Lentil plants having increased resistance to imidazolinone herbicides

Abstract

The present invention is directed to lentil plants having increased resistance to an imidaizolinone herbicide. One such plant described herein is the RH44 lentil variety. The present invention also includes seeds produced by these lentil plants and methods of controlling weeds in the vicinity of these lentil plants.

Claims

.[.1. A lentil plant having increased resistance to an imidazolinone herbicide as compared to a wild type variety of the plant, wherein the lentil plant has an ATCC Patent Deposit Designation Number PTA-3270..].

.[.2. A method for controlling weeds within the vicinity of the lentil plant of claim 1, comprising applying an imidazolinone herbicide to the weeds and to the lentil plant..].

.[.3. The method of claim 2, wherein the imidazolinone herbicide is selected from the group consisting of imazethapyr, imazapic, imazamox, imazaquin, imazethabenz and imazapyr..].

.[.4. The method of claim 2, wherein the imidazolinone herbicide is imazethapyr..].

.[.5. The method of claim 2, wherein the imidazolinone herbicide is imazamox..].

.[.6. A seed of lentil line RH44, representative seed of said line having been deposited under ATCC Patent Deposit Designation Number PTA-3270..].

.[.7. A lentil plant, or a part thereof, produced by growing the seed of claim 6..].

.[.8. A method for producing a hybrid lentil seed wherein the method comprises crossing the plant of claim 7 with a different lentil plant and harvesting the resulting hybrid lentil seed..].

.[.9. A hybrid lentil seed produced by the method of claim 8..].

.[.10. A lentil plant, or a part thereof, produced by growing the seed of claim 9..].

.[.11. A method for controlling weeds within the vicinity of a lentil plant, the method comprising applying an imidazolinone herbicide to the weeds and to the lentil plant, wherein the lentil plant is produced by growing a seed of lentil line RH44, representative seed of said line having been deposited under ATCC Patent Deposit Designation Number PTA-3270..].

.[.12. The method of claim 11 , wherein the imidazolinone herbicide is selected from the group consisting of imazethapyr, imazapic, imazamox, imazaquin, imazethabenz and imazapyr..].

.[.13. The method of claim 11, wherein the imidazolinone herbicide is imazethapyr..].

.[.14. The method of claim 11, wherein the imidazolinone herbicide is imazamox..].

.Iadd.15. A method for controlling weeds within the vicinity of a lentil plant, comprising applying a composition comprising an imidazolinone herbicide to the weeds and the lentil plant, wherein the lentil plant has been obtained by a process comprising crossing lentil line RH44, a representative seed of said line having been deposited under ATCC Patent Deposit Designation Number PTA-3270, with another Lens culinaris variety, wherein the plant comprises an AHAS polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:1. .Iaddend.

.Iadd.16. The method of claim 15, wherein said imidazolinone herbicide comprises at least one of: imazethapyr, imazapic, imazamox, imazaquin, imazethabenz or imazapyr. .Iaddend.

.Iadd.17. The method of claim 15, wherein the imidazolinone herbicide comprises imazethapyr. .Iaddend.

.Iadd.18. The method of claim 15, wherein the imidazolinone herbicide comprises imazamox. .Iaddend.

.Iadd.19. The method of claim 15, wherein said plant is transgenic. .Iaddend.

.Iadd.20. A lentil seed of a lentil plant, said lentil plant having increased resistance to an imidazolinone herbicide as compared to that of a wild type variety of the plant, wherein the lentil plant has been obtained by a process comprising crossing lentil line RH44, a representative seed of said line having been deposited under ATCC Patent Deposit Designation Number PTA-3270, with another Lens culinaris variety, wherein the plant comprises an AHAS polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1. .Iaddend.

.Iadd.21. The lentil seed of claim 20, wherein said lentil seed is transgenic. .Iaddend.

.Iadd.22. The lentil seed of claim 20, wherein said lentil plant is a hybrid. .Iaddend.

.Iadd.23. The lentil seed of claim 20, wherein the process further comprises a step of self pollination. .Iaddend.

.Iadd.24. The lentil seed of claim 20, wherein the lentil plant is true breeding for the herbicide resistance characteristics of lentil line RH44. .Iaddend.

.Iadd.25. A method for selecting a lentil plant comprising applying a composition comprising an imidazolinone herbicide to the lentil plant wherein the lentil plant has been obtained by a process comprising crossing lentil line RH44, a representative seed of said line having been deposited under ATCC Patent Deposit Designation Number PTA-3270, with another Lens culinaris variety, wherein the plant comprises an AHAS polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 1. .Iaddend.

.Iadd.26. The method of claim 25, wherein said imidazolinone herbicide comprises at least one of: imazethapyr, imazapic, imazamox, imazaquin, imazethabenz or imazapyr. .Iaddend.

.Iadd.27. The method of claim 25, wherein the imidazolinone herbicide comprises imazethapyr. .Iaddend.

.Iadd.28. The method of claim 25, wherein the imidazolinone herbicide comprises imazamox. .Iaddend.

.Iadd.29. The method of claim 25, wherein said plant is transgenic. .Iaddend.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a table showing the effects of amino acid substitutions in the AHAS isoenzyme on the herbicide resistance of various plants.

(2) FIG. 2 is a graph showing the percentage injury to RH44 lentil plants and the wild type CDC Richlea lentil variety when sprayed in the seedling stage with various rates of imidazolinone or sulfonylurea herbicides.

(3) FIG. 3 is a graph showing the inhibition of AHAS activity in RH44 and CDC Richlea lentil plants by a negative feedback inhibition assay.

(4) FIG. 4 is a graph showing the inhibition of AHAS activity in RH44 and CDC Richlea lentil plants by increasing concentrations of imazapic.

(5) FIG. 5 is a graph showing the inhibition of AHAS activity in RH44 and CDC Richlea lentil plants by increasing concentrations of imazethapyr.

(6) FIG. 6 is a table showing the agronomic characteristics of RH44 lentil plants and various wild type commercial lentil varieties.

(7) FIG. 7 is a table showing the increased resistance of RH44 lentil plants to ODYSSEY herbicide as compared to various wild type commercial lentil varieties.

(8) FIG. 8 is a table showing the amino acid composition of RH44 lentil plants and various wild type commercial lentil varieties.

(9) FIG. 9 is a table showing the nutritional composition of RH44 lentil plants as compared to other registered commercial lentil varieties.

DETAILED DESCRIPTION

(10) The present invention is directed to lentil plants, lentil plant parts and lentil plant cells having increased resistance to imidazolinone herbicides. In one embodiment, a wild type lentil plant is one which is a member of the Lens genus and does carry the dominant gene for resistance to an imidazolinone herbicide. The present invention also includes seeds produced by the lentil plants described herein and methods for controlling weeds in the vicinity of the lentil plants described herein. It is to be understood that as used in the specification and in the claims, a or an can mean one or more, depending upon the context in which it is used. Thus, for example, reference to a cell can mean that at least one cell can be utilized.

(11) As used herein, the term lentil plant refers to a plant that is a member of the Lens genus of the Leguinosae family. The lentil plants of the present invention can be members of the Lens genus including, but not limited to, Lens culinaris Medikus, Lens orientalis (Boiss.) Hand.-Maz., Lens nigricans (M. Bieb.) Grand., Lens ervoides (Bring.) Grand., Lens odemensis Ladiz., Lens lamoittie Czefranova and Lens tomentosus Ladiz. (Ladizinsky et al. 1984, van Oss et al., 1997) and hybrids thereof. The term lentil plant is intended to encompass lentil plants at any stage of maturity or development as well as any tissues or organs taken or derived from any such plant unless otherwise clearly indicated by context. Plant tissues and organs include, but are not limited to, leaves, seeds, stems, flowers, roots, single cells, gametes, anther cultures, calli cultures, tissue cultures and protoplasts. In particular, the present invention includes seeds produced by the lentil plants of the present invention. In one embodiment, the seeds are true breeding for an increased resistance to an imidazolinone herbicide as compared to a wild type Variety of the lentil plant seed.

(12) The present invention describes a lentil plant having increased resistance to an imidazolinone herbicide as compared to a wild type variety of the plant. The Examples below provide a detailed description of the mutagenesis, breeding and selection of lentil plants having such increased resistance to an imidazolinone herbicide. One plant derived from these procedures is deposited with the ATCC (Patent Deposit Designation Number PTA-3270) and designated herein as the RH44 lentil variety. A deposit of 2500 seeds of the RH44 lentil variety was made with the American Type Culture Collection, Manassas, Va. on Mar. 20, 2001. This deposit was made in accordance with the terms and provisions of the Budapest Treaty relating to the deposit of microorganisms. The deposit was made for a term of at least thirty years and at least five years after the most recent request for the furnishing of a sample of the deposit is received by the ATCC. The deposited seeds were accorded Patent Deposit Designation Number PTA-3270.

(13) The present invention includes the lentil plant having a Patent Deposit Designation Number PTA-3270; a mutant, recombinant, or genetically engineered derivative of the plant with Patent Deposit Designation Number PTA-3270; any progeny of the plant with Patent Deposit Designation Number PTA-3270; and a plant that is the progeny of any of these plants. In a preferred embodiment, the lentil plant of the present invention additionally has the herbicide resistance characteristics of the plant with Patent Deposit Designation Number PTA-3270.

(14) .Iadd.The acetohydroxyacid synthase large subunit (AHASL) gene of the RH44 lentil line was sequenced and found to contain a single mutation that gives rise to the A205V (using the Arabidopsis thaliana AHASL1 amino acid position nomenclature amino acid substitution in the AHASL protein, when compared to wild-type AHASL protein. Thus, a plant of the RH44 lentil line comprises a mutant AHASL gene that encodes an AHASL protein comprising a valine at the position that corresponds to amino acid 205 in the Arabidopsis thaliana AHASL1. In a wild-type AHASL protein, amino acid 205 is known to be alanine. .Iaddend.

(15) Also included in the present invention are hybrids of the RH44 line described herein and another lentil variety including, but not limited to, CDC Richlea, CDC Robin, CDC Sovereign, CDC Glamis, CDC Milestone, CDC Vantage, Eston, Laird, Spanish Brown and French Green. The term variety refers to a group of plants within a species that share constant characters that separate them from the typical form and from other possible varieties within that species. While possessing at least one distinctive trait, a variety is also characterized by some variation between individuals within the variety, based primarily on the Mendelian segregation of traits among the progeny of succeeding generations. A variety is considered true breeding for a particular trait if it is genetically homozygous for that trait to the extent that, when the true-breeding variety is self-pollinated, a significant amount of independent segregation of the trait among the progeny is not observed. In the present invention, the trait arises from a dominant mutation in an AHAS gene of the lentil plant or seed.

(16) In one embodiment of the present invention, the lentil plant having increased resistance to an imidazolinone herbicide comprises an altered AHAS nucleic acid. As used herein, the term altered AHAS nucleic acid refers to an AHAS nucleic acid that is mutated from an AHAS nucleic acid in a wild type lentil plant and that confers increased imidazolinone resistance to a plant in which it is transcribed. In a preferred embodiment, the altered AHAS nucleic acid comprises a serine to asparagine amino acid substitution. In a more preferred embodiment, the altered AHAS nucleic acid comprises a serine to asparagine amino acid substitution in an AHAS gene. In a still further preferred embodiment, the serine to asparagine amino acid substitution corresponds to the serine to asparagine amino acid substitutions found in other AHAS gene paralogs that display imidazolinone resistance. Examples of such mutated AHAS gene paralogs include those found in wheat variety SWP965001, corn variety XI-12 and canola variety PM1, all of which have increased resistance to imidazolinone herbicides and are described in FIG. 1. By AHAS nucleic acid is meant a RNA or DNA sequence that encodes or directs the expression of an AHAS protein, and may include a coding region and its corresponding untranslated 5 and 3 sequence regions; Additionally, AHAS gene refers specifically to a DNA sequence that encodes or directs the expression of an AHAS protein.

(17) It is to be understood that the lentil plant of the present invention can comprise a wild type or unaltered AHAS gene in addition to an altered AHAS gene. As described in Example 3, it is contemplated that the mutation in lentil variety RH44 contains a mutation in only one of two AHAS isoenzymes. Therefore, the present invention includes a lentil plant comprising one or more altered AHAS nucleic acids.

(18) As also used herein, the term AHAS protein refers to an acetohydroxyacid synthase protein and the term altered AHAS protein refers to any AHAS protein that is mutated from a wild type AHAS protein and that confers increased imidazolinone resistance to a plant, plant cell, plant part, plant seed or plant tissue when it is expressed therein. The imidazolinone herbicide can be selected from, but is not limited to, PURSUIT (imazethapyr), CADRE (imazapic), RAPTOR (imazamox), SCEPTER (imazaquin), ASSERT (imazethabenz), ARSENAL (imazapyr) ODYSSEY (imazapyr/imazamox), or a derivative thereof.

(19) In addition to the compositions of the present invention, the present invention provides a method of controlling weeds growing in the vicinity of the lentil plants described above. These methods comprise applying imidazolinone herbicides to weeds in the vicinity of lentil plants having an increased resistance to an imidazolinone herbicide as compared to a wild type variety of the plant. In a preferred embodiment, the lentil plant comprises a altered AHAS nucleic acid. In a more preferred embodiment, the altered AHAS nucleic acid comprises a serine to asparagine amino acid substitution in an AHAS gene. In a still further preferred embodiment, the serine to asparagine amino acid substitution corresponds to the serine to asparagine amino acid substitutions found in other AHAS gene paralogs that display imidazolinone resistance.

(20) As described above, the present invention teaches compositions and methods for increasing the imidazolinone resistance of a lentil plant or seed as compared to a wild-type variety of the plant or seed. In a preferred embodiment, the imidazolinone resistance of a lentil plant or seed is increased such that the plant or seed can withstand an imidazolinone herbicide application of preferably approximately 1-28 ounces, more preferably approximately 3-14 ounces, and most preferably approximately 6, 7, or 8 ounces of active ingredient per acre.

(21) By providing for lentil plants having increased resistance to imidazolinone, a wide variety of formulations can be employed for protecting lentil plants from weeds, so as to enhance plant growth and reduce competition for nutrients. An imidazolinone herbicide can be used by itself for post-emergence control of weeds in areas surrounding the lentil plants described herein or an imidazolinone herbicide formulation can be used that contains other additives. Such additives include other herbicides, detergents, adjuvants, spreading agents, sticking agents, stabilizing agents, or the like. The imidazolinone herbicide formulation can be a wet or dry preparation and can include, but is not limited to, flowable powders, emulsifiable concentrates and liquid concentrates. The imidazolinone herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.

(22) It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. Additionally, all references cited herein are hereby expressly incorporated herein by reference.

EXAMPLES

Example 1

(23) Mutagenesis of Mixed Lentil Seed and Selection of RH44 Lentil Variety Having Increased Resistance to Imidazolinone Herbicides,

(24) Lentil line RH44 was derived from a bulk of mixed F.sub.3 lentil (Lens culinaris) seed developed via conventional crossing followed by self-pollination. Five kilograms of bulk, mixed seed were treated in EMS solution and then planted in the field as the M.sub.1 generation. M.sub.2 seeds were harvested from the field-grown M.sub.1 plants. M.sub.2 plants were advanced to the M.sub.3 generation. M.sub.3 seed was planted on 1 hectare (ha). Prior to flowering, the field was sprayed with 2ODYSSEY herbicide. At harvest, approximately 300 surviving plants were harvested and threshed. Approximately 150 plants were selected for further evaluation by planting a sample of seed from each plant in pots in growth rooms. These plants were sprayed with 2ODYSSEY herbicide at four weeks after emergence. Eight pots were selected for field evaluation. Seed of each line was sown in a small field plot and then sprayed with 2ODYSSEY. Line RH44 was selected as having imidazolinone resistance derived from a population of approximately one million M.sub.3 seeds. Since the original mutagen dose was small, the M.sub.3 population size was large, and the modification rate was relatively low (1:150,000), it is unlikely that multiple modifications occurred in RH44 to contribute to any deleterious effects (Konzak, 1987 Induced mutations in wheat improvement. In: Heyne, E. G. (ed.) Wheat and Wheat Improvement. American Society of Agronomy, Madison, Wis. pp. 428-443). FIG. 2 shows the increased imidazolinone resistance of the RH44 line as compared to the CDC Richlea lentil variety. FIG. 2 also shows that the RH44 line has little resistance to sulfonylureas such as EXPRESS, REFINE and REFINE EXTRA. The results in FIG. 2 reflect tolerance readings taken two weeks after application of the herbicide. The designations 1 and 2 refer to commercial application rates of those products.

Example 2

(25) Analysis of Mutation in Lentil Variety RH44

(26) The available data strongly indicates that a mutation in a single gene similar to that observed in wheat, corn and canola is responsible for the observed imidazolinone tolerance in the RH44 variety of lentil. In wheat, the AHAS isozymes have been labeled as genes A, B and C. The mutation responsible for imidazolinone tolerance in wheat is due to a point mutation of AGC to AAC at a single site in AHAS gene A (FIG. 1). This site corresponds to the maize amino acid position 621 previously identified in X1-12 (imidazolinone-tolerant maize). This single nucleotide change of a guanine to an adenine results in a single amino acid change serine to asparagine (FIG. 1). Additionally, the imidazolinone tolerance trait in CLEARFIELD canola is controlled by two semi-dominant genes PM1 and PM2. Resistance to the PM1 mutant is conferred through a guanine to adenine point mutation in the AHAS1 gene. This results in a similar single amino acid change from serine to asparagine. A different point mutation is responsible for the resistance observed in the PM2 mutant. A guanine to threonine change in AHAS3 results in a tryptophan to leucine amino acid change.

(27) The serine to asparagine amino acid substitution results in a plant that is tolerant to imidazolinone herbicides, but not sulfonylureas (Newhouse et al. 1992 Plant Physiol. 100: 882 -886). This is true of wheat, corn and canola. Similarly, the RH44 lentil variety is similarly not tolerant to sulfonylurea herbicides, suggesting that a similar guanine to adenine point mutation produced a serine to asparagine amino acid substitution similar to the other CLEARFIELD D varieties X1-12, corn, PM1 canola and SWP965001 wheat (FIG. 1).

Example 3

(28) Herbicide Resistance of the RH44 Lentil Variety

(29) The responses of the RH44 lentil variety and the CDC Richlea variety were identical in a negative feedback inhibition assay. As described earlier, AHAS is an enzyme active in the biosynthesis of leucine and valine. Under conditions of excess leucine and/or valine, the activity of AHAS is diminished. The ability of leucine and valine addition to inhibit AHAS activity was the same in the RH44 lentil and CDC Richlea lentil varieties, indicating very similar levels of expression and a lack of any discernible difference in AHAS function (FIG. 3).

(30) The AHAS activity in the wild type CDC Richlea lentil variety was inhibited by imazapic (CADRE)) (FIG. 4) and imazethapyr (PURSUIT) (FIG. 5) representative of all imidazolinone herbicides. The AHAS activity in the RH44 lentil variety was also inhibited by the addition of the two imidazolinone herbicides, but not to the same extent as for the wild type lentil CDC Richlea.

(31) These results suggest the existence of a semi-dominant AHAS isozyme in the RH44 lentil variety that is encoded by genes similar to those in X1-12 corn, SWP965001 wheat and PM1 canola. It is therefore likely that the gene primarily responsible for AHAS activity in the RH44 lentil has undergone a point mutation similar to that observed in corn, wheat and canola. This has made the isozyme coded for by this sequence resistant to imidazolinone inhibition. The wild type isozyme has no such mutation and therefore is susceptible to imidazolinone herbicide. Such a scenario would explain why AHAS activity drops 30 to 40% with the addition of maximum concentrations of imidazolinone herbicides. The resistance trait was determined to be stable in the mutagenized line RH44 lentil variety indicating that the resistance trait is semi-dominant, also similar to the imidazolinone resistant trait in canola.

Example 4

(32) Agronomic Characteristics of the RH44 Lentil Variety

(33) The agronomic traits (yield, height, time to maturity) of the RH44 lentil variety were similar to most of the commercial wild type cultivars of lentil. The time to flowering was approximately 58 days, the time to maturity was approximately 100 days and the mean height was 30 to 35 cm (FIG. 6). Both the RH44 lentil variety and CDC Richlea lentil variety are relatively high yielding, however, they are both susceptible to ascochyta blight and anthracnose.

(34) Initial field evaluation of RH44 lentil indicates that the seed yield is similar to that of CDC Richlea and superior to some commercial varieties (FIG. 7). In field trials, the RH44 lentil has commercially viable phenotypic and agronomic properties. Harvested RH44 lentil seeds were mixed in appearance. Some seeds had a mottled seed coat while some remained clear. Selection of seeds on the basis of appearance resulted in a mixed harvest in subsequent generations, which is a phenotypic trait similar to the Eston lentil variety.

Example 5

(35) Amino Acid Composition of Imidazolinone-Tolerant Lentil

(36) An analysis of the amino acid composition was conducted to compare the RH44 lentil variety with existing registered wild type lentil varieties in order to determine if any significant differences existed (FIG. 8). The seed for analysis was obtained from trials conducted in the year 2000 where RH44 lentil and several other lentil varieties were grown under the same conditions. Amino acid composition was determined using AOAC Method 982.30 D,E,F by Woodson-Tenent Laboratories, Inc. The results of this analysis demonstrated the similarity in amino acid composition among all varieties of lentil, many of which have very different phenotypic characteristics. This analysis also demonstrated that the mutation leading to imidazolinone tolerance in lentil produces no change outside the range of natural variability in the amino acid composition of lentils.

(37) .Iadd.The lentil plant of the present invention, the seeds of which have been deposited under the Patent Deposit Designation Number PTA-3270, comprise the amino acid sequence (SEQ ID NO: 1) of the mature herbicide-tolerant AHAS protein in RH44 lentil plant from amino acid residue 87 to amino acid 661. These amino acids correspond to amino acid residues 96-670 of Arabidopsis thaliana AHAS. .Iaddend.

Example 6

Nutritional Analysis of Imidazolinone-Tolerant Lentil

(38) A proximate analysis was conducted to compare the RH44 lentil variety with existing registered wild type lentil varieties in order to determine if any significant differences in nutritional value existed (FIG. 9). The seed for analysis was obtained from trials conducted in the year 2000 where RH44 lentil and several other lentil varieties were grown under the same conditions. The results of this analysis demonstrate the similarity in many nutritional characteristics among varieties of lentil, several of which have very different phenotypic characteristics. This analysis also demonstrates that the mutation leading to imidazolinone tolerance in RH44 lentil produces no subsequent change in the moisture, fat, fiber or protein content of lentil seed. The RH44 lentil is changed in its AHAS activity, but is unchanged in nutritional/food safety attributes when compared to commercial wild type lentil varieties in Canada.