阅读说明：本技术 多个突变对提高水稻中的除草剂抗性/耐受性的作用 (Effect of multiple mutations on increasing herbicide resistance/tolerance in Rice ) 是由 D·伯纳基 C·克内珀 于 2018-01-05 设计创作，主要内容包括：描述了耐受/抗AHAS/ALS抑制剂的水稻,因为在提供除草剂抗性/耐受性中协同作用的许多突变。抗性/耐受性是由于在水稻中存在组合突变,导致AHAS/ALS酶中的氨基酸取代(A205V和G654E)。本发明还公开了所述水稻在杂草控制中的用途以及制备耐受/抗性水稻的方法。(Rice resistant/resistant to AHAS/ALS inhibitors is described because of a number of mutations that act synergistically in providing herbicide resistance/tolerance. Resistance/tolerance is due to the presence of combinatorial mutations in rice, resulting in amino acid substitutions in the AHAS/ALS enzyme (a205V and G654E). The invention also discloses the use of the rice in weed control and a method for preparing tolerant/resistant rice.)

1. A monocotyledonous plant having tolerance/resistance to an AHAS/ALS inhibitor at a level significantly higher than that tolerated by a plant without a mutation in the amino acid sequence of the AHAS enzyme, wherein the tolerance/resistance is associated with a plurality of nucleic acid sequence mutations resulting in combined amino acid substitutions a205V and G654E in the AHAS/ALS enzyme, and wherein there is a synergistic effect in the tolerance/resistance to said inhibitor associated with the combined substitutions.

2. The monocot plant of claim 1 is a rice plant.

3. the rice plant of claim 2 selected from the group consisting of plants produced from representative seeds deposited under ATCC accession nos. PTA-123859, PTA-123860 and PTA-123861, progeny and derivatives thereof.

4. A rice plant tolerant/resistant to an AHAS/ALS inhibitor, wherein said tolerance/resistance is associated with at least 2 nucleic acid sequences in the plant genome encoding amino acid substitutions in the AHAS/ALS enzyme selected from the group consisting of a205V, G654E and combinations thereof.

5. Seeds of the rice plant of claim 2 or 4 deposited under ATCC accession numbers PTA-PTA-123859, PTA-123860 and PTA-123861.

6. A method of controlling weeds in a rice field, the method comprising:

a. Planting rice in a field, wherein the rice is resistant to one or more AHAS/ALS inhibitors;

b. Contacting a rice field with at least one herbicide, said rice being resistant to a level of said herbicide known to kill weeds.

7. The method of claim 6 wherein said rice is resistant to AHAS/ALS inhibiting herbicides due to a synergistic effect resulting from amino acid substitutions A205V and G654E in the enzyme.

8. The method of claim 7, wherein the plant is the rice plant of claim 2 or 4.

9. The AHAS/ALS inhibitor of claims 1, 4, and 7, comprising at least one imidazolinone herbicide.

10. The imidazolinone herbicide of claim 9, selected from the group consisting of imazethapyr, imazetopic, imazamethabenz, imazaquin, and combinations thereof.

11. Progeny of the rice plant of claim 1 or claim 4.

12. A method of making a rice plant resistant to treatment with an AHAS/ALS inhibitor at a level suitable for weed control, the method comprising combining a rice genome comprising a nucleic acid that causes an a205(179) V substitution in the amino acid sequence of the AHAS/ALS enzyme with a rice genome comprising a nucleic acid that encodes a G654(628) E substitution in the amino acid sequence of the AHAS/ALS enzyme.

Background

Sequence listing

This application contains a sequence listing submitted electronically in ASCII form and is incorporated herein by reference in its entirety. The ASCII copy name created on day 27/12/2017 was 269608_ SEQ _ st25.txt, size 65,409 bytes.

Mutant rice plants are disclosed that (1) are resistant/tolerant (at relatively high concentrations of the inhibitor) to AHAS/ALS inhibitors, particularly imidazolinone ("IMI") herbicides, and (2) wherein the mutation produces a synergistic response to the herbicide in the rice. Methods of weed control are disclosed utilizing rice with herbicide resistance/tolerance of these mutations as crops in agricultural fields. The invention also discloses a method for preparing herbicide resistant/tolerant rice.

Increase the value of rice crops

Rice is an ancient agricultural crop and is now one of the major food crops in the world. There are two rice cultivars: rice (Oryza sativa L.), asian rice, and palea (Oryza glaberrima Steud), african rice. Asian species constitute almost all cultivated rice in the world and are the species grown in the united states. There are three major rice producing areas in the united states: missippi delta (Acken, Missippi, northeastern Louisiana, southeast Melsulia), Mexico gulf coast (southwestern Louisiana, southeast Texas), and California Central canyon. Other countries, particularly in south america and the east, are the major rice producers.

Rice is one of the few crops that can grow in shallow water because it has a unique structure that allows for gas exchange between the roots and the atmosphere through the stem. Growth in shallow water results in optimal yield and is the reason that rice is usually grown in heavy clay or soil with an impermeable hard layer under the soil surface. These soil types are generally not suitable for other crops, or at best, crop yields are poor.

Continuous improvement of rice is an urgent necessity to provide essential nutrients for the growing world population. Most of the world's population has rice as their primary source of nutrition and crops must thrive under various environmental conditions, including competition with weeds and attack by undesirable substances. Rice improvement is carried out by conventional breeding practices, but also by recombinant genetic techniques. Although it appears straightforward to those outside of this discipline, crop improvement requires acute scientific and artistic skills, and the results are generally unpredictable.

Although specific breeding goals vary from rice production region to rice production region throughout the world, increasing yield is a major goal in all projects.

Plant breeding begins with the analysis and definition of the strengths and weaknesses of existing cultivars, followed by the establishment of project goals, improving the debilitating aspects to produce new cultivars. Specific breeding goals include combining improved combinations of desired traits from parental sources in a single cultivar. The desired trait may be introduced as a result of spontaneous or induced mutations. Desirable traits include higher yield, resistance to environmental stress, disease and insects, better stems and roots, tolerance to low temperatures, better agronomic characteristics, nutritional value and grain quality.

for example, a breeder first selects and crosses two or more parental lines and then selects for a desired trait in a number of new genetic combinations. Breeders can theoretically generate billions of new and different genetic combinations through crosses. Breeding by using crosses and selfs does not imply direct control at the cellular level. However, that type of control can be achieved in part using recombinant genetic techniques.

Pedigree breeding is commonly used for the improvement of self-pollinating crops such as rice. For example, two parents with good complementary traits were crossed to produce F1 generation. One or both parents may themselves represent F1 from a previous cross. Subsequently, segregating populations are generated by growing seeds resulting from selfing one or more of F1 if both parents are inbred lines, or by growing seeds resulting from the initial cross directly if at least one parent is F1. Selection of the best individual genome may begin at the first segregating population or F2; then, starting at F3, the best individual in the best family is selected. "best" is defined according to the goal of a particular breeding program, e.g., to improve yield, fight disease. In general, because of genetic interactions, a multifactorial approach is used to define "optimal". A desired gene in one genetic background may differ in different backgrounds. In addition, the introduction of genes may disrupt other favorable genetic characteristics. Repeated testing of families can begin at generation F4 to improve the effectiveness of selection for low heritability traits. At the late stage of inbreeding (i.e., F6 and F7), the best line or a mixture of phenotypically similar lines were tested for potential release as new parental lines.

Backcross breeding has been used to transfer highly heritable traits of genes into desired homologous cultivars or inbred lines, which are the backcross parents. The source of the trait to be transferred is called the donor parent. The resulting plants are expected to have the attributes of the backcross parent (e.g., cultivar) and the desired traits transferred from the donor parent. After the initial cross, individuals with the desired phenotype of the donor parent are selected and repeatedly crossed (backcrossed) with the backcross parent. This process is used to restore all the beneficial characteristics of the backcrossed parents, adding new traits provided by the donor parent.

Promising advanced breeding lines were tested in full and compared for at least 3 years or more with appropriate standards in the environment representing the commercial target area. The best line is a candidate for a new commercial variety or hybrid parent; those that still have some trait defects can be used as parents to generate new populations for further selection.

These processes, which lead to the final steps of marketing and distribution, typically take 8-12 years from the time of the first cross, and may rely on the development of improved breeding lines as precursors. Therefore, the development of new cultivars is not only a time consuming process, but requires accurate prospect planning, efficient use of resources, and minimal changes in direction. The results include new genetic combinations not found in nature.

some rice improvements through breeding may be limited by natural genetic changes in rice and by hybrid species such as wild rice. Introduction of new changes in breeding programs is typically by such hybridization programs as pedigree or backcross breeding. However, it has been found that occasional natural mutations lead to the introduction of new traits such as disease resistance or high degrees of alteration. Breeders have also developed new traits into the rice genome by inducing mutations (small changes in DNA sequence). Some of these mutations or combinations of genes are not found in nature. Typically, EMS (ethyl methanesulfonate) or sodium azide plus MNU (N-methyl-N-nitrosourea) are used as mutagens. These chemicals randomly induce single base changes in DNA, usually G and C to a and T. The overall effect is unpredictable. Most of these changes have no effect on crops because they are outside the gene coding region or do not alter the amino acid sequence of the gene product. However, some produce new traits or incorporate new DNA changes into previous lines.

The breeder has no direct control of the mutation sites in the DNA sequence. The identification of available changes is generally due to the random possibility of inducing a significant mutation, and the breeder will recognize the phenotypic effect of the change and be able to select rice with the mutation for production. Seeds were treated with mutagenic chemicals and immediately planted for growth. The resulting plants were designated M0 as the starting mutagenized population. In self-pollinated crops such as rice, with or without selection, M0 can be selfed to produce M1 progeny, which subsequently produce M2, M3, etc., as the population develops. M2 seeds will carry many new changes; thus, no two experiments would yield the same combination. In these changes, new traits that were not previously present in rice and were not previously available to plant breeders can be discovered and used for rice improvement.

To discover new traits, breeders must use efficient and targeted selection strategies because the process is completely random and has a very low frequency of new combinations available. Of the thousands of induced new genetic variants, only one may have the desired new trait. The optimal selection system will screen thousands of new variants and allow the detection of some or even a single plant that may carry new traits. After identifying or discovering possible new traits, breeders must develop new cultivars by pedigree or backcross breeding, and extensive testing to verify that the new traits and cultivars are of stable and heritable value to rice producers. After the mutation is identified by any means, it can be transferred to rice by recombinant techniques.

Herbicide resistance in rice

Weeds in the field compete for resources and greatly reduce crop yield and quality. Weeds in crops have been controlled by the application of selective herbicides that kill the weeds but do not harm the crop. Often the selectivity of herbicides is based on biochemical changes or differences between the crop and the weed. Some herbicides are non-selective, meaning that they kill all or almost all plants. Non-selective or broad spectrum herbicides can be used on crops only if the crop has a genetic mechanism that confers herbicide tolerance. Crops can be converted to "herbicide tolerance" (HT) if a new gene is inserted that expresses a specific protein that confers tolerance or resistance to herbicides. Resistance to herbicides has also been achieved in crops by genetic mutations that alter proteins and biochemical processes. These mutations may occur naturally, but in most crops identified, they are produced by breeders.

In some cases, particularly with repeated use of a particular herbicide, weeds develop resistance through unintended selection of natural mutations that provide resistance. When weeds become resistant to a particular herbicide, the herbicide is no longer useful for weed control. The development of resistance in weeds can be delayed by alternating the use of herbicides with different modes of action to control the weeds, interrupting the development of resistant weeds.

Rice production suffers from difficult to control broad-leaved and gramineous (grass) weeds, one of which is particularly difficult to control is called "red rice". One difficulty arises because red rice is genetically similar to cultivated rice (they occasionally pollinate each other), and no selective herbicides are available that target red rice but do not harm cultivated rice. Control is currently provided in commercial rice production by the development of mutations found in rice that render rice resistant to a broad spectrum of herbicides such as imidazolinone and sulfonylurea herbicides. There is a need for rice crops that are resistant to herbicides that inhibit other harmful plants, such as broadleaf plants.

Finding new mutations in rice that increase herbicide resistance would greatly facilitate rice production. Genes, and combinations of genes, that increase herbicide resistance are obtained and incorporated into the rice genome while maintaining favorable characteristics to maintain or improve health (fitness) are challenging, unpredictable, time consuming, and expensive, but are essential to meet the increasing food demand in the world.

Summary of The Invention

Novel and unique rice lines and hybrids having unique resistance to herbicides, particularly AHAS/ALS-inhibiting herbicides, are described and disclosed herein. 1 when rice plants are challenged with IMI herbicides, the resistance conferred to rice plants by the substitutions a205V/G6254E in the AHAS/ALS amino acid sequence is significantly higher than that exhibited by rice plants carrying a205V alone or G654E alone. This uniqueness is due to the combination of genetic mutations, resulting in a synergistic increase in herbicide tolerance/resistance in rice with the combination of genetic mutations compared to tolerance/resistance caused by the genetic mutations alone.

Various mutations in the nucleic acid coding sequence of the AHAS/ALS2 enzyme cause amino acid substitutions that when expressed in rice result in an enzyme that is resistant to IMI-tolerant herbicides. FIG. 1 shows an alignment of nucleic acid sequences encoding AHAS/ALS amino acid sequences in 2 rice lines, one (0001-2-2) having a mutation resulting in substitution A205V and the other (80034-5) resulting in substitution G654E. The sequences in wild type rice (P1003) and parental patent line R0146 are also shown. The encoded specified amino acid substitutions are listed in the box below the mutated codon. FIG. 2 shows the positions of some of the AHAS/ALS known amino acid substitutions [205(179),653(627),654(628) ]. 3

Superposition of two genetic mutations (designated RTC1 and RTC2)4 by genomic exemplification of seed from 3 hybrid lines deposited under Budapest treaty and designated PTA-123859, PTA-123860 and PTA-123861 at the ATCC respectively

Unexpectedly, the synergy shown in IMI herbicide tolerance was significantly greater than the sum of the dose/tolerance shown for AHAS proteins carrying a single a205V or a single G654E mutation. Using data from multiple experiments, (identified as 15SA-T11,16-T7,16 GH-T7) for more than 2 years, specific interactions of two single mutations RTC1+ RTC2 were estimated at multiple locations with multiple different imidazolinone herbicides (imazetapyr, imazamox, and imazetapic) and multiple lines, when working together (RTC1-RTC2), and expressed as an additional avoidance of% injury relative to the mutations alone, which can be estimated as a-25% reduction in injury rate at 1X dose, a-55% reduction in injury at 2X, a-65% reduction at 3X, and a-67% reduction at 4X (fig. 10A).

As disclosed herein, the discovery of herbicide resistance/tolerance that results from the combined effects of mutations at two different substitutions in the target enzyme AHAS/ALS is a surprising and unexpected result. It is particularly unexpected that the combination produces a synergistic response-the percentage of injury due to herbicide exposure is less in rice with the combination than in rice with either mutation alone. Importantly, yield was not as adversely affected as that reported for herbicide resistant/tolerant rice with some mutations (attribute).

Because these mutations affect the same enzyme and in the same gene, one might expect at best additive effects, and possibly negative effects on the enzyme. Unexpectedly, the synergistic effect of herbicide tolerance levels had no detrimental effect on other traits (tables 4, 5).

The mechanisms of herbicide tolerance are roughly divided into two groups: herbicide tolerance to targeted and untargeted sites. Herbicide tolerance at the target site results from preventing the herbicide from binding to the target enzyme, resulting from point mutations that occur in the target. The molecular mechanism of herbicide tolerance at the target site is primarily regulated by a single gene encoding the target enzyme that contains a point mutation. The hybrid lines disclosed herein have a combination of 2 point mutations.

Disclosed herein is a method of controlling weeds in a rice field, wherein the rice in the field comprises plants that are resistant to IMI herbicides. The method comprises the following steps:

a. Herbicide resistant/tolerant rice is used in the field; and

b. Contacting the paddy field with at least one herbicide or a plurality of herbicides, e.g. as being likely to belong to

Any herbicide of the family of chemicals known as AHAS/ALS-inhibitors, or may belong to

Any herbicide of the class of herbicides known as AHAS/ALS-inhibiting herbicides.

A method of growing herbicide resistant/tolerant rice plants comprising: (a) planting resistant rice seeds; (b) allowing the rice seeds to germinate; (c) one or more herbicides are applied to rice sprout (sprout) at a level of herbicide that would normally inhibit the growth of a rice plant.

The method of producing a herbicide tolerant rice plant may also use a transgene or transgenes. One embodiment of such a method is to transform cells of a rice plant with a transgene, wherein the transgene encodes 2 different mutations, each resulting in rice against an IMI inhibitor. Any suitable cell may be used in the practice of these methods, for example, the cell may be in the form of a callus. Specific mutations disclosed herein include those that result in substitutions a205V and G654E in the AHAS/ALS enzyme. With this combination, resistance synergy occurs.

Drawings

FIG. 1 shows an alignment of nucleic acid sequences between wild type rice line P1003(SEQ ID NO:1), patent parental line R0146(SEQ ID NO:3), and one line having a mutation encoding substitution A205(179) V (SEQ ID NO:2) and another line having G654(628) E (SEQ ID NO: 4).

FIG. 2 shows an AHAS/ALS amino acid sequence alignment of substitutions at positions 205(SEQ ID NO:5), 653(SEQ ID NO:6) and 654(SEQ ID NO: 7); wherein P1003(SEQ ID NO:10) and R0146(SEQ ID NO:11) are non-mutated rice lines; nipponbare (SEQ ID NO:12) and Arabidopsis (SEQ ID NO:13) are also shown. In order of appearance, FIG. 2 also discloses SEQ ID NOS: 8-9(Cyp-MT-653, Cyp-WT), respectively. The blank display does not match.

FIGS. 3A-3I illustrate the results of example 1: FIG. 3A control; FIG. 3B 0.5 Ximazethapyr; FIG. 3C 1X imazethapyr; FIG. 3D 2X imazethapyr; FIG. 3E 4X imazethapyr; FIG. 3F1X imazethapyr +0.5 Ximazethapyr; FIG. 3G 1 Ximazethapyr +1X imazamox; FIG. 3H1X imazethapyr +2 Ximazethapyr; FIG. 3I 1 Ximazethapyr +4X imazamox; pots L to R: LF2-RTC2/LM1-RTC1, LF1-RTC2/LM1-RTC1, LF3-RTC2\ LM4-RTC1, mutant strain P1003A205V, mutant strain R0146G654E, sensitive control commercial variety Cypress and commercial hybrid line CLXL 745.

FIG. 4 graphically illustrates the results of example 1.

FIGS. 5A-5D illustrate the results of example 2: FIG. 5A; comparison; FIG. 5B 8X; FIG. 5C 1X; FIG. 5D; 0.25X; planting sequence L to R: P1003A205V, R0146G654E, P1003, LF2-RTC2/LM1-RTC1, LF3-RTC2\ LM4-RTC 1.

Figure 6 graphically illustrates the results of example 2.

FIG. 7 graphically illustrates the results of example 3.

Figure 8 is a graphical illustration of percent injury 4 weeks after pre-waterflood application.

Figure 9 is a graphical comparison of yields after various doses of IMI herbicides: the control was compared to various rice lines and hybrid lines.

FIGS. 10A and 10B show the average percent injury 3 weeks after the last herbicide application across the test [15SA-T11,16-T7,16GH-T7], which includes independent experiments with single active ingredients imazetaphyr, imazethapyr or imazetapic and combinations used sequentially; average effect data across experiments for each strain, RTC1 only, RTC2 only or RTC1-RTC 2; the synergistic effect of RTC1+ RTC2 interaction is expressed as observed RTC1+ RTC 2% damage minus expected RTC1+ RTC 2% damage, calculated as [ (RTC1+ RTC2) - (RTC1XRTC2/100) ]; figure 10A shows the average% injury 3 weeks after herbicide application, and the RTC1-RTC2 synergistic effect was calculated as described above; figure 10B graphically shows the percent average% damage at different applied doses for homozygous single mutant lines RTC1 and RTC2 and biszygote RTC1/RTC 2.

Detailed Description

Rice lines with different herbicide resistance genes pyramidically or stacked in the same genetic background, or as a single product used alternately in crop rotation by farmers, represent a key tool or strategy to extend the useful life of herbicides, as these practices slow down the development of herbicide resistant variants in the targeted weeds. Several approaches can deploy these resistances into hybrid lines or varieties for weed control, as well as selection in hybrid seed production. The rice lines described herein represent novel methods of weed control in rice, and can be deployed in any possible strategy to control weeds and provide for long-term use of these and other weed control methods.

Novel effect of double mutations on AHAS protein

AHAS is an important enzyme in plants and microorganisms that catalyzes the first step in the biosynthesis of acetolactate, the amino acids valine and isoleucine, from pyruvate. Functional protein complexes may have a homodimeric or homotetrameric structure and exhibit large catalytic subunits and small regulatory subunits.

The regulatory subunit stimulates the activity of the catalytic subunit and confers sensitivity to feedback inhibition by branched-chain amino acids. Because the crystal structure of AHAS is well characterized in Arabidopsis thaliana (Arabidopsis thaliana), it has been possible to (1) identify AHAS-herbicide binding sites and (2) establish and understand the molecular interactions between AHAS, its cofactors and the herbicides that affect it. The AHAS catalytic site is deep within the channels of the protein, but it is noteworthy that AHAS herbicides do not bind within the catalytic site. Instead, they bind through the herbicide binding domain across the entrance of the channel, thereby preventing the substrate from entering the catalytic site and disrupting normal enzyme metabolism, resulting in plant death. Across this domain, many amino acid residues are involved in herbicide binding. Structurally different AHAS herbicides are positioned differently in the herbicide binding domain, resulting in different levels of interaction for any given substitution. Specific amino acid substitutions within the herbicide binding domain may confer resistance to some (but not others) AHAS herbicides (see review in Powles and Yu, 2010).

Despite a large number of well-characterized single induced or spontaneous mutations spanning many amino acid residues, no one has reported simultaneous substitutions that describe 2 or more active substitutions at key amino acid residues of the AHAS gene. Moreover, given the proximity of these different active herbicide binding sites, it is also not possible to stack these from a single mutant donor into a single background genome by sexual reproduction. This limitation exists simply because of the extremely unlikely nature of such specific recombination events. Thus, the RTC1-RTC2 product is novel in the hybrid rice disclosed herein, as it effectively overcomes the unlikely combination of multiple mutations in the AHAS protein. Hybrid lines were generated by combining two different mutant alleles of the AHAS locus, which in turn produced a functional protein complex comprising two forms of subcomponents. Surprisingly, the result is that synergistic herbicide tolerance is conferred to rice when compared to the effect of a single mutation in pure form.

Application of rice with 2 different mutations

Cells derived from herbicide resistant seeds, plants grown from such seeds and cells derived from such plants, progeny of plants grown from such seeds and cells derived from such progeny are all within the scope of the present disclosure. The growth of plants produced from the deposited seeds and the progeny of such plants are typically resistant/tolerant to herbicides (e.g., IMI inhibitors) at herbicide levels that would normally inhibit the growth of the corresponding wild-type plant. Some natural (non-inducible) levels of tolerance to some herbicides, but they do not protect plants at commercially useful levels.

A method of controlling the growth of weeds in the vicinity of a herbicide resistant/tolerant rice plant is also within the scope of the present disclosure. One example of such a method is the application of one or more herbicides to the paddy field at a level of herbicide that normally inhibits the growth of rice plants. For example, at least one herbicide inhibits AHAS/ALS activity.

To maximize weed control in rice fields, different herbicides may be needed to cover the spectrum of weeds present, and accordingly, several applications in a crop cycle may be required for any particular herbicide, depending on the overlap between the window of effective control provided by a single application and the window of time over which its target weeds can germinate, which is generally longer than the protection provided by a single herbicide application. Temperature and soil moisture conditions are key factors that affect the herbicide efficacy window, the time window for weed germination and growth. Based on these factors, herbicide control patterns often involve repeated applications in sequence through the crop cycle.

In a standard herbicide tolerance system, such as the one currently used commercially in rice for resistance to imidazolinone herbicides, the first application of the herbicide is applied at the 2-leaf stage and the second application is after a minimum of 10 days, just before permanent flooding is established at the tillering of the plant. The purpose of the second application is to eliminate weeds that may germinate after the first application before they can be effectively suppressed by impoundment of water. In some traits, including "IMI" inhibitor herbicides, the time of herbicide application is critical not only for effective weed control, but also for the level of tolerance observed in the plant itself. The observed plant damage in response to herbicide application can closely coincide with the plant stage. In some rice lines, very early post-emergence applications caused very high damage at the 1-leaf stage, with the observed damage decreasing at each plant growth stage throughout the first tillers. Some herbicide tolerance traits do not even exhibit tolerance to pre-emergence applications, even though post-emergence tolerance is excellent. This variable herbicide response associated with the growth stage of the plant requires careful testing to establish a margin for safe use of the new herbicide tolerant products.

The plurality includes, for example, at least 2 "IMI" herbicides.

Given the combination of different herbicide resistance genes, whether the combination includes two or more different modes of action on the same herbicide, or two or more genes of herbicides of different families or functions, antagonistic or synergistic interactions resulting from gene-gene interactions can be observed, as demonstrated by some embodiments described herein. The combination of the novel mutant genes disclosed herein against AHAS/ALS inhibiting herbicides results in herbicide tolerance that is far superior to the additive resistance of the two genes acting alone, demonstrating synergistic effects. (FIGS. 3-6; examples 1-4).

High-yielding rice production requires specific weed control measures. Some herbicides are applied pre-emergence (premergent), after planting but before emergence; other post-emergence (postemergent) applications. In the case of rice, the post-emergence application can be before, or after, the crop is soaked in water. Preferred applications are usually multiple, as defined by the number of open leaves in the growing plant, depending on the developmental stage of the crop (Table 3: developmental stage of rice). The time of herbicide application is an important factor, not only from the standpoint of maximizing weed control efficiency, but also from the standpoint of minimizing the effect on herbicide-tolerant crops. This consideration stems from the fact that mutagenic, naturally occurring or transgenic herbicide resistance is generally not completely independent of dose and application time effects. Different genes of herbicide resistance have different dose responses, as well as application time responses, so typically the phytotoxicity in resistant crops increases with increasing dose beyond a certain level, or for a given herbicide dose, the phytotoxicity to resistant crops varies with time of application.

The evaluation of the new herbicide resistance gene (subject of the present application) is carried out with a range of suitable herbicide doses covering the application rates normally used in rice agricultural operations, while also taking into account possible deviations from the manufacturer's recommended doses. Consider 1X, recommended manufacturer or best practice recommended dose, the most commonly evaluated additional doses are 2X and 4X, with some experiments including other values.

The RTC1 line was derived from rice line P1003. RTC2 was derived from R0146-Chinese derived patent strain. Both lines did not have any tolerance to imidazolinone herbicides. After backcrossing to fix the mutation and to remove unwanted effects, the trait has been introgressed into many inbred rice lines to produce a range of hybrid rice varieties suitable for a range of different commercial requirements for herbicide tolerance.

The a179(205) V mutation was developed by EMS mutagenesis from line P1003, also known as Lemont, which is a public variety named Cypress. An independently developed mutation G628(654) E was obtained from chinese-derived patent strain R0146 by a chemical mutagenesis procedure (sodium azide + MNU). These mutations were independently fixed by inbreeding during line optimization after mutagenesis and early detection, and thus were available in stable homozygous structures in the mutant derived inbred lines. These two independent mutations are located in the same expressed gene, at positions contained within the same protein, and do not overlap in inbred stocks. Fortunately, the hybrid product with one dose of each allele and expressing both modifications exhibited higher herbicide tolerance than the homozygous line for either gene and was therefore the product of interest.

All genetic material used for the development of these mutants or derived therefrom is the property of RiceTec. All tags used were developed internally from available public sequences or sequence information derived from the same material. Standard IMI commercial herbicides were selected for the screening process, using labeling guidelines to determine herbicide use parameters. The herbicide response was determined using the rate of plant injury (see table 2).

Table 4 is a comparison of the morphological-physiological/grain quality attributes of RTC1-RTC2 hybrid rice lines compared to their non-mutated counterparts to highlight that the IMI-tolerant rice lines of these mutations are agronomically identical to their non-mutated counterparts. In summary, no commercially relevant differences were identified between the RTC1-RTC2 rice hybrid lines and their non-mutated controls (same original lines without mutations). Hybrid lines containing both mutations also showed little statistically significant difference compared to single mutations and to control lines, neither of which were biologically relevant. It is known that mutagenesis treatments often result in multiple alterations in the regenerated plant, but these lines have been repeatedly backcrossed and transformed into inbred lines, thereby removing unwanted mutations from the germplasm.

The following examples are illustrative of the invention, but are not limiting.

Examples