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WO2008021397A1 - Matériaux et procédés améliorant la qualité et les caractéristiques d'herbes - Google Patents

Matériaux et procédés améliorant la qualité et les caractéristiques d'herbes Download PDF

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WO2008021397A1
WO2008021397A1 PCT/US2007/018065 US2007018065W WO2008021397A1 WO 2008021397 A1 WO2008021397 A1 WO 2008021397A1 US 2007018065 W US2007018065 W US 2007018065W WO 2008021397 A1 WO2008021397 A1 WO 2008021397A1
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grass
seq
bahiagrass
protein
polynucleotide
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Fredy Altpeter
Hangning Zhang
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • Bahiagrass (Paspalum notatutri) represents the prime utility turf along highways in the Southeastern US and is a popular perennial grass for low input residential lawns (http://hortbusiness.ifas.ufl.edu/turfgrass.pdf). Bahiagrass tolerates marginal soil fertility, and its excellent persistence is supported by drought tolerance, heat tolerance, insect- and disease resistance and nematode suppression. However, the turf quality of bahiagrass is compromised by its open-growth habit and the more than 60 cm tall seed heads formed during summer. Improvement of turf quality will necessitate suppression of seed heads and increased number of vegetative tillers per plant. The former will reduce the need for frequent mowing to control seedhead emergence. The latter will also reduce weed encroachment and erosion.
  • plant growth retardants PGR' s
  • PGR' s plant growth retardants
  • plant growth retardants have been used to suppress bahiagrass seedhead production exclusively in low maintenance areas such as highway roadsides, airports, and golf course roughs.
  • new chemicals which may be used in higher maintained commercial situations have been developed.
  • undesirable characteristics which have been associated with application of growth retardants include: phytotoxicity; reduced recuperative potential from physical damage to treated turf; and increased weed pressure due to reduced competition from treated plants (Unruh and Brecke 1999).
  • Transcription factors are key components in the regulation of gene expression and play important roles in plant development and its response to the environment.
  • the functions of an increasing number of plant transcription factors are being elucidated, some of them have been used in genetic engineering for stress tolerance (Valliyodan and Nguyen, 2006; Yamaguchi-Shinozaki and Shinozaki, 2005) and to engineer metabolic pathways (Broun, 2004).
  • Transcriptional regulation of plant development via repression of genes involved in cell elongation, leaf expansion or flowering provides an opportunity for improvement of turf and forage grasses.
  • ATHl Arabidopsis homeobox transcription factor gene
  • ryegrass produced late heading or non-flowering plants with more vegetative tillers, which may be useful to improve fodder quality of perennial ryegrass (van der VaIk et al., 2004).
  • ATHl affects the plant growth as a negative regulator in the light regulated gibberellin biosynthesis pathway (Garcia-Martinez and Gil, 2001; Quaedvlieg et al., 1995).
  • ATHB 16 represses cell elongation in Arabidopsis independent of GA signal transduction (Wang et al., 2003).
  • the ATHB 16 gene encodes a homeodomain leucine zipper class I (HDZip) protein, which acts to regulate plant development as a mediator of plant growth response to light.
  • HDZip homeodomain leucine zipper class I
  • Over-expression of ATHBl 6 in Arabidopsis reduces leaf expansion, leads to reduced shoot length but increased number of shoots, and reduces the sensitivity of flower induction to photoperiod (Wang et al., 2003).
  • the subject invention concerns materials and methods for improving grass quality and characteristics, including suppression or reduction of seedheads, increasing vegetative tillers per plant, increasing forage quality, and increasing resistance to abiotic stress conditions such as drought.
  • Grasses within the scope of the invention include turfgrasses and forage grasses.
  • the turfgrass is bahiagrass.
  • Methods of the invention comprise introducing a nucleic acid encoding a homeodomain leucine zipper class I protein into a grass plant.
  • a grass is transformed with a nucleic acid that encodes Arabidopsis ATHB 16 protein, or a fragment, variant, or homolog thereof that has substantially the same activity as the ATHB 16 protein.
  • the subject invention also concerns grass plants that comprise a heterologous nucleic acid encoding a homeodomain leucine zipper class I protein or that have been modified or engineered to overexpress or constitutively express a nucleic acid encoding a homeodomain leucine zipper class I protein, or a fragment, variant, or homolog thereof.
  • the presented data indicate that over-expression of the Arabidopsis ATHB 16 gene in bahiagrass significantly changes plant architecture of this important low input turfgrass. All transgenic plants investigated produced significant more vegetative and less reproductive tillers, shorter leaves and shorter tillers. Over-expression of ATHB 16 resulted in proportional reduction of leaf width and leaf length. Formation of seedheads under natural photoperiod was delayed in some transgenic lines for approximately 4 weeks. Total root or shoot biomass and seed set were not compromised in semi-dwarf bahiagrass plants over-expressing A THB 16.
  • the present invention provides for changes in plant architecture and flowering of soil and hydroponic grown grass plants, following constitutive over-expression of nucleic acid encoding a leucine zipper class I protein, such as the ATHB 16 transcription factor from Arabidopsis.
  • Figures 1A-1G show generation and molecular characterization of transgenic (ATHB16) bahiagrass plants.
  • Figure IA shows induction of bahiagrass callus from germinating mature seeds of apomictic bahiagrass cultivar "Argentine”.
  • Figure IB shows selection of transgenic bahiagrass callus on paramomycin containing culture medium following biolistic co-transfer of ATHB 16 and nptll expression cassettes.
  • Figure 1C shows regeneration of transgenic bahiagrass plants on paromomycin containing culture medium.
  • Figure ID shows transgenic bahiagrass plants expressing ATHB16 (left) in comparison to transgenic (nptll) plants not-expressing ATHB 16 (right) three weeks after transfer to soil.
  • Figure IE shows PCR analysis of genomic DNA isolated from wildtype plants (WT) or putative transgenic (ATHBl 6) plants (I-4b; I-10b; I-18a; I-28a; I-30a; I-32a) regenerated from paramomycin (50mgr') selection medium in comparison to ATHB 16 plasmid.
  • Figure IF shows RT-PCR analysis for expression of the ATHB 16 gene in wildtype (WT) or transgenic bahiagrass (I-4b; I-10b; I-18a; l-28a; I-30a; I-32a) in comparison to ATHBl 6 plasmid.
  • Figure IG shows southern blot analysis of BamHI restricted genomic DNA from wildtype (WT) or transgenic bahiagrass plants (1-3; I-4b; I-10b; I-18a; I-32a). Signals following hybridization with a full length A THBl 6 cDNA probe are shown.
  • Figures 2 A-2H show transgenic bahiagrass following 4 weeks of propagation from single tillers in hydroponics culture in comparison to wildtype plants.
  • Figure 2A shows establishment of hydroponic bahiagrass culture from single rooted tillers.
  • Figure 2B shows Hydroponic culture four weeks after establishment.
  • Figure 2C shows side view of transgenic bahiagrass lines (I-4b; I- 10b; I-32a) in comparison to wildtype bahiagrass (WT) following 4 weeks of propagation from single tillers in hydroponics culture.
  • Figure 2D shows number of tillers of transgenic bahiagrass lines (I-4b; I-10b; I-32a) and wildtype bahiagrass (WT) following 4 weeks of propagation from single tillers in hydroponics culture.
  • Figure 2E shows length of the longest root of transgenic bahiagrass lines (I-4b; I- 10b; I-32a) and wildtype bahiagrass (WT) following 4 weeks of propagation from single tillers in hydroponics culture.
  • Figure 2F shows length of tillers from crown to leaf tip of transgenic bahiagrass lines (I-4b; I- 10b; I-32a) and wildtype bahiagrass (WT) following 4 weeks of propagation from single tillers in hydroponics culture.
  • Figure 2G shows root biomass dry weight of transgenic bahiagrass lines (I-4b; I-10b; I-32a) and wildtype bahiagrass (WT) following 4 weeks of propagation from single tillers in hydroponics culture.
  • Figure 2H shows shoot biomass dry weight of transgenic bahiagrass lines (I-4b; I-10b; I-32a) and wildtype bahiagrass (WT) following 4 weeks of propagation from single tillers in hydroponics culture.
  • Figures 3A-3F show transgenic bahiagrass grown in soil in comparison to wildtype plants.
  • Figure 3 A shows number of tillers.
  • Figure 3B shows length of tillers from crown to leaf tip.
  • Figure 3 C shows width of leaves.
  • Figure 3D shows length of leaves of transgenic bahiagrass lines (I-4b; I- 10b; I-32a) and wildtype bahiagrass (WT) following two months of vegetative propagation of single tillers in the greenhouse.
  • Figure 3E shows top view of transgenic bahiagrass lines (I-4b; HOb; I-32a) in comparison to wildtype bahiagrass (WT) following a 2 months propagation period of single tillers.
  • Figure 3F shows side view of transgenic bahiagrass line I- 10b in comparison to wildtype bahiagrass (WT) following 17 weeks of vegetative propagation from single tillers in the greenhouse.
  • Figure 4 shows RT-PCR analysis for expression of the ATHB 16 gene in transgenic I lines. (NC: negative control, PC: positive control, and WT: wild type).
  • Figures 5A-5C show propagation and field establishment of transgenic lines.
  • Figure 5A shows propagation of plants under greenhouse conditions.
  • Figure 5B shows establishment of field plots, and
  • Figure 5C shows field plots 4 weeks after transplanting.
  • Figure 7 shows chlorophyll content measured with a Minolta SPAD 502 meter 4 weeks after transplanting (diagonal cross bar column), 8 weeks after transplanting (filled-in column), and 12 weeks (open column) after transplanting. Bars of the same observation timepoint with different letters indicate significant difference at P ⁇ 0.05.
  • Figures 8A-8B show comparison transgenic lines four weeks after transplanting.
  • Figure 8 A shows 1 10 on the right and wildtype on the left.
  • Figure 8B shows 132 on the right) and wild type on the left.
  • Figure 9 shows number of tillers produced by transgenic lines and wild-type in a 10x10 cm area 8 weeks after transplanting. Bars of the same observation timepoint with different letters indicate significant difference at P ⁇ 0.05.
  • Figures HA and HB show freshly mowed transgenic lines Hl and 1-10 (Figure HA) compares to wild-type (Figure 1 IB) following weekly mowing at 8cm mowing height and fourteen weeks after transplanting.
  • Figure 12A shows establishment of transgenic lines and wild-types in treepots.
  • Figure 12B shows transgenic lines and wild-types after six weeks under non-irrigated conditions.
  • Figure 12C shows transgenic lines and wild-types after twelve weeks under non- irrigated conditions.
  • Figures 13A-13B show transgenic lines and wild-types three weeks after re-hydration.
  • Figures 14A-14C show Relative Water Content of transgenic lines and wild-types.
  • Figure 14A shows Relative Water Content of transgenic lines and wild-types under well-watered conditions.
  • Figure 14B shows Relative Water Content of transgenic lines and wild-types six weeks after withholding irrigation.
  • Figure 14C shows Relative Water Content of transgenic lines and wild-types eight weeks after withholding irrigation (• indicates significant difference from the wild-type bahiagrass (WT-AB) and * indicates significant differences from St. Augustinegrass (WT-SA) atP ⁇ 0.05).
  • Figure 15 shows Volumetric Soil Water Content measured under well-watered conditions (line with filled-in diamonds), six weeks after withholding irrigation (line with filled-in squares), and eight weeks after withholding irrigation (line with filled-in triangles) (• indicates significant difference from the wild-type bahiagrass (WT-AB) and * indicates significant differences from St. Augustinegrass (WT-SA) at P ⁇ 0.05).
  • Figure 16A shows visual scores one week after re-hydration.
  • Figure 16A shows visual scores two weeks after re-hydration (• indicates significant difference from the wild-type bahiagrass (WT-AB) and * indicates significant differences from St. Augustinegrass (WT-SA) at P ⁇ 0.05).
  • Figure 17A shows shoot growth of transgenic lines and wild-types three weeks after re-hydration.
  • Figure 17B shows shoot total biomass (dead and green).
  • Figure 17C shows root biomass of transgenic lines and wild-types three weeks after re-hydration (• indicates significant difference from the wild-type bahiagrass (WT-AB) and * indicates significant differences from St. Augustinegrass (WT-SA) at PO.05).
  • SEQ ID NO: 1 is a nucleic acid sequence of ATHB16 gene in Arabidopsis thaliana that can be used according to the present invention.
  • SEQ ID NO: 2 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 1.
  • SEQ ID NO: 3 is a PCR primer that can be used according to the present invention.
  • SEQ ID NO: 4 is a PCR primer that can be used according to the present invention.
  • SEQ ID NO: 5 is a PCR primer that can be used according to the present invention.
  • SEQ ID NO: 6 is a PCR primer that can be used according to the present invention.
  • SEQ ED NO: 7 is a nucleotide coding sequence for the ATHB16 protein shown in SEQ ID NO: 2.
  • SEQ ID NO: 8 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 9 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ED NO: 10 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 9.
  • SEQ ED NO: 11 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 12 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 11.
  • SEQ ED NO: 13 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ED NO: 14 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 13.
  • SEQ ED NO: 15 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 16 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 15.
  • SEQ ID NO: 17 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 18 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 17.
  • SEQ ID NO: 19 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 20 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 19.
  • SEQ ID NO: 21 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 22 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 21.
  • SEQ ID NO: 23 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 24 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 23.
  • SEQ ID NO: 25 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 26 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 25.
  • SEQ ID NO: 27 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 28 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 27.
  • SEQ ID NO: 29 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 30 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 29.
  • SEQ ID NO: 31 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 32 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 31.
  • SEQ ID NO: 33 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 31.
  • SEQ DD NO: 34 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 35 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 34.
  • SEQ ID NO: 36 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 37 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 36.
  • SEQ ED NO: 38 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 39 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 38.
  • SEQ ED NO: 40 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ED NO: 41 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ED NO: 42 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 41.
  • SEQ ED NO: 43 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ED NO: 44 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 43.
  • SEQ ED NO: 45 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 46 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 45.
  • SEQ ED NO: 47 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 48 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 47.
  • SEQ ID NO: 49 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 50 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 49.
  • SEQ ID NO: 51 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 52 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 51.
  • SEQ ID NO: 53 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 54 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 55 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 54.
  • SEQ ID NO: 56 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 57 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 56.
  • SEQ ID NO: 58 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 59 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 58.
  • SEQ DD NO: 60 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 61 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 60.
  • SEQ ID NO: 62 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 63 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 62.
  • SEQ ID NO: 64 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 65 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 66 is an amino acid sequence encoded by the nucleic acid of SEQ ID NO: 65.
  • SEQ ED NO: 67 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 68 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 67.
  • SEQ ED NO: 69 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ED NO: 70 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 69.
  • SEQ ED NO: 71 is a nucleic acid sequence that can be used according to the present invention.
  • SEQ ID NO: 72 is an amino acid sequence encoded by the nucleic acid of SEQ ED NO: 71.
  • the subject invention concerns materials and methods for improving quality and characteristics of grasses, including suppressed or reduced numbers of seedheads, delayed or later seedhead production, increased number of vegetative tillers per plant, shorter tillers, shorter leaves, finer leaves, reduced senescence, increased forage quality, and increased resistance to abiotic stress conditions such as drought and temperature ⁇ e.g., resistance to cold and/or heat stress conditions).
  • Grasses included within the scope of the invention are both turfgrasses and forage grasses.
  • Turfgrasses contemplated within the scope of the invention include, but are not limited to, Bahiagrass, Brachiariagrass, St.
  • the turfgrass is bahiagrass.
  • Forage grasses contemplated within the scope of the invention includes, but are not limited to, Bahiagrass, Bachiariagrass, Stargrass, Bermudagrass, Tall Fescue, Perennial Ryegrass, Annual Ryegrass, Rye, Wheat, Pennisetum, and Limpograss.
  • transgenic bahiagrass up to 76% and 41% respectively.
  • Formation of normal flowers and seed set was not compromised in transgenic bahiagrass constitutively over-expressing ATHB 16 in contrast to constitutive over-expression of gibberellin catabolizing enzyme GA 2-oxidase (Sakamoto et al., 2001).
  • Most of the warm season turfgrasses are vegetatively mass-propagated and installed as sod, eliminating the need for seed production.
  • seed production is not desired to prevent introduction of genetic variability by open pollination which might compromise the uniformity of the turf and might result in transgene dispersal by pollen.
  • ATHl affects the plant growth as a negative regulator in the light regulated gibberellin biosynthesis pathway (Garcia-Martinez and Gil, 2001; Quaedvlieg et al., 1995). In contrast, Axabidopsis ATHBl 6 represses cell elongation independent of GA signal transduction (Wang et ⁇ l., 2003). Target genes downstream of ATHB 16 have still to be identified. Potential target genes for the ATHBI6 repressor include receptors for auxin-dependent cell expansion (Jones et ⁇ l., 1998; Schwob et ⁇ l., 1993). HDzip genes like ATHB16 represent a large gene family with 42 members in Arabid ⁇ psis.
  • ATHB16 can interact with other HDzip proteins especially with ATHB6 (Wang, 2001).
  • ATHB6 is known to be upregulated in response to water- deficit conditions and to treatment of abscisic acid, and has been proposed to function as a regulator of growth and development in response to limited water conditions (Himmelbach et ⁇ l., 2002; S ⁇ derman et ⁇ l., 1999). This implies that over-expression of ATHB 16 can, optionally in concert with ATHB 6, reduce plant growth during water deficit, which can enhance the drought tolerance of transgenic plants over-expressing ATHB 16.
  • Bahiagrass cultivar "Argentine” is also a very popular forage grass in subtropical regions. Since total biomass production does not seem to be negatively impacted in transgenic bahiagrass plants, over-expressing ATHB 16 can also improve the forage quality of bahiagrass by higher production of better digestible young, vegetative tillers on expense of poorly digestible seedheads. In floral stems, low digestible compounds such as lignin and cell wall compounds cross-linked with lignin accumulate, which reduce the palatability and therefore fodder quality of the grass. Digestibility and productivity can be analyzed using field grown transgenic bahiagrass.
  • Argentine bahiagrass has no leaf tissue freezing tolerance which compromises its performance in regions with seasonal freezing temperatures, e.g. Northern Florida.
  • the altered growing pattern with shorter shoots and higher tiller density can also affect the micro- environment in the field and reduce the leaf tissue damage during short freezing periods, which are typically experienced in the Northern parts of the subtropical regions where bahiagrass is grown.
  • a grass plant, plant tissue, or plant cell is transformed with a nucleic acid encoding a homeodomain leucine zipper class I protein, or a biologically active fragment, variant, or homolog.
  • nucleic acids, and homeodomain leucine zipper protein encoded thereby included within the scope of the invention are disclosed at Genbank database under accession numbers DQ226915.1, AY336103.1, AF268422.1, AY101610.1, AB092574.1, AF443620.1, AF184277.2, AF402604.1, AP006364.1, CT571261.1, AC145165.9, AB028072.1, AC139840.1, AC135288.1, AC142505.1, AC145120.1, D26578.1, AM486924.2, AB028076.1, AB084623.1, AB028078.2, EF025304.1, CR954197.2, AB028080.2, AK247553.1, CU
  • Transformed plant, plant tissue or plant cell incorporating the nucleic acid in its genome can be selected for and a transgenic grass produced therefrom.
  • a grass is transformed with a nucleic acid that encodes Arabidopsis ATHB 16 protein, or a fragment, variant, or homolog thereof that has substantially the same activity as the ATHB 16 protein.
  • Methods for screening for and obtaining fragments, variants, and homologs of a protein are known in the art.
  • Variants and homologs of ATHB 16 proteins from other plant species are contemplated within the scope of the present invention.
  • the nucleic acid comprises the protein coding sequence of the nucleotide sequence of the ATHB 16 gene (Genbank Accession No.
  • a nucleic acid of the invention used to transform a grass comprises the nucleotide sequence shown in SEQ DD NO: 1, or a fragment, variant, or homolog thereof.
  • the nucleic acid comprises a nucleotide sequence that encodes a protein having the amino acid sequence shown in SEQ ID NO: 2, or a fragment, variant, or homolog thereof that has substantially the same activity as the protein of SEQ ID NO: 2.
  • the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 7 which encodes the protein having the amino acid sequence shown in SEQ ID NO: 2.
  • the nucleic acid comprises a nucleotide sequence that encodes a protein having an amino acid sequence shown in one or more of SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:
  • SEQ ID NO: 39 SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and/or SEQ ID NO: 72.
  • the nucleic acid can comprise a nucleotide sequence show in one or more of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 34, SEQ DD NO: 36, SEQ ID NO:
  • a transformed plant, plant tissue, or plant cell can comprise in its genome one or more copies of a nucleic acid of the invention.
  • a plant, plant tissue, or plant cell is transformed with or comprises multiple copies of one or more nucleic acids of the invention.
  • the nucleic acid is provided in an expression construct that results in overexpression or constitutive expression of the homeodomain leucine zipper class I protein, or a fragment, variant, or homolog thereof.
  • the nucleic acid is provided in a construct that results in. overexpression of the nucleic acid in the plant.
  • Expression constructs of the invention can include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed.
  • Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.
  • operably linked refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner. In general, operably linked components are in contiguous relation.
  • An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence of the invention.
  • the promoter is one that provides for overexpression of a polynucleotide of the invention.
  • Promoters useful for overexpression of an operably linked nucleic acid sequence are known in the art. Promoters can be incorporated into a polynucleotide sequence or an expression construct using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention.
  • a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity.
  • a transcription start site is typically included in the expression construct.
  • plant viral promoters such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Patent No. 5,106,739)) or a CaMV 19S promoter can be used.
  • the promoter is a CaMV 35S promoter.
  • Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA 1'- or 2'-promoter of A.
  • tumefaciens polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR- Ia promoter, ubiquitin promoter (U.S. Patent Nos. 6,528,701 and 6,054,574), actin promoter (e.g., from rice), alcA gene promoter, pin2 promoter (Xu et ah, 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Patent No.
  • alfalfa His 3 promoter for example fruit-specific promoters, such as the E8 promoter of tomato (accession number: AF515784; Good et al. (1994)), a hybrid E4/E8 promoter (U.S. Patent No. 6,118,049), the LeExp-1 promoter (U.S.
  • Patent No. 6,340,748) and the polygalacturonase- ⁇ subunit promoter of tomato (U.S. Patent No. 6,127,179) can be used.
  • Flower organ-specific promoters can be used with an expression construct of the present invention for expressing a polynucleotide of the invention in the flower organ of a plant. Examples of flower organ-specific promoters include any of the promoter sequences described in U.S. Patent Nos. 6,462,185; 5,859,328; 5,652,354; 5,639,948; and 5,589,610.
  • Seed-specific promoters such as the promoter from a ⁇ -phaseolin gene ⁇ e.g., of kidney bean) or a glycinin gene (e.g., of soybean), and others, can also be used.
  • Root-specific promoters such as any of the promoter sequences described in U.S. Patent No. 6,455,760 or U.S. Patent No. 6,696,623, or in published U.S. patent application Nos. 20040078841; 20040067506; 20040019934; 20030177536; 20030084486; or 20040123349, can be used with an expression construct of the invention.
  • Constitutive promoters such as a CaMV, ubiquitin, actin, or NOS promoter
  • developmentally-regulated promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • inducible promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements.
  • Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination.
  • a signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment.
  • Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct.
  • Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35 S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element.
  • CMV cytomegalovirus
  • Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken- 1 enhancer element (Clancy and Hannah, 2002).
  • DNA sequences which direct polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, and include, but are not limited to, an octopine synthase or nopaline synthase signal.
  • the expression constructs of the invention can also include a polynucleotide sequence that directs transposition of other genes, i.e., a transposon.
  • Expression constructs can also include one or more dominant selectable marker genes, including, for example, genes encoding antibiotic resistance and/or herbicide-resistance for selecting transformed cells.
  • Antibiotic-resistance genes can provide for resistance to one or more of the following antibiotics: hygromycin, kanamycin, bleomycin, G418, streptomycin, paromomycin, neomycin, and spectinomycin.
  • Kanamycin resistance can be provided by neomycin phosphotransferase (NPT II).
  • Herbicide-resistance genes can provide for resistance to phosphinothricin acetyltransferase or glyphosate.
  • markers used for cell transformation screening include genes encoding ⁇ -glucuronidase (GUS) 5 ⁇ -galactosidase, luciferase, nopaline synthase, chloramphenicol acetyltransferase (CAT), green fluorescence protein (GFP), or enhanced GFP (Yang et ⁇ l, 1996).
  • GUS ⁇ -glucuronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescence protein
  • enhanced GFP Yang et ⁇ l, 1996.
  • the subject invention also concerns polynucleotide vectors comprising a polynucleotide sequence of the invention that encodes a homeodomain leucine zipper class I polypeptide of the invention.
  • Unique restriction enzyme sites can be included at the 5' and 3' ends of an expression construct or polynucleotide of the invention to allow for insertion into a polynucleotide vector.
  • vector refers to any genetic element, including for example, plasmids, cosmids, chromosomes, phage, virus, and the like, which is capable of replication when associated with proper control elements and which can transfer polynucleotide sequences between cells.
  • Vectors contain a nucleotide sequence that permits the vector to replicate in a selected host cell.
  • a number of vectors are available for expression and/or cloning, and include, but are not limited to, pBR322, pUC series, pGEM series, M 13 series, and pBLUESCRIPT vectors (Stratagene, La Jolla, CA).
  • Polynucleotides of the present invention can be composed of either RNA or DNA. Preferably, the polynucleotides are composed of DNA.
  • the subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.
  • polynucleotide sequences can encode a homeodomain leucine zipper class I polypeptide of the present invention (e.g., an ATHDB 16 protein), or a fragment, variant, or homolog thereof.
  • a table showing all possible triplet codons (and where U also stands for T) and the amino acid encoded by each codon is described in Lewin (1985).
  • U also stands for T the amino acid encoded by each codon
  • references to "essentially the same" sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the functional activity of the polypeptide encoded by the polynucleotides of the present invention.
  • amino acids other than those specifically exemplified or naturally present in a polypeptide of the invention are also contemplated within the scope of the present invention.
  • non-natural amino acids can be substituted for the amino acids of a polypeptide, so long as the polypeptide having the substituted amino acids retains substantially the same functional activity as the polypeptide in which amino acids have not been substituted.
  • non-natural, amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4- diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, ⁇ - amino butyric acid, ⁇ -amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, ⁇ - butylglycine, ⁇ -butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C-methyl amino acids, N-
  • Non-natural amino acids also include amino acids having derivatized side groups.
  • any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form.
  • Allelic variants of a protein sequence of a polypeptide of the present invention are also encompassed within the scope of the invention.
  • Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide encoded by a polynucleotide of the present invention having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polynucleotide encoding the substitution still retains substantially the same functional activity as the polynucleotide that does not have the substitution. Polynucleotides encoding a polypeptide having one or more amino acid substitutions in the sequence are contemplated within the scope of the present invention. Table 2 below provides a listing of examples of amino acids belonging to each class. Single letter amino acid abbreviations are defined in Table 3.
  • the subject invention also concerns variants of the polynucleotides of the present invention that encode polypeptides of the invention.
  • Variant sequences include those sequences wherein one or more nucleotides of the sequence have been substituted, deleted, and/or inserted.
  • the nucleotides that can be substituted for natural nucleotides of DNA have a base moiety that can include, but is not limited to, inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, and tritylated bases.
  • the sugar moiety of the nucleotide in a sequence can also be modified and includes, but is not limited to, arabinose, xylulose, and hexose.
  • the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, and/or thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.
  • Polynucleotides and proteins of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein.
  • the sequence identity will typically be greater than about 60%, preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 90%, and can be greater than about 95%.
  • the identity and/or similarity of a sequence can be about 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
  • the subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the sequences exemplified herein so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis, T. et al., 1982).
  • stringent conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6x SSPE, 5x Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.
  • Tm melting temperature
  • Washes are typically carried out as follows:
  • polynucleotide sequences that hybridize under stringent conditions with the sequence of SEQ ID NO: 1 or SEQ ID NO: 7.
  • Examples include sequences disclosed at Genbank accession numbers DQ226915, AY336103, and AYlOl 610.
  • nucleic acid and “polynucleotide” refer to a deoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide and ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides.
  • the polynucleotide sequences include the DNA strand sequence that is transcribed into RNA and the strand sequence that is complementary to the DNA strand that is transcribed.
  • the polynucleotide sequence includes both the sense and antisense strands either as individual strands or in the duplex.
  • the subject invention also concerns polynucleotides, and their use in the methods of the present invention, that encode a fragment, variant, or homolog of a leucine zipper class I polypeptide of the invention that have substantially the same activity as the leucine zipper class I polypeptide from which the fragment, variant, or homolog is derived.
  • polypeptide fragments of ATHB 16 protein typically comprise a contiguous span of about or at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118
  • Polypeptide fragments of ATHB16 protein can be any integer in length from at least about 25 consecutive amino acids to 1 amino acid less than the sequence shown in SEQ ID NO: 2.
  • a polypeptide fragment can be any integer of consecutive amino acids from about 25 to 293 amino acids.
  • integer is used herein in its mathematical sense and thus representative integers include: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,
  • Each polypeptide fragment of ATHB 16 protein can also be described in terms of its N- terminal and C-terminal positions. For example, combinations of N-terminal to C-terminal fragments of about 25 contiguous amino acids to 1 amino acid less than the full length polypeptide of SEQ ID NO: 2 are included in the present invention.
  • a 25 consecutive amino acid fragment could correspond to amino acids of SEQ ID NO: 2 selected from the group consisting of 1-25, 2-26, 3-27, 4-28, 5-29, 6-30, 7-31, 8-32, 9-33, 10-34, 11-35, 12-36, 13-37, 14-38, 15-39, 16-40, 17-41, 18-42, 19-43, 20-44, 21- 45, 22-46, 23-47, 24-48, 25-49, 26-50, 27-51, 28-52, 29-53, 30-54, 31-55, 32-56, 33-57, 34- 58, 35-59, 36-60, 37-61, 38-62, 39-63, 40-64, 41-65, 42-66, 43-67, 44-68, 45-69, 46-70, 47- 71, 48-72, 49-73, 50-74, 51-75, 52-76, 53-77, 54-78, 55-79, 56-80, 57-81,
  • amino acids corresponding to all other fragments of sizes between 26 consecutive amino acids and 293 consecutive amino acids of SEQ ED NO: 2 are included in the present invention and can also be immediately envisaged based on these examples. Therefore, additional examples, illustrating various fragments of the polypeptides of SEQ ID NO: 2 are not individually listed herein in order to avoid unnecessarily lengthening the specification.
  • Polypeptide fragments comprising: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
  • c is an integer between 25 and the number of amino acids of the full length polypeptide sequence (294 for SEQ ID NO: 2) and "n” is an integer smaller than "c" by at least 24.
  • n is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
  • n is a value less than "c" by at least 24. Every combination of "n” and “c” positions are included as specific embodiments of polypeptide fragments of ATHB 16 protein of the invention. All ranges used to describe any polypeptide fragment .embodiment of the present invention are inclusive unless specifically set forth otherwise.
  • a grass plant comprises a nucleic acid encoding a leucine zipper class I polypeptide, or a fragment, variant, or homolog thereof.
  • a grass plant comprises a nucleic acid that encodes an Arabidopsis ATHB 16 protein, or a fragment, variant, or homolog thereof.
  • the ATHB 16 protein comprises the amino acid sequence shown in SEQ ID NO: 2, or a fragment, variant, or homolog thereof.
  • a grass plant comprises a nucleic acid comprising the nucleotide sequence shown in SEQ ED NO: 1 or SEQ ID NO: 7.
  • a grass plant comprises any of the nucleic acids having any of the sequences disclosed herein or that encode a protein having any of the sequences disclosed herein, or a fragment, variant, or homolog thereof.
  • the nucleic acid, or the protein encoded thereby such as ATHB 16 is overexpressed or constitutively expressed in the plant.
  • Methods for transforming plants with heterologous nucleic acid are known in the art and any suitable method can be used with the present invention.
  • Turfgrasses contemplated within the scope of the invention include Bahiagrass, St.
  • grasses contemplated within the scope of the invention include Bahiagrass, Brachiariagrass, St. Augustine, Bermudagrass, Bentgrass, Zoysia, Tall fescue, Perennial Ryegrass, Kentucky Bluegrass, Buffalograss, Carpetgrass, Seashore Paspalum, and Centipedegrass.
  • Forage grasses contemplated within the scope of the invention include Bahiagrass, Brachiariagrass, St. Augustine, Bermudagrass, Bentgrass, Zoysia, Tall fescue, Perennial Ryegrass, Kentucky Bluegrass, Buffalograss, Carpetgrass, Seashore Paspalum, and Centipedegrass.
  • the grass is transgenic.
  • Polynucleotides of the present invention can be introduced directly into plants, e.g., by biolistic or Agrobacterium-mediated transformation (see, for example, U.S. Patent No. 7,057,090), and transformed and transgenic plant lines prepared therefrom. Plants containing a polynucleotide of the invention can also be prepared through conventional breeding from a transformed or transgenic breeding line. Agrob ⁇ cterium containing a polynucleotide of the invention can be used to transform plant cells with the polynucleotide according to standard methods known in the art. For Agrob ⁇ cterium transformation, polynucleotide vectors of the invention can also include T-DNA sequences.
  • Polynucleotides can also be introduced into plant cells by a biolistic method (Carrer, 1995), by electroporation, by direct gene injection, and by other methods known in the art. Plants can also be transformed with polynucleotides of the present invention using marker-free transformation techniques (Zuo et ⁇ l, 2002).
  • the coding region of ATHB 16 gene was amplified from Arabidopsis ecotype Columbia cDNA using primers A16F: 5 -AGGATCCACGCGT
  • ATGAAGAGACTAAGCAGCTCO 1 SEQ ID NO: 3
  • A16R 5'-AGAGCTC TCAAGTCCAATGATCTGAAG-S 1 (SEQ ID NO: 4)
  • the primer were designed based on the ATHB 16 gene coding DNA sequence (cds) present in GenBank under the accession number of AF076641.
  • MS medium Physical Engineering medium
  • the RNEASY Plant Mini Kit Qiagen, Valencia, CA
  • ISCRIPT cDNA Synthesis kit BioRad, Hercules, CA
  • the amplified 885 bp fragment was cloned in the pDrive vector (Qiagen, USA), sequenced and inserted into an expression vector between CaMV 35 S promoter (Odell et al, 1985)with HSP70 intron (Rochester et al, 1986) and NOS (nopaline synthase) 3' terminator (Bevan, 1984; Fraley et al, 1983) following excision with the restriction enzymes Barri ⁇ l and Sad.
  • the fragment of 2.6 kb containing the entire expression cassette was isolated from the vector backbone for biolistic gene transfer following restriction digest and gel purification.
  • Fig. IA Mature seeds of bahiagrass 'Argentine', a tetraploid, apomictic cultivar, were used to initiate callus induction for genetic transformation.
  • the tissue culture and gene transformation procedures were as described by Altpeter and James (2005).
  • the gene npt II neomycin phosphotransferase D
  • ATHB 16 expression cassette was co-transformed with ATHB 16 expression cassette.
  • the ATHB 16 and npt II expression cassettes were mixed in a 2:1 ratio, and precipitated on 1.0 ⁇ m diameter gold particles as described previously (Sanford et al, 1991) and delivered to ernbryogenic calli of Argentine bahiagrass using a DuPontPDS- 1000/He device (Sanford et al, 1991) at 1100 psi.
  • Transgenic callus (Fig. IB) and plantlets (Fig. 1C) were selected on medium containing 50 mgl "1 paromomycin sulfate. Rooted transgenic plants were transferred to soil and propagated under controlled environment conditions at 27°C/20°C day/night with 12 hour photoperiod and 800 InEnV 2 S "1 light.
  • PCR Polymerase chain reaction
  • RT-PCR reverse transcription PCR
  • genomic DNA was extracted from approx. 100 mg leaf tissue using a DNEASY Plant Mini Kit (Qiagen, Valencia, CA), and about 100 ng genomic DNA was used as templates. PCR was carried out in an Eppendorf MASTERCYCLER (New York City, USA). Samples were denatured at 95 0 C for 15 min; followed by 30 cycles at 95 0 C for 30sec, 6O 0 C for 30sec, 72 0 C for lmin; and final extension at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 1.5% agrose gel.
  • the primer pair with sense 5'-TGGGTCTATCGGAGAAGAAG-S' (SEQ ID NO: 5) and anti- sense 5'-TTGGAGAAGGGAATCATTGT-S' (SEQ ID NO: 6) was designed to amplify a 278 bp fragment from the gene ATHB 16, the same primer pair was used for RT-PCR expression analysis.
  • total RNA was extracted from 100 mg leaves using the RNEASY Plant Mini Kit (Qiagen, Valencia, CA); 500 ⁇ g of total RNA was used for cDNA synthesis via reverse transcription with the ISCRJDPT cDNA Synthesis kit (BioRad, Hercules, CA) in a reaction volume of 20 ⁇ l. 2 ⁇ l of the cDNA were used as a template to detect the transcripts of the gene ATHB 16 by PCR with the same primer pair as described above for PCR from genomic DNA.
  • Total genomic DNA was isolated from leaves of transgenic and wild type plants as described by Saghai-Maroof et al (Saghai-Maroof et al., 1984). 20 ⁇ g of genomic DNA, fully digested with Bam ⁇ I were separated with electrophoresis using a 1% agrose gel and then blotted onto a Hybond-N+ membrane (GE Healthcare (formerly Amersham Biosciences), Pistcataway, NJ). The amplified 897 bp fragment from the ATHB 16 gene was used as probe. Hybridization and detection were performed according to the manufacturers' instructions.
  • Vector backbone sequences of both the constitutive ATHB 16 expression cassette and the nptll expression cassette were removed by restriction digestion and gel purification. Unlinked expression cassettes were precipitated on gold particles in a 2:1 (ATHB 16: nptll) molar ratio, introduced into mature seed derived callus by biolistic gene transfer and transgenic events were selected by growth and regeneration on paromomycin containing culture medium as described earlier (Altpeter and James 2005). A total of twenty-one independent paramomycin-resistant bahiagrass plants were regenerated from 300 bombarded callus pieces. PCR ( Figure 2A) with primers annealing to the ATH 16 coding region revealed that 18 of them had at least one copy of the ATHB 16 transgene.
  • ATHB 16 expression was correlated with an increased number of vegetative tillers and a proportional semi-dwarfing (shorter and finer leaves), while transgenic lines without detectable transcript displayed a phenotype like the wildtype "Argentine" bahiagrass.
  • Three lines expressing ATHB 16 and differing in the severity of the semi-dwarf and dense phenotype were selected for more detailed morphological analysis.
  • Mass propagation was achieved by using rooted tillers of equal size to initiate soil or hydroponics culture.
  • the hydroponic grown transgenic lines (I-4b, 10b and 32a) showed a significantly increased number of tillers (Fig. 2C and 2D), reduced tiller length ( Figure 2F). None of the transgenic lines produced less root (Fig.
  • Transgenic line I- 10b showed the highest density with 77% more tiller ' s generated than wild type (Fig. 2D) and 87% more shoot biomass than wildtype (Figure 2H) with no significant difference in root biomass to wildtype but 20% longer roots.
  • the length of the transgenic tillers were 29%; 18% or 17% less than wild type, for I-4b; I-10b or I-32a respectively (Fig. 2F).
  • 1-32 represents the transgenic line that is most similar to wildtype in tiller length (Figure 2F) but produced 34% more shoot (Fig. 2H) and 32% more root biomass (Fig. 2G) dry weight and 39% longer roots (Fig. 2E) than wildtype.
  • I-4b the line with the shortest tillers (Figure 2F) did not show any significant difference to wildtype in shoot (Figure 2H) or root biomass (Figure 2G) dry weight, or length of the longest root ( Figure 2E).
  • AU soil grown transgenic lines (I-4b, 10b and 32a) showed a significantly increased number of tillers (Figure 3A), reduced tiller length (Figure 3B) resulting from shorter leaves (Fig. 3D) and a shorter tiller base (data not shown), narrower leaves (Figure 3C) than the wild type plants.
  • Transgenic line I-10b showed the highest density with 38% more tillers generated than wild type ( Figure 3A).
  • the length and width of the leaves of I- 10b were 36% (Fig. 3B) and 17% (Figure 3C) less than wild type, respectively.
  • I-10b displayed delayed flowering compared to the wildtype and had significantly less and shorter seedheads (Figure 3F; Table 1).
  • Line I-32a represented the transgenic line which was most similar to the wildtype but also displayed significant differences with 15% more tillers generated than wild type (Figure 3A). The length and width of the leaves of I-32a were 26% (Figure 3B) and 20% less (Figure 3C) than wild type, respectively. Transgenic lines produced a large number of roots similar or better than wildtype as shown for I-10b ( Figure 3F). While all transgenic plants produced seedheads with normal flower morphology. Heading was delayed for more than a month in transgenic lines I-4b and I-10b (Table 1).
  • Plants from three transgenic lines (1-4, 1-10 and 1-32) expressing ATHB16 (Figure 4) were propagated under greenhouse conditions along with wildtype "Argentine” bahiagrass and St. Augustine grass “Floratam” ( Figure 5A).
  • Transgenic and wild-type plants were established ( Figure 5B) and evaluated at the UF-IFAS Plant Research and Education Center in Citra, Florida (USDA permit 05-364-0Ir) in a randomized block design with a total of 24 replications evaluated in small field plots (Figure 5C). Data on establishment, turf density, chlorophyll content, and seed-head production and length was gathered during three months of growth after transplanting.
  • Transgenic I- 10 lines showed overall faster establishment with more vegetative tillers than other lines and wild type bahiagrass and St. Augustine grass ( Figure 6).
  • the turf quality of wild-type bahiagrass is compromised by its open growth habit.
  • the sparse looking lawn will affect its aesthetic value and facilitates weed encroachment.
  • the transgenic line 1-10 displayed the highest turf density (Figure 8) as a consequence of significantly more tillers per area then wild-type ( Figure 9).
  • Transgenic lines also displayed proportional dwarfing ( Figure 8).
  • NTEP National Turf Evaluation Program
  • transgenic bahiagrass lines over-expressing ATHBl 6 displayed improved turf quality under field conditions including higher turf density and delayed or reduced seed- head formation.
  • the low-input characteristics of bahiagrass, including ease of establishment and persistence were maintained in the transgenic plants.
  • EXAMPLE 4 EVALUATION OF BAHIAGRASS LINES CONTAINING ATHB 16 FOR DROUGHT STRESS RESPONSE IN COMPARISON TO WILD-TYPE BAHIAGRASS PLANTS AND ST. AUGUSTINEGRASS UNDER CONTROLLED ENVIRONMENT CONDITIONS
  • Transgenic lines and the wild-types were established in top soil using single uniform tillers per pot (15 cm diameter) in five replications. The plants were allowed to grow under greenhouse conditions until closed canopy was achieved. The plants were then transplanted into 15x15 cm wide and 41 cm deep treepots using top soil ( Figure 12A). The pots were placed in two bins in a completely randomized design with five replications. Following transplanting, the plants were allowed to grow in the bins for four weeks with daily irrigation.
  • WT-SA St. Augustinegrass
  • transgenic line 14 Six weeks after withholding irrigation, transgenic line 14 had the highest soil moisture and was significantly different from both wild-type bahiagrass and St. Augustinegrass (P ⁇ 0.05; Figure 15). At the end of eight weeks under non-irrigated conditions, there were no significant differences among the transgenic lines, wild-type bahiagrass and St. Augustinegrass (P ⁇ 0.05; Figure 15).
  • transgenic lines were measured by estimating the difference between the total biomass (dead and necrotic) and dry weight of the shoots of transgenic lines and wild-types.
  • Line 132 had the highest differential between freshweight and dry weight biomass and was significantly higher than wild-type bahiagrass and St. Augustinegrass (PO.05; Figure 17A) indicating better regrowth after drought stress.
  • Total biomass of shoot dry weight for line 132 was also the highest and differed significantly from the wild-type bahiagrass and St. Augustinegrass (P ⁇ 0.05; Figure 17B).
  • the dry weight of the roots of transgenic lines was not significantly different from wild-type bahiagrass and St. Augustinegrass (P ⁇ 0.05; Figure 17C).
  • auxin-dependent cell expansion mediated by overexpressed auxin-binding protein 1 Science. 282:1114-1117.

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Abstract

Matériaux et procédés améliorant la qualité et les caractéristiques d'herbes, y compris la suppression ou la réduction de tiges porte-graines, l'augmentation de talles végétatives par plante, l'augmentation de la qualité de fourrage et l'augmentation de la résistance aux conditions de stress abiotique. Selon une variante, l'herbe est de l'herbe de Bahia. On peut transformer l'herbe avec un acide nucléique codant une protéine de classe I à fermeture éclair à leucines, par exemple une protéine ATHB16 d'Arabidopsis, y compris un fragment, variant ou homologue correspondant ayant sensiblement la même activité que cette protéine ATHB 16. L'acide nucléique peut être fourni dans une construction qui donne une surepxression ou une expression constitutive de la protéine.
PCT/US2007/018065 2006-08-15 2007-08-15 Matériaux et procédés améliorant la qualité et les caractéristiques d'herbes Ceased WO2008021397A1 (fr)

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Cited By (2)

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WO2010139993A1 (fr) * 2009-06-01 2010-12-09 Plant Bioscience Limited Procédés et compositions pour la tolérance au stress chez les végétaux
CN119082136A (zh) * 2024-10-22 2024-12-06 西北农林科技大学深圳研究院 调控多年生黑麦草株型的LpBPM5.6基因、低表达载体、构建方法及其应用

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US5559223A (en) * 1991-08-09 1996-09-24 E. I. Dupont De Nemours And Company Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants
US6355863B1 (en) * 1996-06-05 2002-03-12 The Regents Of The University Of California Seed plants exhibiting inducible early reproductive development and methods of making same
US6476212B1 (en) * 1998-05-26 2002-11-05 Incyte Genomics, Inc. Polynucleotides and polypeptides derived from corn ear
US6683230B1 (en) * 1998-02-20 2004-01-27 Syngenta Limited Hybrid seed production
JP2005535295A (ja) * 2002-03-27 2005-11-24 カウンシル・オブ・サイエンティフィック・アンド・インダストリアル・リサーチ 渇水ストレス寛容茶植物からの遺伝子および水ストレス寛容性を導入する方法

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Publication number Priority date Publication date Assignee Title
US5559223A (en) * 1991-08-09 1996-09-24 E. I. Dupont De Nemours And Company Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants
US6355863B1 (en) * 1996-06-05 2002-03-12 The Regents Of The University Of California Seed plants exhibiting inducible early reproductive development and methods of making same
US6683230B1 (en) * 1998-02-20 2004-01-27 Syngenta Limited Hybrid seed production
US6476212B1 (en) * 1998-05-26 2002-11-05 Incyte Genomics, Inc. Polynucleotides and polypeptides derived from corn ear
JP2005535295A (ja) * 2002-03-27 2005-11-24 カウンシル・オブ・サイエンティフィック・アンド・インダストリアル・リサーチ 渇水ストレス寛容茶植物からの遺伝子および水ストレス寛容性を導入する方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010139993A1 (fr) * 2009-06-01 2010-12-09 Plant Bioscience Limited Procédés et compositions pour la tolérance au stress chez les végétaux
CN102449154A (zh) * 2009-06-01 2012-05-09 利托瑞尔国立大学 植物中用于胁迫耐性的方法和组合物
US20120185964A1 (en) * 2009-06-01 2012-07-19 Julieta Virginia Cabello Methods and compositions for stress tolerance in plants
AU2010255488B2 (en) * 2009-06-01 2013-10-10 Consejo Nacional De Investigaciones Cientificas Y Tecnicas Methods and compositions for stress tolerance in plants
CN102449154B (zh) * 2009-06-01 2015-07-01 利托瑞尔国立大学 植物中用于胁迫耐性的方法和组合物
CN119082136A (zh) * 2024-10-22 2024-12-06 西北农林科技大学深圳研究院 调控多年生黑麦草株型的LpBPM5.6基因、低表达载体、构建方法及其应用

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