US 200701.80574A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0180574 A1 Penner et al. (43) Pub. Date: Aug. 2, 2007 (54) METHODS FOR BREEDING GLYPHOSATE Publication Classification RESISTANT PLANTS AND COMPOSITIONS THEREOF (51) Int. Cl. AOIH 5/00 (2006.01) (76) Inventors: Donald Penner, Williamston, MI (US); CI2N 5/82 (2006.01) Marulak Simarmata, Lansing, MI (52) U.S. Cl. ............................................ 800/278; 800/320 (US) (57) ABSTRACT Correspondence Address: A plant comprising SEQ. ID. NO. 2 or a functional portion HARNESS, DICKEY & PIERCE, P.L.C. thereof, wherein SEQ ID NO. 2 is not native to said plant. P.O. BOX 828 A glyphosate resistant grass of economic value comprises a BLOOMFIELD HILLS, MI 48.303 (US) nucleic acid molecule that encodes a EPSPS enzyme. In Some embodiments, the nucleic acid molecule comprises a (21) Appl. No.: 11/656,817 sequence of SEQ. ID. NO. 1, or a functional portion thereof. In some embodiments, the EPSPS enzyme can be a polypep (22) Filed: Jan. 23, 2007 tide molecule comprising an amino acid sequence that is essentially of SEQ. ID. NO. 2, or portion thereof. Embodi Related U.S. Application Data ments include a DNA construct comprising a SEQ. ID. NO. 1 or a functional portion thereof and transgenic methods for (60) Provisional application No. 60/761.216, filed on Jan. inserting the DNA construct into a plant. Some embodi 23, 2006. Provisional application No. 60/761,710, ments include non-transgenic glyphosate resistant turf filed on Jan. 23, 2006. grasses. Patent Application Publication Aug. 2, 2007 Sheet 1 of 4 US 2007/0180574 A1 80 6 O 4 O 2 O f : . K. Control O. lx lx 10x 100X Glyphosate dose (X = 1.12 kgha") FIG. 1 100 8 O 6 O 4O 2 O O Control l 10 100 1000 10,000 Glyphosate (M) FIG 3 Patent Application Publication Aug. 2, 2007 Sheet 2 of 4 US 2007/0180574 A1 poly A mRNA CDNA dT-anchor FP1-> 1- RP1 A-tail a- RP2 --> --- FP2 Anchor primer dT-anchor RP3 -> 1H Anchor primer RP4 (5' end fragment) (main fragment) (3' end fragment) Primer Abreviation. FP1 5'-GAT GCC AAG GAG GAA GTA AAG-3' FP2 5'-TGC TAT CAGAGA TGT TGC GTC CTG-3' RPI 5-TCT CTG GTG GCG TGA TAATG-3 RP2 5'-AAC AGG TGG GCA GTC AGT G-3' RP3 5-ATAGGA CGCTCC CTCATT CTTGGT-3 RP4 5 TTT CCA CCA GCA GCT ACT ACA GCA-3' dT-anchor 5'-GAC CAC GCG TAT COSA TGT CGA CTTTTTTTTTTT-3' Anchor Printer 5'-GAC CAC GCG TAT COGA TGT CGA C-3' FIG 2 Patent Application Publication Aug. 2, 2007 Sheet 3 of 4 US 2007/0180574 A1 100 80 60 40 20 (S 0.5x) (0.5 - 8x) (R 8x) Resistance Class FIG. 4 100 8O 60 40 (S 0.25x) (0.5 - 8x) (R 8x) Resistance Class FIG. 5 Patent Application Publication Aug. 2, 2007 Sheet 4 of 4 US 2007/0180574 A1 100 8 O 60 40 O lx 2x 4x 6x 8x 10x 12x Glyphosate(x = 1.12 kg aiha) FIG. 6 0. 3 0. 2 0.1 Glyphosate (mM) FIG. 7 US 2007/0180574 A1 Aug. 2, 2007 METHODS FOR BREEDING GLYPHOSATE 0006 The present disclosure also provides a glyphosate RESISTANT PLANTS AND COMPOSITIONS resistant plant comprising a non-transgenic glyphosate-re THEREOF sistant Lolium rigidum germplasm. In some embodiments, the present disclosure provides germplasm deposited in the CROSS-REFERENCE TO RELATED ATCC as Penner ryegrass Assession No. XXXX. In some APPLICATIONS embodiments the germplasm comprises a nucleic acid mol ecule of sequence of SEQ. ID. NO. 1, or a functional portion 0001) This application claims the benefit of U.S. Provi thereof. The germplasm can comprise a polypeptide mol sional Application No. 60/761.216 filed on Jan. 23, 2006. ecule of a glyphosate resistant, plant EPSPS enzyme. In This application claims the benefit of U.S. Provisional Some embodiments, the germplasm comprises a polypeptide Application No. 60/761,710 filed on Jan. 23, 2006. The molecule comprising an amino acid sequence of SEQ. ID. disclosures of the above applications are incorporated herein NO. 2, or a functional portion thereof. In some embodi by reference. ments, the germplasm comprises two different glyphosate resistant genes. In some embodiments, the germplasm com INTRODUCTION prises at least one glyphosate resistant protein. The germ plasm can be used in a breeding program or crossed with a 0002 Glyphosate, N-(phosphonomethyl)glycine, is the sexually compatible plant. world's most widely used herbicide. It is sold commercially under such names as RoundUp(R) and Touchdown(R). Gly 0007. In various embodiments, the present invention pro phosate is a foliar non-selective herbicide and has no activity vides grasses or other plants that are in in a golf course in the soil. Glyphosate can be used pre-plant to control fairway, a golf course rough, a golf course tee box, a lawn, emerged weeds in a no-tillage planting system or post an athletic field, a park, a roadside, a right of way for a utility emergence by spot and direct application to control an or a railway, a trail, seed, or Sod. In various embodiments, extensive range of weeds, as well as to control weeds in Such plants are plants of economic value, including glypho glyphosate resistant crops such as, for example, soybeans, sate resistant turfgrass. The turfgrass can be, for example, corn, canola, and cotton. Typically, glyphosate resistant selected from ryegrass, fescue, combinations thereof, and crops are designed using genetic engineering. Naturally hybrids thereof. In some embodiments, the present disclo occurring glyphosate resistant plants are very rare and Such Sure provides glyphosate resistant forage grass. In some rare plants are typically weed varieties. There is a need for embodiments, then present disclosure provides glyphosate more glyphosate resistant crop and ornamental plants and, resistant cereal crops such rye and wheat. especially, glyphosate resistant turf and forage grasses. 0008. The present disclosure also provides seed of gly phosate resistant plants, as well as the mixtures comprising SUMMARY Such seed. The present disclosure further provides progeny, 0003. The present disclosure provides various embodi vegetative sprigs, tillers, tissues cultures, cell protoplast, ments of technology regarding plants having resistance to clones, and/or germplasm of the plants. glyphosate. Various embodiments relate, for example, to grasses having economic value and other plants that com 0009. The present disclosure further provides methods prise genetic material comprising certain amino acid for creating a lawn that is substantially free of a weed variety sequences, comprise certain enzymes, are bred using certain to which the lawn is susceptible. The method includes non-transgenic methods, comprise aspects of certain depos providing a lawn comprising a non-transgenic glyphosate resistant perennial turf grass and applying to the lawn a ited germplasm, or are produced using transgenic methods. herbicidally amount of a glyphosate compound. 0004 For example, the present disclosure provides a plant comprising a polypeptide molecule of a glyphosate DRAWINGS resistant EPSPS enzyme, the polypeptide molecule compris ing an amino acid sequence of SEQ. ID. NO. 2, or a 0010. The skilled artisan will understand that the draw functional portion thereof. In some embodiments, the ings, described herein, are for illustration purposes only. The EPSPS enzyme is not natural to the plant. SEQ. ID. NO. 2 drawings are not intended to limit the scope of the present or functional portion thereof can be encoded by a nucleic disclosure in any way. acid sequence comprising SEQ. ID NO. 1 or a functional portion thereof. In some embodiments, the polypeptide 0011 FIG. 1 is a graph illustrating a dose response curve molecule has about 80% or greater identity with SEQ. ID. for plant injury of resistant, sensitive, and intermediate NO. 2. In some embodiments, the nucleic acid sequence has California rigid ryegrass; about 93% or greater identity with SEQ. ID, NO. 1. 0012 FIG. 2 illustrates a scheme of 5"/3' Random Ampli 0005 The present disclosure also provides a glyphosate fication cDNA End Polymerase Chain Reaction (RACE resistant grass of economic value comprising a nucleic acid PCR) of an EPSPS gene from rigid ryegrass; molecule that encodes a EPSPS enzyme. In some embodi 0013 FIG. 3 is a graph illustrating EPSPS activity of ments, the nucleic acid molecule comprises a sequence of glyphosate resistant (R) and sensitive (S) biotypes of the SEQ. ID. NO. 1, or a functional portion thereof. In various California rigid ryegrass in the presence of glyphosate; embodiments, the nucleic acid molecule has greater than 97% identity with SEQ. ID, NO. 1. In some embodiments, 0014 FIG. 4 is a graph illustrating the distribution of the EPSPS enzyme can be a polypeptide molecule compris glyphosate resistant classes in a F population of plants from ing an amino acid sequence that is essentially of SEQ. ID. a cross between perennial ryegrass and glyphosate resistant NO. 2, or portion thereof. rigid ryegrass; US 2007/0180574 A1 Aug. 2, 2007 0.015 FIG. 5 is a graph illustrating the distribution of the stated features. Specific examples are provided for glyphosate resistant classes in a single Fabc population of illustrative purposes of how to make and use the composi 371 plants derived from a backcross between a glyphosate tions and methods of this technology and, unless explicitly resistant F hybrid plant with a sensitive perennial ryegrass; stated otherwise, are not intended to be a representation that 0016 FIG. 6 is a graph illustrating a glyphosate sensi given embodiments of this technology have, or have not, tivity of the resistant (R) clone from Fabc hybrid 3 and 4 been made or tested.