dimethyl-β-cyclocitral, dihydroactinidiolide (DHAD), α-ionone, pseudoionone, and combinations thereof.
In one embodiment, the apocarotenoid of the disclosure is β-cyclocitral. β-cyclocitral is particularly useful in methods to improve growth of the plant.
In one embodiment, the apocarotenoid of the disclosure is selected from the group consisting α-ionone, β-ionone, safranal, dimethyl-β-cyclocitral, pseudoionone, or a combination thereof.
In certain embodiments, the apocarotenoid of the disclosure is present in the composition in a concentration of about 0.01 μM to about 100 mM. For example, in certain embodiments, the apocarotenoid of the disclosure is present in the composition in a concentration of about 0.01 μM to about 50 mM, or about 0.01 μM to about 50 mM, or about 0.01 μM to about 10 mM, or about 0.01 μM to about 5 mM, or about 0.01 μM to about 2 mM, or about 0.01 μM to about 1 mM, or about 0.01 μM to about 500 μM, or about 0.1 μM to about 100 mM, or about 0.1 μM to about 50 mM, or about 0.1 μM to about 50 mM, or about 0.1 μM to about 10 mM, or about 0.1 μM to about 5 mM, or about 0.1 μM to about 2 mM, or about 0.1 μM to about 1 mM, or about 0.1 μM to about 500 μM, or about 1 μM to about 100 mM, or about 1 μM to about 50 mM, or about 1 μM to about 50 mM, or about 1 μM to about 10 mM, or about 1 μM to about 5 mM, or about 1 μM to about 2 mM, or about 1 μM to about 1 mM, or about 1 μM to about 500 μM. Moreover, the apocarotenoid of the disclosure can be present in the composition in a concentration of any of the minimums and maximums provided above.
In certain embodiments, the apocarotenoid of the disclosure is present in the composition in a concentration of about 0.01 μM to about 1 mM, or about 0.01 μM to about 500 μM, or about 0.1 μM to about 100 μM, or about 0.05 μM to 0.15 μM, or about 50 μM to about 150 μM, or about 5 μM to about 15 μM. In certain embodiments, such concentrations are useful for improving plant growth. In certain embodiments, the concentration of about 0.05 μM to 0.15 μM is useful in improving the growth of Arabidopsis. In certain embodiments, the concentration of about 50 μM to 150 μM is useful in improving the growth of tomato. In certain embodiments, the concentration of about 5 μM to 15 μM is useful in improving the growth of rice.
In certain embodiments, the apocarotenoid of the disclosure is present in the composition in a concentration of more than about 1 mM, or more than about 10 mM, or more than about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 20 mM, or about 1 mM to about 50 mM, or about 5 mM to about 10 mM, or about 5 mM to about 15 mM, or about 5 mM to about 20 mM, or about 10 mM to about 100 mM, or about 10 mM to about 20 mM. In certain embodiments, such concentrations are useful for reducing plant growth.
The compositions of the disclosure may further comprise one or more substances formulated for at least one agricultural application. For example, the compositions of the disclosure further comprise one or more of agriculturally acceptable carrier, excipient, and/or diluent. Examples include, but are not limited to fertilizers (e.g., calcium, nitrogen, potassium, phosphorous), micronutrients (e.g., metal ions), insecticides, fungicides, nematocides, bactericides, acaricides, herbicides, plant nutrients, rooting stimulants, chemosterilants, semiochemicals, repellents, attractants, pheromones, feeding stimulants, microbial inocula or entomopathogenic bacteria, viruses, fungi, and other signal compounds including apocarotenoids, flavonoids, jasmonates and strigolactones (Akiyama et al., Nature, 2005, 435:824-827; Harrison, Ann. Rev. Microbiol., 2005, 59:19-42; Besserer et al., PLoS Biol., 2006, 4(7):e226; International Patent Publication no. WO 2009/049747). These compounds can also be formulated into mixtures or multi-component formulations. Agricultural applications are understood to include, but not be limited to, yield improvement, pest control, disease control, weed control, and resistance to abiotic environmental stress.
The compositions described herein can be produced by known processes, for example, as a formulation of one or more of the compounds described herein. In some embodiments, the compounds may be optionally mixed with further active ingredients, additives and/or customary formulation auxiliaries, which are then applied in a customary manner, such as, diluted with water, or as what are called tank-mixes by co-dilution of the separately formulated or partially separately formulated component(s) with water. In other embodiments, the compositions are formed in a dry mixture that is added to the soil in, on and/or around the plant. Such formulations may dissolve when exposed to water and thereby be taken up by the roots of the plant. Likewise possible is the application at different times (split application) of the separately formulated or partially separately formulated one or more compounds (e.g., the application of an apocartenoid followed by an additional component, e.g., herbicide, fungicides, etc.). It is also possible, for example, to apply one or more compounds in several portions (sequential application), for example pre-emergence applications followed by post-emergence applications, or early post-emergence applications followed by post-emergence applications at an intermediate or late stage.
In the methods of the disclosure, the composition disclosed herein is contacted with the plant. Any part of the plant may be contacted with the compositions of the disclosure, including tubers, roots, stems, leaves, flowers, and fruits. The composition may be applied directly to seeds or plants, or may be placed in soil in the vicinity of a seed or plant prior to or at the time of planting. In one embodiment, the composition is sprayed on seeds, tubers, or foliage. Seedlings, as well as more mature plants, may be treated. Flowers and fruits may also be treated by spraying. Roots of transplants may be sprayed or dipped in the composition prior to planting. In certain embodiments, the composition of the disclosure may applied, for example, by contacting the unwanted plants (for example harmful plants such as mono- or dicotyledonous weeds or unwanted crop plants), the seed (for example grains, seeds or vegetative propagation organs such as tubers or budded parts of shoots), or the area on which the plants grow (for example the area under cultivation).
The composition of the disclosure, and any additional compounds, can be deployed together (for example as a ready-made formulation or by the tank-mix method) or successively in any sequence, for example by application by irrigating, spraying, watering and sprinkling, or by granule scattering, or by soil injection.
According to their properties, the compositions of the disclosure may be used for pretreatment of the seed of the crop plant (for example for dressing of the seed), or introduced into the seed furrows prior to sowing, or employed alone or together with an additional compound(s) prior to or after emergence of the plants. Pre-emergence treatment includes both the treatment of the area under cultivation (including any water present in the area under cultivation, for example in the case of applications to rice) prior to sowing and the treatment of the area under cultivation in which seeds have been sown but which is not yet covered by growing plants.
In other embodiments, the composition of the disclosure may be applied directly to the plant. In other embodiments, the compositions of the disclosure and any additional compounds are applied directly to the roots of the plant. In yet other embodiments, the compositions of the disclosure and any additional compounds are applied directly to soil around the plant (for example, by soil injection). In yet other embodiments, the compositions of the disclosure and any additional compounds are dissolved in water and applied directly to the plant and/or soil of the plant.
In certain embodiments, contacting the composition to the plant is accomplished, for example, by irrigation, watering, sprinkling, spraying, or broadcasting.
For application, the formulations in commercial form are, if appropriate, diluted in a customary manner, for example in the case of wettable powders, emulsifiable concentrates, dispersions and water-dispersible granules with water. Dust-type formulations, granules for soil application or granules for broadcasting, and sprayable solutions are not normally diluted further with other inert substances prior to application.
The composition of the disclosure may be applied to monocot or dicot plants, and to legumes and non-legumes. In one embodiment, the composition is applied to field-grown plants. In another embodiment, the composition is applied to greenhouse-grown plants. In one embodiment, for example, the composition may be applied to tomatoes, rice, corn, cotton, canola, wheat, barley, sugar beet, turf grass, soybeans, peas, chickpeas, dry beans, peanuts, clover, or alfalfa.
One of skill in the art will recognize that the amounts of the composition (and any additional compounds) which are optimal in each case depend on the nature of the apocarotenoid used and any additional compounds used and on the nature and development stage of the plant stock to be treated, and can be determined in each individual case by simple, routine preliminary tests. In one embodiment, the effective amount of one or more apocarotenoids is at least about 100 nM per m3 of soil, or at least about 10 mg per m3 of soil.
Certain aspects of the disclosure are now explained further via the following non-limiting examples.
Materials and Methods
Plant Growth and Treatment Conditions:
Arabidopsis thaliana:
All seeds were in the Columbia-0 background. Seeds were sterilized with chlorine gas, incubated in 4° C. water for 2 days, and plated on 1% (wt/vol) Murashige and Skoog (1% MS) media with 1% agar. They were exposed to 100-130 μmol/(m2s1) illumination and grown vertically under long-day conditions at 22° C. Plants were analyzed at 7 days after stratification (das), unless noted otherwise. D15-treated media was made using D15 (N-(4-fluorobenzyl)-methoxycinnamic hydroxamic acid) synthesized by LeadGen Labs, LLC solubilized in DMSO. 100 mM D15 stock solution was diluted directly into 1% MS media for final concentrations ranging from 1 μM to 100 μM. For initial experiments testing the ability of apocarotenoids to rescue D15, plants were seeded on 1% MS media containing 30 μM D15 and 100 nM apocarotenoid (diluted from 1 mM stock solutions dissolved in DMSO). For subsequent experiments characterizing the effect of β-cyclocitral, plants were exposed to volatile analytical grade β-cyclocitral (≥97%, Sigma Aldrich) using 100×15 mm partitioned petri dishes. One side of the partition was filled with 25 μM β-cyclocitral diluted in 1% MS media. Seeds were plated on the other side of the partition, on 1% MS media. This media did not contact the β-cyclocitral, so seedlings were exposed to only volatile β-cyclocitral. For norflurazone treatments, norflurazone (Chem Service) dissolved in DMSO was diluted to a final concentration of 500 nM norflurazone in 1% MS media. For all experiments, including those performed on tomatoes and rice, plants were never exposed to DMSO concentrations that exceeded 0.1%.
Tomato:
Heinz tomatoes were used for all tomato experiments. Seeds were sterilized using 40% liquid bleach for 30 min, then plated on 1% MS media in 120×120×17 mm square petri dishes. Seeds were kept in the dark at room temperature for 3 days, and then exposed to 100-130 μmol/(m2s1) illumination and grown vertically under long-day conditions at 22° C. For treating tomatoes, β-cyclocitral, norflurazone, and D15 were added directly to the media, at the concentrations described in the figures. Norflurazone was diluted to a final concentration of 1 μM in media. Tomato phenotypes were analyzed at 6 das.
Rice:
Azucena (tropical japonica) seeds were dehulled and germinated in the dark for 3 days at 30° C. Germinated seeds were transferred to Yoshida's nutrient solution, solidified with 0.25% gelzan (Caisson Laboratories, Inc.), in 2 L glass cylinders, as described previously (Topp, C. N., et al., Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(18): p. E1695-E1704.). Rice roots were imaged at 12 das.
Root Phenotyping:
To measure lateral root capacity, the primary root apical meristem of each seedling was sterilely excised at 7 days. The plants were given 3 days to grow out lateral roots, and then each emerged lateral root was counted using a dissection microscope, as described previously (Van Norman et al., Proceedings of the National Academy of Sciences of the United States of America, 2014. 111(13): p. E1300-E1309). Luciferase activity was measured as previously described, using exposure times of 7 min (Moreno-Risueno, M. A., et al., Science, 2010. 329(5997): p. 1306-1311). WOX5+ and EN7+ primordia were counted using a stereo zoom microscope (Zeiss). Lateral and primary root lengths for Arabidopsis thaliana and tomato were measured using ImageJ (National Institutes of Health) software. 3D images of rice root architecture were captured using a 360° revolving platform and these images were analyzed using GiA Roots (www.giaroots.org), as described previously by Topp (2013). Primary root lengths were determined by dissecting the root and measuring the length with a ruler. Shoot mass was measured after removing the shoots and drying them for 10 days at 60° C.
First, the effect of D15 on lateral root branching was characterized and is shown
D15 did not affect lateral root development after primordia initiation, as measured by comparing its effect on WOX5 and EN7 positive primordia. Additionally, it did not inhibit the emergence of lateral root primordia, as compared to control roots (
Endogenous apocarotenoids were screened for their ability to rescue D15 inhibition of lateral root branching. Most apocarotenoids tested, including ABA and GR24 (a synthetic strigolactone analogue), further decreased lateral root capacity when combined with D15 (
In addition, at certain concentrations, a combination of β-cyclocitral and DHAD increased lateral root production and shoot mass in plants not subject to D15 inhibition (
The effect of β-cyclocitral on QC development in lateral root primordia was examined. Surprisingly, β-cyclocitral did not rescue WOX5+ primordia initiation in D15-treated plants (
It is difficult to specifically inhibit β-cyclocitral production in plants because there are nine carotenoid cleavage dioxides (CCDs), which are responsible for producing apocarotenoids that appear to be functionally redundant. Due to the challenge of further characterizing the role of β-cyclocitral through genetics, it was determined that β-cyclocitral's effect on root growth was conserved across plant species. Tomatoes were treated with β-cyclocitral, and a significant increase in lateral root length in a dose-dependent manner was found (
To determine if β-cyclocitral has an effect on root growth in monocots, β-cyclocitral was applied exogenously to rice plants, and changes were measured in root architecture. β-cyclocitral has a striking role in modifying root growth and architecture in Azucena, a japonica cultivar (
β-Cyclocitral enhances primary root growth in a conserved manner across species. It additionally promotes lateral root growth in Arabidopsis and tomato, and crown root growth in rice. In certain embodiments, exogenous modification of these traits in varying environmental conditions helps plants survive different types of stress. In particular, the dramatic ability of β-cyclocitral to generate deeper and more compact rice root systems, in one embodiment, enhances drought tolerance. In one embodiment, β-cyclocitral improves plant performance, especially in harsh environmental conditions.
At high concentrations (10 mM) of β-cyclocitral or DHAD, root length, shoot mass, and lateral root growth were inhibited. 10 mM β-cyclocitral, applied only to the root tip, was sufficient to reduce root growth (
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be incorporated within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated herein by reference for all purposes.
This application claims the benefit of U.S. Provisional Patent Application No. 62/338,590, filed May 19, 2016, which is incorporated herein by reference in their entireties.
This invention was made with United States government support awarded by NIH Grant No. R01-GM043778. The United States government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/033547 | 5/19/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62338590 | May 2016 | US |
