Patent Application
20130125257
Current fertility recommendations for corn were developed over a long period of time with traditional corn susceptible to insect attack, or with corn protected from insects via application of chemical insecticides. Subject to the price of chemical fertilizers, typical current practice is for farmers to over-saturate their fields with fertilizers to a lesser or greater degree.
This invention is in the field of genetically engineered plants having improved yield.
The subject invention concerns the surprising discovery that transgenic corn that produces Bacillus thuringiensis (Bt) insecticidal toxins to provide in-plant protection against feeding damage by above-ground and below-ground insect pests exhibits desirable agronomic characteristics apart from the protection against insect feeding damage.
The subject invention relates in part to the use of insect-protected transgenic corn to modify fertility recommendations for a given yield target. Specifically, it is found that transgenic corn commercially adopted as HERCULEX-XTRA; thus containing genes that encode Cry34Ab1, Cry35Ab1, and Cry1F insecticidal proteins, produces increased grain yield, as measured by kernel weight and number, when grown in field conditions and compared to isogenic control populations. It is additionally found that these enhanced yields are obtained with a further advantage of increased efficiency of nitrogen fertilizer utilization.
The present invention also includes methods of modulating nitrogen utilization efficiency (NUE) in a plant cell, comprising: (a) introducing into a plant cell a recombinant expression cassette comprising a Cry protein operably linked to a promoter that drives expression in a plant; and (b) culturing the plant cell under plant cell growing conditions; wherein the nitrogen uptake in the plant cell is modulated.
Other methods include increasing yield in a plant, wherein such methods comprise the steps of: (a) introducing into a plant cell a construct comprising a Cry protein operably linked to a promoter functional in a plant cell, so as to yield transformed plant cells; and, (b) regenerating a transgenic plant from said transformed plant cell, wherein said Cry protein is expressed in the cells of said transgenic plant at levels sufficient to increase yield in said transgenic plant; wherein increased yield comprises enhanced root growth, increased seed size, increased seed weight, seed with increased embryo size, increased leaf size, increased seedling vigor, enhanced silk emergence, increased ear size, nitrogen utilization or chlorophyll content.
Seeds and plants made from these methods may also be included.
SEQ ID NO:1: cry34 plant-optimized gene sequence
SEQ ID NO:2: cry35 plant-optimized gene sequence
SEQ ID NO:3: truncated cry1F sequence encoding core toxin
SEQ ID NO:4: truncated/core-toxin Cry1F protein sequence
SEQ ID NO:5: native/full-length cry1F sequence
SEQ ID NO:6: native/full-length Cry1F protein sequence
SEQ ID NO:7: native cry34 sequence
SEQ ID NO:8: Cry34 protein sequence
SEQ ID NO:9: native cry35 sequence
SEQ ID NO:10: Cry35 protein sequence
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. The following is presented by way of illustration and is not intended to limit the scope of the invention.
The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Langenheim and Thimann, (1982) Botany: Plant Biology and Its Relation to Human Affairs, John Wiley; Cell Culture and Somatic Cell Genetics of Plants, vol. 1, Vasil, ed. (1984); Stanier, et al., (1986) The Microbial World, 5.sup.th ed., Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant Pathology Methods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: A Laboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization, Hames and Higgins, eds. (1984); and the series Methods in Enzymology, Colowick and Kaplan, eds, Academic Press, Inc., San Diego, Calif.
As used herein “operably linked” includes reference to a functional linkage between a first sequence, such as a promoter, and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. As used herein, the term “plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants, which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum. Plants of the invention include, but are not limited to, rice, wheat, peanut, sugarcane, sorghum, corn, cotton, soybean, vegetable, ornamental, conifer, alfalfa, spinach, tobacco, tomato, potato, sunflower, canola, barley or millet Brassica sp., safflower, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, palm, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beet, sugarcane, buckwheat, triticale, spelt, linseed, sugar cane, oil seed rape, canola, cress, Arabidopsis, cabbages, soya, pea, beans, eggplant, bell pepper, Tagetes, lettuce, Calendula, melon, pumpkin, squash and zucchini or oat plant. A particularly preferred plant is Zea mays.
As used herein, “yield” may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15% typically for maize, for example), and the volume of biomass generated (for forage crops such as alfalfa, and plant root size for multiple crops). Grain moisture is measured in the grain at harvest. The adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated.
As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue preferred.” A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “regulatable” promoter is a promoter, which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development. Tissue preferred, cell type specific, developmentally regulated, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter, which is active under most environmental conditions, for example, the ubiquitin gene promoter Ubil.
Transgenic corn varieties with insect protection traits have been available for several years. Multiple events are available for control of above-ground and below-ground feeding insects. These events are transformed with genes that produce Bt proteins and are marketed under names such as HERCULEX, YIELDGARD and AGRISURE. They are widely recognized to be efficacious against insect damage and are recommended for planting by University field extension services, and by their manufacturers, in locations where insect damage is expected to adversely affect corn yields. The growing recommendations (e.g. fertilizer application rates) for all of these insect protected corn varieties are identical to those recommended for nonprotected corn varieties.
While insect-protected corn varieties such as HERCULEX-XTRA are currently planted in many corn growing regions globally, farming methods of growing them with reduced fertilizer recommendations were not previously taught or suggested and are a subject of the present invention. Fertilization rates, ranges, and amounts as described elsewhere herein and as specifically exemplified in the Examples can be used to define methods of the subject invention. Various units and rates can be used to express such rates and/or ranges—some of which are used in the Examples (e.g., pounds of nitrogen fertilizer per acre).
Nitrogen fertilizer inputs are the third most costly input (behind land and seed) in corn production, and the cost may vary greatly depending on the price of natural gas required to produce nitrogen fertilizer. Nitrogen use efficiency (NUE) represents an important target for maize (corn) breeding programs. Previous research demonstrates that genetic variability exists for NUE and its components, N uptake efficiency (NUpE) and N utilization efficiency (NUtE). Thus, widespread adoption of the subject invention, having a result of less natural gas devoted to fertilizer production, can reduce costs for farmers as well as for consumers who use natural gas for heating and cooking.
Grain yield in corn is largely affected by the ability of the plant to take up nitrogen (N) from the soil and utilize it for growth and reproduction. Plant responses to nitrogen fertilizer are observed across a wide range of application rates. The subject invention demonstrates that grain yield and its components, kernel weight and number, are increased across a range of N application rates by the presence of the transgenic insecticidal proteins Cry34Ab1, Cry35Ab1 and Cry1F. These enhancements are accompanied by prolonged stay-green after flowering, and they correlate with increased N uptake efficiency before and after flowering. Further, it is surprisingly seen that the plants of the subject invention have increased grain yield per unit of plant nitrogen, both within and between N application rates. The effect is not seen with nontransgenic isogenic lines protected from insect attack by application of chemical insecticides, thus demonstrating that these benefits may be in addition to the root protection afforded by the transgenic insect control.
The subject invention stems in part from our observation that yields of genetically similar corn lines, with and without the insect resistance traits, unexpectedly respond differently to inputs, particularly nitrogen fertilizer. The insect-protected plants have less root damage from below-ground insect feeding. In addition, when damaged by root feeding insects such as corn root worm, the damaged plants regrow quicker and produce a larger root mass and have improved overall plant health. This combination of factors allows the rate of nitrogen and other fertilizers to be reduced to obtain the same amount of yield as is obtained with non insect-protected corn lines grown with higher fertility amounts.
The present invention can comprise transgenic plants that accumulate the insecticidal proteins Cry34Ab1 and Cry35Ab1 as well as CryF1. These proteins protect the root system of maize from damage from corn rootworm feeding and facilitate improved N uptake and utilization, thereby providing increased grain yield. Fertilizer application rates can be determined from the subject disclosure. Exemplary application rates, included within the subject invention, are demonstrated. Some preferred embodiments are also further specified in the Claims. Additionally, Herculex can be used in other crop species such as canola, wheat, rice, barley and other non-legume crops.
The present invention also includes methods of increasing nitrogen uptake efficiency in a plant by administering to a plant an expression cassette containing at least one Bacillus thuringiensis insect-resistance gene which functions to improve expression of at least one insecticidal portion of a protein or amino acid sequence variant thereof from a nucleic acid coding sequence in a plant cell. Such method can further increase in the yield of the plant. The increase in yield can include an increase in the kernel number per plant and/or an increase in the kernel mass per plant. Nitrogen utilization can also be modulated. Additionally, the increase in nitrogen can occur during the flowering and grain filling periods of development.
The Bacillus thuringiensis insect-resistance gene used can include a Cry protein. The insect-resistance gene can be selected from the group consisting of, e.g., a cry34 gene, a cry35 gene, a cry1F gene, and a cry3A gene. The Bacillus thuringiensis insect-resistance gene can comprise, e.g., Cry34Ab, Cry35Ab, and/or Cry1F. Sequences of the relevant proteins and genes of HERCULEX products are readily determinable. Unless otherwise indicated herein, the Cry1F protein and gene are as described in U.S. Pat. No. 6,218,188 (preferably the truncated, plant-optimized version described therein), and the Cry34/35 genes and proteins are as described in U.S. Pat. No. 6,340,593 (preferably the 149B1 genes and proteins). Other genes and proteins that can be used according to the subject invention are also known in the art. See e.g. U.S. Pat. Nos. 7,179,965; 7,524,810; 7,939,651; 6,893,872; and 6,900,371. The Crickmore et al. website of the official Bacillus thuringiensis nomenclature committee is also well-known in the art and provides links to many, publically available Cry protein and gene sequences. Truncated and/or core-toxin fragments of Cry1F, for example, can be used, as is known in the art. Variants thereof are included. The official nomenclature committee uses boundaries of at least 45% identity (e.g. Cry1F), 78% identity (e.g. Cry1Fa), and 95% protein sequence identity (Cry1Fa1) as primary, secondary, and tertiary ranks, respectively. Such boundaries, as well as 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and/or 99% sequence identity (with an exemplified or suggested protein or gene sequence) can be used according to the subject invention in some embodiments.
Either monocotyledonous plants or dicotyledonous plants may be used in the present invention. The present invention also includes method of growing transgenic corn plants having an increased yield by using a reduced amount of fertilizer, wherein the transgenic corn is insect resistant due to expression of an insect-resistance gene, and wherein the reduced amount of fertilizer is relative to fertilizer recommended for use on non-transgenic corn, wherein said non-transgenic corn is optionally protected by granular chemical insecticide to control rootworms. The increase in year can include an increase in the kernel number per plant or can include an increase in the kernel mass per plant. Such transgenic corn plants can yield corn comparable to corn yield from said non-transgenic corn grown using said recommended amounts of fertilizer. The fertilizer used can be a nitrogenous fertilizer. The nitrogenous fertilizer can be applied at any rate of less than 150 pounds per acre. Alternatively the nitrogenous fertilizer can be applied at a rate of less than 50, 60, 70, 75, 80, 90, 100, 110, 120, 125, 130, 140 pounds per acre. In general, it may be preferable to have some nitrogenous fertilizer added to a crop. The nitrogenous fertilizer can be applied to a field after planting said corn plants in the field but prior to emergence.
The present invention also includes methods of increasing yield of monocotyledonous plants or dicotyledonous plants due to nitrogen utilization.
Likewise, by means of the present invention, other agronomic genes can be expressed in plants of the present invention. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:
1. Genes that Confer Resistance to Pests or Disease and that Encode:
2. Genes that Confer Resistance to an Herbicide:
3. Genes that Confer or Contribute to a Value-Added Trait, Such as:
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
Unless specifically indicated or implied, the terms “a”, “an”, and “the” signify “at least one” as used herein.
The present invention is explained in greater detail in the Examples that follow. These examples are intended as illustrative of the invention and are not to be taken are limiting thereof. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Corn hybrids derived from the IBMRIL population (Lee, M.; Sharopova, N.; Beavis, W. D.; Grant, D.; Katt, M.; Blair, D.; and Amel Hallauer, A. (2002) Expanding the genetic map of maize with the intermated B73×Mo17 (IBM) population. Plant Molec. Biol. 48:453-461) were crossed to HERCULEX-XTRA and non-HERCULEX-XTRA isogenic testers. One hundred female inbreds were used in this experiment. These inbreds consisted of 99 recombinant inbred lines from the intermated B73×Mo17 population (IBMRILs) and one Dow AgroSciences proprietary inbred (DASM7). The genotypes were consistent between 2008 and 2009 with the exception of MO329 which was grown only in 2008 and MO379 which was grown only in 2009. Furthermore, the hybrids formed from the parental inbred lines, B73 and Mo17, were only grown in 2009. The IBMRILs were chosen based on previous data obtained in 2006 and 2007, and were selected to minimize any confounding effects due to differences in maturity. Each of the female lines was crossed to both DASV8 and its near isogenic line, DASV8XT, which contains the HERCULEX-XTRA traits, to create a total of 200 hybrids.
A split˜block design with three replications was used in which N rate and female parent were the whole plot treatments. Male parent was included as a split˜plot within each N rate/female parent subplot. Plots were planted on May 7, 2008 and May 21, 2009 on the University of Illinois Cruse Farm in Champaign, Ill. All plots received an in˜furrow application of chlorpyrifos (Lorsban 15G) at a rate of 1.3 lbs a.i. per acre in 2008 and tefluthrin (Force 3G) at a rate of 0.099 lbs a.i. per acre in 2009. N was applied as ammonium sulfate ((NH4)2SO4) in a diffuse band after emergence and incorporated between V2 and V3 plant growth stage. The N rates used in the study were 0 and 225 lbs per acre. Each experimental unit consisted of two rows spaced 2.5 feet apart. The rows were 15 feet long in 2008 and 17.5 feet long in 2009. Plots were thinned to an approximate density of 32,000 plants acre. At flowering, four (2008) or five (2009) representative plants were sampled and weighed. A shredded aliquot was dried to constant weight and ground in a Wiley mill to pass through a 20 mesh screen. Dried, ground stover samples were analyzed for total N concentration using a combustion technique (NA2000 N˜Protein, Fisons Instruments, San Carlos, Calif.). A similar sampling approach was used at the R6 plant growth stage except that the ears were removed, dried, and shelled to allow for calculation of per-plant grain weight at physiological maturity. At harvest, all plants within a single row of the two row plot were harvested. A subsample of the grain was analyzed for protein concentration using near-infrared transmittance spectroscopy (Foss 1241 NIT grain analyzer; FOSS, Eden Prairie, Minn.). Three-hundred kernels from each plot were counted using an electronic seed counter and weighed to obtain an estimate of individual kernel weight.
The phenotypic data were analyzed using the MIXED procedure of SAS. Nitrogen rate, female parent, and male parent were treated as fixed effects while replication was considered random.
Genetic utilization (GU) is defined as grain weight (kg) per unit of plant N (kg) under nonfertilized conditions, and has units of kg/kg plantN. Nitrogen Use Efficiency (NUE) was calculated as the ability to produce grain (kg) per unit of added fertilizer N (kg) and has units of kg/kgN. Nitrogen Uptake Efficiency (NUpE) was calculated as the difference in plant nitrogen content at high and low N application levels, divided by the difference in the applied levels of N. This is a measure of the efficiency of fertilizer N uptake into the plant per unit of fertilizer N (kg) and has units of kg plantN/kgN. Nitrogen Utilization Efficiency (NUtE) is defined as the grain weight (kg) per unit of N taken up by plant and has units of kg/kg plantN
The fields in which these hybrids were grown were treated with chemical pesticides at rates recommended by the manufacturer to control corn rootworms. Thus control of root damage was not dependent upon efficacy of the Cry34Ab1 and Cry35Ab1 HERCULEX-XTRA genes. The data summarized in Table 1 show that when averaged across the 99 IBMRIL female parents, in both 2008 and 2009, the hybrids with the HERCULEX-XTRA trait (DASV8XT) had significantly better Nitrogen Uptake Efficiency (NUpE) than did the hybrids which did not contain the HERCULEX-XTRA genes.
The values of Nitrogen Use Efficiency (NUE) and Nitrogen Utilization Efficiency (NUtE) were found not to correlate with the presence/absence of the HERCULEX-XTRA traits in 2008 and 2009 (Table 1)
aGenetic Utilization (GU). Defined as grain weight (kg) per unit of plant N (kg) under nonfertilized conditions. Units are kg/kgplantN.
bN Use Efficiency (NUE) Defined as ability to produce grain (kg) per unit of added fertilizer N (kg). Units are kg/kgN.
cN Uptake Efficiency (NUpE). Defined as efficiency of fertilizer N uptake into the plant (kg) per unit of fertilizer N (kg). Units are kgplantN/kgN.
dN Utilization Efficiency (NUtE). Defined as grain weight (kg) per unit of N taken up by plant (kg). Units are kg/kgplantN.
The increased Nitrogen Uptake Efficiency seen with plants having the HERCULEX-XTRA traits was reflected in increased plant N content at the R6 physiological state (Table 2) in both 2008 and 2009.
Further, the HERCULEX-XTRA containing hybrids had significantly better yield traits than did the hybrids which did not contain the HERCULEX-XTRA genes (Table 3). Yield was increased based on both an increase in kernel number per plant and an increase in kernel mass Improved yield correlated with higher N content at the R6 stage in both years of the study. The data Table 2 and Table 3 taken together demonstrate that improved yield correlated with higher N content at the R6 stage. Thus, one skilled in the field of crop physiology will realize that the HERCULEX-XTRA traits are effective in causing an increase in N uptake during the flowering and grain filling periods on development.
Ma et al. (Ma, B. L.; Meloche, F.; and Wei, L: (2009) Agronomic assessment of Bt trait and seed or soil-applied insecticides on the control of corn rootworm and yield. Field Crops Research. 111th edition: 189-196) showed little to no yield effect of Cry3Bb1 events (active against corn rootworms) in a commercial hybrid compared with an isogenic non-transgenic hybrid treated with Force 3G insecticide to control corn rootworms. Likewise, Vigna (Vigna, M. M. (2008) Comparison of the effect of corn rootworm technology and seed-applied insecticide (clothianidin) to nitrogen status in corn. MA Thesis, Iowa State University, Ames) found no difference in yield or N content of transgenic hybrids containing either Cry3Bb1 or Cry34Ab1+Cry35Ab1 (event DAS59122-7) with isogenic non-transgenic controls treated with Poncho 1250. Thus, the HERCULEX-XTRA transgenes may have broader effects than simply limiting rootworm damage.
Increasing nitrogen uptake efficiency and yield by production of HERCULEX-XTRA proteins in plants could have a dramatic effect on agriculture if also effective in crop species such as canola, wheat, rice, barley and other non-legume crops.
The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/44505 | 7/19/2011 | WO | 00 | 1/18/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61365538 | Jul 2010 | US |
