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WO2004035797A2 - Promoteurs specifiques aux tissus issus de plantes - Google Patents

Promoteurs specifiques aux tissus issus de plantes Download PDF

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Publication number
WO2004035797A2
WO2004035797A2 PCT/EP2003/011038 EP0311038W WO2004035797A2 WO 2004035797 A2 WO2004035797 A2 WO 2004035797A2 EP 0311038 W EP0311038 W EP 0311038W WO 2004035797 A2 WO2004035797 A2 WO 2004035797A2
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Prior art keywords
plant
promoter
expression cassette
seq
product
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WO2004035797A3 (fr
Inventor
Klaus K. Nielsen
Claus H. Andersen
Ingo Lenk
Klaus Petersen
Thomas Didion
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Riso National Laboratory
DLF Trifolium AS
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Riso National Laboratory
DLF Trifolium AS
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Priority to AU2003293602A priority Critical patent/AU2003293602A1/en
Priority to NZ539360A priority patent/NZ539360A/en
Publication of WO2004035797A2 publication Critical patent/WO2004035797A2/fr
Anticipated expiration legal-status Critical
Publication of WO2004035797A3 publication Critical patent/WO2004035797A3/fr
Ceased legal-status Critical Current

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    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters

Definitions

  • the present invention relates to tissue-specific promoters in plants and, in particular, to tissue-specific promoters that are capable of directing the expression of an associated gene in floral determined tissue from plants, for example grasses such as Lolium perenne L.
  • grasses such as Lolium perenne L.
  • the value of grass as forage is limited by the fact that the grass stems, in particular, contain high amounts of low digestible compounds, mainly lignins and cell wall compounds coupled to lignin. Ruminants can only eat a limited amount of grass per day and therefore if the nutritional value can be improved, grass would account for a larger portion of the daily nutritional need.
  • SAM shoot apical meristem
  • the SAM During vegetative growth of perennial ryegrass (Lolium perenne L), the shoot apical meristem (SAM) gives rise to primordia that develop into leaves. Upon a switch to reproductive growth, the SAM is reprogrammed to form primordia that develop into inflorescence. This transition from vegetative to reproductive development is triggered by a vernalization period (primary induction) followed by increased temperature and day length (secondary induction) and is accompanied by transcriptional up- or down-regulation of flowering-related genes. This transcriptional up- or down-regulation is the consequence of transcription factor activities, initiated after the transition from vegetative to reproductive growth.
  • MADS box genes are transcription factors controlling a wide range of developmental features, associated with the process of flowering and flower formation but also completely unrelated processes such as root and trichome formation, and seed germination. Plants typically contain about 100 different MADS box genes with different temporal and spatial expression patterns and functions.
  • WO 97/30162, WO 98/13503 and WO 00/55172 describe the use of MADS box promoters specific to reproductive tissues for enhancing vegetative growth in Pinus and Eucalyptus trees.
  • evidence for conserved function of specific MADS box genes between trees and grasses has not yet been shown.
  • recent publications from Johansen et al. 2002 and Martin-Trillo and Martinez-Zapater 2002 show that sequence homologues do not necessarily have the same expression pattern or function in species of different genus. It is an object of the present invention to provide promoters that direct expression of a gene product in floral determined tissue in plants such as grasses.
  • the present invention is based on the identification of novel promoters from monocot plants, such as Lolium perenne L, which display tissue-specific and/or temporal expression patterns, and the use of such promoters to express genes in floral determined tissue.
  • the present invention provides a method of controlling expression of a product from an associated polynucleotide sequence in floral determined cells, said method comprising: transforming a plant cell with an expression cassette, said expression cassette comprising a promoter sequence and an associated polynucleotide sequence, wherein said promoter sequence is capable of controlling preferential expression of a product from said associated polynucleotide sequence in floral determined cells, and expressing the associated polynucleotide sequence in floral determined cells.
  • the present invention provides tissue-specific expression by enabling a particular product to be preferentially expressed from a polynucleotide fragment in floral determined tissue of a plant but not generally in other tissues of the plant.
  • the expressed product may be a protein whose site of action is only in floral determined or reproductive cells, or may be an antisense RNA to a protein which is expressed only in floral determined or reproductive cells.
  • the levels of expression of a product lethal to other vegetative tissues, as well as floral determined tissue would necessarily require to be at sub-lethai levels in these vegetative tissues for the growth of these tissues and hence the plant.
  • the product is expressed in non-target tissues at a level of less than 30% of the target tissues, preferably at a level of less than 20%, more preferably at a level of less than 10% and most preferably at a level of less than 5%.
  • the plants that can be used in the present invention may, for example, be monocots, such as Poaceae, such as Phleum spp., Dactylis spp., Lolium spp., Festulolium spp., Festuca spp., Poa spp., Bromus spp., Agrostis spp., Arrhenatherum spp., Phalaris spp., and Trisetum spp., for example, Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, Festulolium loliaceum, Festulolium holmbergii, Festulolium pabulare, Festuca pratensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Fest
  • Floral determined cells refers to . any shoot apical cells and/or any cells or tissue derived therefrom, which will differentiate into and comprises any floral or reproductive structures or tissues and stems upon exposure to floral inductive environmental stimuli.
  • inflorescence refers to the total number of reproductive tissues or structures that the shoot apical cells differentiate into.
  • polynucleotide sequence and “polynucleotide fragment” as used herein refer to a chain of nucleotides such as deoxyribose nucleic acid (DNA) and transcription products thereof, such as RNA.
  • the polynucleotide fragment can be isolated in the sense that it is substantially free of biological material.
  • the isolated polynucleotide fragment may be cloned to provide a recombinant molecule comprising the polynucleotide fragment.
  • polynucleotide fragment includes double and single stranded DNA, RNA and polynucleotide sequences derived therefrom, for example, sub sequences of said fragment and which are of any desirable length. Where a nucleic acid is single stranded then both a given strand and a sequence or reverse complementary thereto is within the scope of the present invention.
  • promoter and “promoter sequence” used herein refers to a segment of DNA which has the ability and function to promote and/or regulate and/or modify expression of a product resulting from a polynucleotide fragment associated with said promoter sequence inside a host cell.
  • the host cell may be a plant, plant cells or a transgenic plant regenerated from the plant cells. It is intended to encompass functional equivalents of the sequences disclosed herein.
  • the promoter sequence of the expression cassette may be active, that is, capable of expressing the product of the polynucleotide fragment, in the shoot apical cells, or any cells derived therefrom, which will differentiate into floral structures at a very early time point. Differentiation occurs once the floral transition has been initiated by floral inductive environmental stimuli such as vernalization, followed by an increase in temperature and day length, that is, during the process of floral morphogenesis and before fertile floral organs have differentiated.
  • the promoters may continue to be active in later stages, including flowering. This is expected to provide the controlled expression of polynucleotide fragments in stems and flowers.
  • associated polynucleotide sequence or fragment refers to a polynucleotide sequence wherein the expression of a product from said polynucleotide sequence is under the control of the promoters of the present invention.
  • the polynucleotide sequence or fragment is associated with the promoter by being ligated to a site downstream from the promoter in order to express the product of the polynucleotide fragment.
  • the product to be expressed by the promoter of the expression cassette may be a polypeptide chain lethal to the plant cell it is expressed in, a polypeptide that enhances production of a particular protein, for example, a flowering repressor, or a polynucleotide sequence that is anti-sense to at least at portion of a gene, such as a flowering activator gene.
  • polynucleotide sequences to express in sense and/or antisense orientation may be flowering time genes such as FLOWERING LOCUS T, SOC1 (AGL20), FRIGIDA (FRI), FLOWERING LOCUS C (FLC) (Samach et al.
  • the expression "lethal product” includes, but is not limited to, a polypeptide product of the polynucleotide fragment, a ribonucleic acid sequence antisense to a particular gene, or a ribozyme or other non peptide which significantly disrupts a target cell leading thereby to the arrested growth or death of the target cell, such as a floral determined cell, or even the whole plant, if appropriate. The death of the target cell is therefore expected to prevent differentiation of the target cell into floral or reproductive tissue.
  • a polynucleotide sequence that produces a lethal product may be selected from those that encode ribonucleases such as Barnase (Hartley, 1988), from B.
  • RNase T1 Amyloliquefaciens
  • RNase T1 Mentioni et al., 1990; Mariani et al., 1992; Reynaerts et al., 1993
  • bovine RNase A Carsana et al., 1988
  • RNase I Zahu LQ et al., 1990
  • RNase H Wu H. et al., 1998) from E.coli and set of plant RNases (family of S-proteins) (Norioka et al., 1996).
  • the polynucleotide sequence that encodes a lethal product may be selected from nucleases such as the family of restriction endonucleases, diphtheria toxin A chain (DTA) (Collier, 1975), enzymes including glucanases such as ⁇ -1-4-glucanases or ⁇ -1-3- glucanases (U.S. Pat. No. 6,096,946; Worrall et al., 1992), ubiquitins (Gausing and Barkardottir, 1986), acid pyrophosphatases (Kieber. and Signer, 1991 ), and inhibitors of plant cell wall synthesis (U.S. Pat. No. 6,184,440).
  • nucleases such as the family of restriction endonucleases, diphtheria toxin A chain (DTA) (Collier, 1975), enzymes including glucanases such as ⁇ -1-4-glucanases or ⁇ -1-3- glucanases (U.S. Pat. No. 6,096,94
  • Ribozymes mentioned herein are referring to molecules cleaving mRNAs (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987) for defined proteins to inhibit the translation of these defined proteins. Ribozymes can be designed from gene sequences encoding defined proteins.
  • ribozymes mentioned herein include any ribozymes which can cleave mRNAs for defined proteins to inhibit the translation of these defined proteins (Probst 2000) regardless of their types such as hammer-head-type ribozymes (Prody et al., 1986; Palukaitis et al., 1979; Symons, 1981 ), hairpin-type ribozymes (Berzal-Herranz et al., 1992; Chowrira et al., 1993), delta-type ribozymes (U.S. Pat. No. 5,625,047).
  • the promoters of the present invention are suitable for preventing the development and dispersion of pollen and seeds, and may be particularly suitable for preventing dispersion of pollen and seeds from genetically modified plants.
  • the product to be expressed by the promoter of the expression cassette may, for example, be an insect resistance gene (Frutos et al., 1999), a bacterial disease resistance gene (Sharma et al., 2000), a fungal disease resistance gene (Jongedijk et al., 1995), a viral disease resistance protein (WO9807875-A1 ), a herbicide resistance gene (Rathore et al., 1993), a male sterility gene (Jagannath et al., 2001 ), a selectable marker gene (Ziemienowicz, 2001 ), a screenable marker gene (Stewart, 2001), a negative selectable marker gene (Koprek et al., 1999), a reporter gene (Ziemienowicz, 2001 ), a gene which expresses a protein affecting plant agronomic characteristics including plant morphology (Wang et al., 2000), plant architecture (Venglat et al., 2002), and seed
  • the promoter sequence may be any of the MADS box gene promoters which preferentially expresses the product of an associated polynucleotide fragment in the floral determined tissue of plants.
  • the promoter sequence may be a subset of the MADS box gene promoters, such as those promoters which control a subset of MADS box genes that share a common sequence motif.
  • the promoter sequence may comprise or consist essentially of sequences selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, fragments or derivatives thereof.
  • the promoter sequences themselves have been identified as bases 1-2381 in SEQ ID NO. 1 , bases 1-1751 in SEQ ID NO. 2, bases 1-2390 in SEQ ID NO. 3 and bases 1-3443 in SEQ ID NO. 4. It has been observed that the first intron identified as bases 2672-4617 in SEQ ID NO. 1 , bases 1996-4595 in SEQ ID NO. 2, bases 2672-5902 in SEQ ID NO. 3 and bases 3841-5681 in SEQ ID NO. 4 may be involved in promoting expression of the gene normally associated with the promoter.
  • the first intron associated with the promoters of the present invention are not essential for the tissue specific expression of the polynucleotide sequences associated with them, it has been observed that the intron associated with one of the promoters (SEQ ID NO. 2, also known as MADS5) contains regulatory elements of importance for specifying the level of gene expression in the floral determined tissue, that is, both enhancer and inhibitor regions. Inclusion of the whole intron has been found to significantly enhance the tissue specific expression (up to approx. 3 fold). However, deletion of the central region of the intron in SEQ ID NO. 2 (nucleotides 2282 to 4322) gave rise to a further increase in expression level. The present inventors postulate that this further increase may be caused by deletion of inhibitory regions in the intron.
  • the present invention may further include the first intron of each sequence herein identified for use in conjunction with the promoter sequences. Furthermore, the central part of each intron may be further deleted to yield an increase in promoter activity.
  • the inventors have also postulated that it may be possible to use the first intron from one promoter with another promoter, for example the promoter sequence of SEQ ID NO. 1 may be used with the intron of SEQ ID NO. 4. It has been further postulated that the introns may be used in conjunction with non-MADS promoters.
  • fragments are defined as any portion of the sequence as shown in SEQ ID NOS: 1 , 2, 3 or 4 which retains the ability to express an associated polynucleotide fragment preferentially only in floral determined tissue.
  • homologues or “homologous” as used herein refers to nucleotide sequences of polynucleotide fragments of the present invention which have 65% identity or above with the sequence disclosed herein, such as 66%, 68%, 70%, 75%, 80%, 83%, 86%, 88%, 90%, 92%, 95%, 97% or 99% identity.
  • identity with respect to nucleotide sequences is defined as the percentage of nucleotides in a polynucleotide sequence which are identical to the nucleotides in the sequence disclosed herein after alignment as determined by using sequence analysis programs.
  • Programs which are used for database searching and sequence alignment and comparison for example, from the Wisconsin Package Version 10.2, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, WI) or public available sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PhytoSeq (Incyte Pharmaceuticals, Palo Alto, CA) may be used to determine sequence identity, Alignment for sequence of comparison may be conducted by the local homology algorithm of Smith and Waterman (1981 : Adv. Appl. Math., 2:482), by the homology alignment algorithm of Needleman and Wunsch (1970: J. Mol. Biol., 48:443), by the search for similarity method of Pearson and Lipman (1988: Proc. Natl. Acad. Sci. USA., 85: 2444), by computerized implementations of these algorithms.
  • Wisconsin Package Version 10.2 such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG
  • isolated polynucleotide fragments similar to the sequences disclosed herein for use in the methods of the present invention may now be obtained from any plant source using standard methods, for example, by employing consensus oligonucleotides and PCR.
  • similar is meant an isolated polynucleotide fragment comprising a nucleotide sequence which is capable of hybridising to a sequence which is complementary to the nucleotide sequence of the inventive polynucleotide fragment.
  • the stringency of the hybridisation is used to determine the degree of similarity between two sequences.
  • stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a erfectly matched sequence (probe).
  • the T m values of the sequences are preferably within 10°C of each other. More preferably hybridisation may be performed under stringent conditions, with either the similar or inventive DNA preferably being supported.
  • a denatured similar or inventive sequence is preferably first bound to a support and hybridisation may be effected for a specified period of time at a temperature of between 50 and 70°C in double strength SSC (2 x NaCI 17.5g/l and sodium citrate (SC) at 8.8g/l) buffered saline containing 0.1% sodium dodecyl sulphate (SDS) followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration.
  • SSC double strength SSC
  • SDS sodium dodecyl sulphate
  • such reduced concentration buffers are typically single strength SSC containing 0.1% SDS, half strength SSC containing 0.1 % SDS and one tenth strength SSC containing 0.1% SDS.
  • Sequences having the greatest degree of similarity are those the hybridisation of which is least affected by washing in buffers of reduced concentration. It is most preferred that the similar and inventive sequences are so similar that the hybridisation between them is substantially unaffected by washing or incubation at high stringency, for example, in one tenth strength sodium citrate buffer containing 0.1% SDS. These similar polynucleotide fragments from plants other than ryegrass are also encompassed by the term "homologues".
  • the promoters of the present invention may also be used to modify flowering of a plant, for example, to accelerate, delay or substantially or completely prevent flowering of a plant.
  • Flowering of a plant may be accelerated by using the promoters of the present invention to, for example, overexpress flowering activators, such as FLOWERING LOCUS T, SOC1 (AGL20), CONSTANS (CO) (Suarez-Lopez et al., 2002; Mouradov et.
  • overexpress flowering activators such as FLOWERING LOCUS T, SOC1 (AGL20), CONSTANS (CO) (Suarez-Lopez et al., 2002; Mouradov et.
  • LFY the API- family (MADS box class of genes) (Simpson and Dean, 2002), and genes involved in plant hormone biosynthesis, sensing or signalling (for example, Gibberellins, Cytokinins and Auxins) (McCourt, 1999), and to express antisense sequences to known flowering repressors, such as TFL and FLOWERING LOCUS C (FLC) (Samach et al., 2000; Simpson and Dean, 2002), homeobox genes such as ATH1 (Proveniers and Smeekens, 1997), floral morphology genes such as MADS box genes of the A,B,C class of function (Ng and Yanofsky, 2000; Lohmann and Weigel, 2002), genes involved in light perception and signalling such as Cryptochromes, Phytochromes, and, genes acting as regulators of the cell cycle (Vandepoele et al., 2002).
  • MADS box class of genes for example, Gibberellins, Cytokinins
  • flowering of a plant may be delayed by the controlled expression of flowering repressors, such as TFL and FLOWERING LOCUS C (FLC) (Samach et al., 2000; Simpson and Dean, 2002), homeobox genes such as ATH1 (Proveniers and Smeekens, 1997) and by antisense expression of flowering activators, such as FLOWERING LOCUS T, SOC1 (AGL20), CONSTANS (CO) (Suarez-Lopez et al., 2002; Mouradov et.
  • flowering repressors such as TFL and FLOWERING LOCUS C (FLC) (Samach et al., 2000; Simpson and Dean, 2002), homeobox genes such as ATH1 (Proveniers and Smeekens, 1997)
  • antisense expression of flowering activators such as FLOWERING LOCUS T, SOC1 (AGL20), CONSTANS (CO) (Suarez-Lopez
  • the present inventors demonstrate that controlled expression of a lethal product from a polynucleotide fragment by the promoters of the present invention may substantially prevent or delay floral determined cells from differentiating into inflorescence.
  • a delay in differentiation is also thought to produce higher nutritional value, digestibility and/or biomass.
  • the expression of a lethal product is thought to result in the marked reduction of plant stems and, in particular, high amounts of low digestible compounds. It is also thought that the energy required for the development of the reproductive organs would now be targeted to the growth of digestible leaves. Therefore, this is thought to result in a higher nutritional value and leaf biomass production of the plant.
  • any product which enhances or delays the time of flowering, modifies plant development, flower morphology or plant architecture such as those described above may be used to substantially prevent or delay flowering.
  • the product need not necessarily be lethal to the floral determined cells, that is, it does not necessarily have to kill the floral determined cells.
  • the present invention provides a method of increasing the nutritional value and/or yield of a plant, said method comprising: transforming a cell from a plant with an expression cassette, said expression cassette comprising a promoter sequence and an associated polynucleotide fragment , wherein said promoter sequence preferentially expresses a product from said associated polynucleotide fragment in floral determined cells of the plant, wherein said polynucleotide fragment expresses a product which substantially or completely prevents or delays floral determined cells of a plant developing into inflorescence, or modifies plant development, flower morphology or plant architecture, and thereafter expressing said product to increase the nutritional value and/or yield of the plant.
  • the product may substantially or completely prevent development of inflorescence through virtue of it being lethal to the floral determined cells.
  • the product may substantially or completely prevent development of inflorescence through virtue of it encoding a flowering repressor which is overexpressed, or an antisense sequence of a flowering activator.
  • the overexpression of genes and the use of anti- sense sequences for reducing or eliminating translation of genes are well known in the art.
  • the above method of the present invention provides a plant where the tissue-specific promoter substantially or completely prevents the floral determined cells from developing into reproductive tissue as a result of the tissue-specific expression of a lethal gene, overexpression of a flowering repressor, expression of sequences antisense to flowering activators, or a combination of any of these.
  • tissue-specific promoter substantially or completely prevents the floral determined cells from developing into reproductive tissue as a result of the tissue-specific expression of a lethal gene, overexpression of a flowering repressor, expression of sequences antisense to flowering activators, or a combination of any of these.
  • this will result in the marked reduction of stems and, in particular, high amounts of low digestible compounds, and will result in a higher nutritional value and/or yield of the plant.
  • the plant may, for example, be any of those previously described hereinabove, and in particular, may be a foraging plant.
  • an expression cassette comprising a promoter sequence which is capable of expressing a product from a polynucleotide fragment associated with said promoter sequence in floral determined cells of a plant, and the polynucleotide fragment from which said product is expressed.
  • sequences are also referred to hereinafter as MADS4, MADS5, MADS6 and MADS7, respectively.
  • the invention further provides a transformed plant cell containing an expression cassette according to the present invention.
  • the expression cassette may be stably incorporated in the genome of the plant by transformation.
  • the invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom. Any transformation method suitable for the target plant or plant cells may be employed, including, but not restricted to, infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation and pollen tube transformation (see generally, Wang et al.
  • the transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way.
  • the promoters MADS4 (SEQ ID NO. 1 ) and MADS6 (SEQ ID NO. 3) have been observed to induce expression in the callus of ryegrass.
  • expression of a lethal product from a polynucleotide fragment associated with the promoters MADS4 or MADS6 during the initial phase of transformation will compromise the initial production of transgenic plants by substantially or completely preventing regeneration.
  • the promoters MADS5 (SEQ ID NO: 2) and MADS7 (SEQ ID NO:4) do not appear to induce expression in the callus and are thus suitable for transformation of the callus.
  • the present invention provides a method of producing a transgenic plant, said method comprising: transforming a callus derived from a plant with an expression cassette said expression cassette comprising a promoter sequence selected from SEQ ID NO:2
  • MADS5 or SEQ ID NO:4 (MADS7), fragments, derivatives or homologues thereof, and a polynucleotide fragment whose expression is under the control of said promoter sequence, and selecting any transformants and vegetatively propagating them.
  • the plant may, for example be any of those previously described hereinabove.
  • MADS6 (SEQ ID NO:3) is still possible by substantially or completely preventing expression of the lethal product at the early stage by using an inducible promoter system to control expression of a polynucleotide fragment whose product counteracts the lethal product.
  • a lethal product and counteracting product may, for example, be the Barnase/Barstar genes discussed above.
  • the present invention also provides a method of producing a transgenic plant, said method comprising: - transforming a callus derived from a plant with a first expression cassette and second expression cassette wherein said first expression cassette comprises a first promoter sequence selected from SEQ ID NO:1 (MADS4) or SEQ ID NO:3 (MADS6), fragments or derivatives thereof, and an associated first polynucleotide fragment, and said second expression cassette comprises a second inducible promoter sequence and an associated second polynucleotide fragment, wherein said first promoter sequence preferentially expresses a first product from said associated first polynucleotide fragment in floral determined cells of the plant, said first polynucleotide fragment substantially or completely preventing said floral determined cells from developing into reproductive tissue, and wherein said second promoter sequence expresses a second polynucleotide fragment product from said associated second polynucleotide fragment, and said second product substantially negates or counteracts the effect of said first product,
  • the plant may, for example, be any of those previously described hereinabove.
  • each expression cassette may be either located on separate polynucleotide molecules, such as plasmids, or may be located on the same polynucleotide molecule.
  • an inducible promoter suitable for the above method may be an ethanol- inducible system (Roslan et al., 2001 ; Salter et al., 1998).
  • tissue ablation approach A problem often associated with a tissue ablation approach is the effect of promoter leakage or expression in other tissues. Accordingly, if the promoter controlling the expression of the coding region expresses an associated product at low levels in tissues other than floral determined tissue, any residual leakage may be overcome by modification of the expression cassette to include appropriate leakage control.
  • a second tissue specific promoter such as a vegetative promoter, may be used to promote expression of an inhibitor to the gene product that substantially or completely prevents floral development in vegetative tissues. This will therefore negate the effect of the polynucleotide fragment (intended for expression in floral determined cells only) in tissues other than floral determined.
  • the present invention provides a transformed plant cell containing a first expression cassette according to the present invention and a second expression cassette containing a second promoter, capable of expression in non-target tissue, that is, tissues other than floral determined tissue, and a second polynucleotide fragment for a control product capable of inhibiting the first polynucleotide fragment product of the first expression cassette of the present invention.
  • This control product will substantially reduce any damaging effects the first product may have on vegetative cells.
  • the inhibitor may be under the control of a weak constitutively active promoter.
  • the inhibitor is expressed at low levels thus substantially inhibiting any damaging effect of the first product, which may be caused by promoter leakage in vegetative tissues.
  • the expression of the product that substantially or completely prevents floral development in vegetative tissues is at such a level that the inhibitor is unable to inhibit the first product in these tissues.
  • the present invention provides a transformed plant cell containing a first expression cassette according to the present invention and a second expression cassette containing a second promoter capable of weak constitutive expression of an associated second polynucleotide fragment which encodes an inhibitor capable of inhibiting the first product of the first expression cassette of the present invention in vegetative tissues.
  • the promoters of the present invention may be useful in systems for biological containment.
  • a plant comprising a promoter of the present invention which controls the expression of a polynucleotide fragment, which substantially or completely prevents the floral, determined tissue from developing or differentiating into inflorescence will not produce sexual organs. This is expected to prevent the dispersion of pollen and seeds from the plant since the sexual organs will not develop. This is also known as "biological containment".
  • This system of biological containment can be used in conjunction with a second, inducible system, such as the ethanol-inducible system as disclosed above, which can counteract the effects of the product under the control of a promoter of the present invention.
  • a second, inducible system such as the ethanol-inducible system as disclosed above, which can counteract the effects of the product under the control of a promoter of the present invention.
  • Figure 1 is a FSD (Family Specific Differential display) profile gel specific for MADS box genes.
  • Apices meristematic regions dissected from Lolium perenne shoots (hereafter "apices” refers to the shoot apical meristem and/or any floral determined structures derived thereof:
  • Figure 2 illustrates the expression patterns of LpMADS1-7 and 9 obtained from real- time RT-PCR during Lolium perenne development and in various organs.
  • Stems stems tissue without intercalary meristems (knees) and leaves
  • Figure 3 illustrates a phylogenetic tree of fifteen LpMADS proteins.
  • the MIK-regions from 15 LpMADS proteins were aligned by the CLUSTALX package.
  • a similarity matrix was obtained by PRODIST, using the PAM matrix of amino acid transition and a phylogenetic tree was then derived by the PHYLIP phylogeny inference package.
  • the similarity matrix was analysed by the neighbour-joining algorithm (NJ).
  • NJ neighbour-joining algorithm
  • the data set was also bootstrapped (100 times, 100 jumble) and a consensus tree obtained, also by NJ.
  • Figure 4 illustrates the I37 vector used for Promoter::GUS expression, and the L50 vector used for Promoter.::Barnase expression.
  • Figure 5 illustrates the nucleotide sequence of the MADS4 promoter (SEQ ID NO:1 ).
  • Figure 6 illustrates the nucleotide sequence of the MADS5 promoter (SEQ ID NO: 2).
  • Figure 7 illustrates the nucleotide sequence of the MADS6 promoter (SEQ ID NO: 3).
  • Figure 8 illustrates the nucleotide sequence of the MADS7 promoter (SEQ ID NO: 4).
  • Figure 9 illustrates the expression cassette with the LpMADS4promoter-Actin intron::GUS fusion (CON ID NO: 1 ) and the LpMADS4promoter-intron::GUS fusion (CON ID NO: 2).
  • Figure 10 illustrates the expression cassette with the LpMADS5promoter-Actin intron::GUS fusion (CON ID NO: 3) and the LpMADS5promoter-intron::GUS fusion (CON ID NO: 4).
  • Figure 11 illustrates the expression cassette with the LpMADS6promoter-Actin intron::GUS fusion (CON ID NO: 5) and the LpMADS6promoter-intron::GUS fusion (CON ID NO: 6).
  • Figure 12 illustrates the expression cassette with the LpMADS7promoter-Actin intron::GUS fusion (CON ID NO: 7) and the LpMADS7promoter-intron::GUS fusion (CON ID NO: 8).
  • Figure 13 illustrates the expression cassette with the LpMADS4promoter-Actin intron::Barnase fusion (CON ID NO: 9) and the LpMADS4promoter-intron::Barnase (CON ID NO: 10).
  • Figure 14 illustrates the expression cassette with the LpMADS5promoter-Actin intron::Barnase fusion (CON ID NO: 11) and the LpMADS5promoter-intron::Bamase (CON ID NO: 12).
  • Figure 15 illustrates the expression cassette with the LpMADS6promoter-Actin intron::Bamase fusion (CON ID NO: 13) and the LpMADS6promoter-intron::Barnase (CON ID NO: 14).
  • Figure 16 illustrates the expression cassette with the LpMADS7promoter-Actin intron::Barnase fusion (CON ID NO: 15) and the LpMADS7promoter-intron::Bamase (CON ID NO: 16).
  • Figure 17 illustrates the expression cassette with the LpMADS5promoter::GUS fusion
  • Figure 18 illustrates transient expression studies on the influence of the first intron of LpMADS ⁇ on expression in different tissues from L. perenne using different deletion derivatives of the LpMADS5 genomic region. Values above the bars indicate the number of independent shots.
  • Figure 19 illustrates a comparison of the relative expression levels of three different LpMADS constructs. Values above the bars indicate the number of independent shots.
  • Figure 20 illustrates the expression of CON ID No:18 in different tissues from L. perenne transiently transformed by particle bombardment- a) young leaf, b) secondary induced leaf, c) - f) floral primordial tissue.
  • Figure 21 illustrates the expression of CON ID NO 4 (LpMADS5promoter-intron::GUS fusion) in stably transformed L. perenne plants.
  • Panel A shows a part of an inflorescence 7 weeks after secondary induction expressing the GUS reporter gene. Spikelets consisting of several flowers showed blue staining in each of the flowers.
  • Panel B and C illustrate opened flowers with the detached ovary and stigma, where clearly most of the GUS staining is seen in the ovary at this time point.
  • Figure 22 illustrates the expression of CON ID NO 8 (LpMADS7promoter-intron::GUS fusion) in stably transformed L. perenne plants.
  • Panel A depicts the developing inflorescence of a transgenic L. perenne line expressing the GUS reporter gene. The GUS staining was performed 3 weeks after secondary induction. Blue staining is observed in the developing flowers on the developing spikelets.
  • Panel C shows an enlargement of three developing spikelets. Gus staining is in addition detected in a distinct region below the developing inflorescence in the junction zone to the stem (peduncle) as shown in panel B.
  • Figure 23 - panel A to C illustrate the non-flowering phenotype of 3 independent stably transformed Festuca rubra lines expressing the cytotoxic Barnase gene under the control of the
  • L. perenne LpMADS7 promoter (CON ID NO 16) in comparison to a flowering control plant. All plants had previously been subjected to floral inductive stimuli (12 weeks 6°C, short day - 8 hours light, followed by 20°C, long day - 18 hours light).
  • nucleotide codes used in the sequences disclosed in the Figures and the specification are: A, a - adenine; C, c - cytosine, G, g - guanine; T, t - thymine; U, u - uracil; Y, y - c or t(u); R, r - a or g; M, m -a or c; K, k -g or t(u); S, s - g or c; W, w - a or t(u); H, h - a, c or t(u); and B, b - g, t(u) or c.
  • Plant material Plant material Plant material Plant apices from Lolium perenne L. (cv Tetramax, DLF-Trifolium Ltd.) grown as described previously (Jensen et al., 2001) were harvested at 8 different developmental stages, frozen in liquid nitrogen and stored at -80°C prior to RNA extraction. The developmental stages of the apices were recorded corresponding to their age. Apices from the first stage were harvested from vegetative growing plants. The next two stages were harvested from 6 and 12 weeks primary induction (6°C, short day - 8 hours light) plants and the final 5 stages were harvested from 3, 7, 14, 21 and 35 days secondary induced (20 °C, long day - 18 hours light) plants.
  • Leaf, stem, knee, flower, seedling and root tissues were also sampled and frozen in liquid nitrogen immediately upon harvest.
  • FSD Family Specific Domain differential display
  • PCR was performed with 0.5 ⁇ l aliquots of the cDNA templates using a primer designed to be family-specific for MADS box genes, that is, a primer designed to anneal to a common conserved domain found in all monocot MADS box genes.
  • the FSD primer 5'-CTCAAGAAGGC(G/C)(C/T)ACGAG-3' was based on conserved domains from an alignment of different monocot MADS box genes from the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov) GenBank database.
  • a further primer was used annealed to the DNA adapter sequence.
  • Amplified PCR products were run on a 4% denaturing polyacrylamide gel using HR-1000TM Gel Reagent and a genomyxLRTM Programmable DNA sequencer (Genomyx, Foster City, CA, USA).
  • Bands of the expected size that is, bands which were a size similar to the original differential display band, were purified using QIAGEN Gel Extraction Kit (QIAGEN) and cloned into the pCR 2.1-TOPO vector (TOPO TA Cloning Kit, Invitrogen Corp.). Plasmids were prepared using the QIA-Spin Plasmid Mini Kit (QIAGEN). Heterogeneous inserts for each clone with the same size were digested with Sau3A restriction enzyme and clones containing different inserts of the expected size were sequenced.
  • a shoot apex cDNA library of Lolium perenne L. variety Green Gold was constructed from extracted RNA isolated from apices at different growth stages after floral induction, using the ZAP-cDNA/Gigapacklll Gold Cloning Kit (Stratagen, La Jolla, CA, USA). The cDNA library containing approximately 700,000 plaques was screened with 32 P-labelled MADS box probes constructed downstream of the MADS box region. Isolated MADS box cDNA clones were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmner Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 377 DNA sequencer (Perkin- Elmner Applied Biosystems).
  • Relative levels of mRNA transcripts corresponding to each MADS box gene were determined using real-time kinetic quantification of RT-PCR reactions using the Rotorgene 2000 instrument (Corbett Research, Australia)
  • Single-stranded cDNA was transcribed from mRNA isolated from 5 ⁇ g of total RNA (DNA-free) using Superscript reverse transcriptase (Gibco-BRL) according to the manufacturer's instructions.
  • PCR primers unique for each of the LpMADS1-7 and 9 sequences were designed using sequence alignments of many MADS box genes in order to ensure specificity to the gene of interest. The sequences of the primers were:
  • KP068 5'-CAG CTC GCA CGG TGC TTC-3'
  • KP069 5'-GAA ACT GAG CAG AAC AGA-3'
  • LpMADS2 KP070: 5'-CTT CAT GAT GAG GGA TCA-3'
  • KP071 5'-AGG TAC GAT CAC CAG CAT-3'
  • LpMADS3 KP072: 5'-GAG CAG ACG AAT GGA GCA-3' KP073: 5'-ACT GAT GGT GCG GAG CAT-3' LpMADS4: KP074: 5'-CAA CAG CTT CAG GGC GAT-3' KP075: 5'-TCG ATG GCA AGT GAC CAG-3'
  • LpMADS5 KP076: 5'-ACT TAC TCA GCT ACG AAC-3' KP077: 5'-TCC ATC ACT CAG AAG TAG-3'
  • KP053 5'-CTC AAG CGT AAG GAA CAA-3'
  • KP054 5'-CAC ACT TAG ATA GTT CAC-3"
  • KP051 5'-AGA GGA CGC AAA CTT GAC-3'
  • KP052 5'-CAG GAC AGT AGG ACA CAC-3'
  • LpMADS9 KP078: 5'-CAG CTG AAC AAC AAA GAC-3' KP067: 5'-GTC CTT ACA ATT CCT CCA-3' PCR reactions were performed with the Rotorgene 2000 instrument (Corbett Research, Australia) using SYBR Green and the supplied quantification software according to the manufacturer's instructions. Using a set of serially-diluted cDNA templates as standards, the relative levels of MADS box gene transcripts were calculated based on the "crossing-point" of each reaction (the PCR cycle in the reaction enters the log-linear phase) according to the manufacturer's instructions.
  • Genomic DNA library of Lolium perenne L. variety F6 was constructed from extracted genomic DNA isolated from leaf material. The DNA was digested with BamHI and cloned in the cloning vector EMBL SP6/T7. The DNA library containing 1.5 x 10 6 independent clones of an average size of 14 kb and more than 10 9 pfu/ml was screened with 32 P-labelled MADS box cDNA probes corresponding to the differentially expressed fragments identified by FSD (see details in "Results").
  • Isolated MADS box genomic DNA clones were sequenced through primer walking strategies using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin- Elmner Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 377 DNA sequencer (Perkin-Elmer Applied Biosystems).
  • Promoters were PCR amplified with primers that had been restricted in order to ligate them into either I37 or L50 (see Figure 3).
  • the Actin intron from I37 and L50 was first removed with restriction enzymes Sacl and Ncol.
  • the first intron includes the 5'-UTR, the first exon, the first intron and a part of the second exon in order to be in frame to the coding sequence of the udiA reporter or barnase gene.
  • the DA56 and DA18 primers were restricted with Sacl and the PCR fragment of the LpMADS4 promoter was cloned into Sacl in both I37 and L50.
  • LpMADS ⁇ , 6 and 7 promoter or promoter-intron were restricted with Sacl and Ncol in order to clone the PCR fragment of LpMADS4 promoter-intron into I37 (without Actin intron) and L50 (without Actin intron).
  • LpMADS ⁇ , 6 and 7 promoter or promoter-intron were restricted into 137 and L50.
  • LpMADS ⁇ promoter was restricted with Clal and Sacl and LpMADS ⁇ promoter-intron with Clal and Ncol.
  • LpMADS6 promoter was restricted with EcoRI and Sacl and LpMADS6 promoter-intron with EcoRI and Ncol.
  • LpMADS7 promoter was restricted with EcoRI and Sacl and LpMADS7 promoter-intron with Sacl and Ncol. All clonings were carried out according to Sambrook et al. (1989).
  • the sequences of the primers are: LpMADS4 promoter:
  • DA56 5'- GAG CTC GGT AAA TCC CAT GAA TCG GTG -3' DA18: 5'-CGA GCT CGG CTC TAG CTA GCT AGC -3'
  • DA61 5'- AAA CCA TGG CAT TGT AGC AGA GTG TTG G -3' LpMADS ⁇ promoter: MADSpromClalA: 5'-GGA ATC ATC GAT TGA AGG TGA TGT GGA GAC-3'
  • MADS5UTR5' 5'-GTT CCT CCA TGG ATG C-3' LpMADS ⁇ promoter and 1 st intron:
  • MADS ⁇ promClalA ⁇ '-GGA ATC ATC GAT TGC AGG TGA TGT GGA GAC-3'
  • MADS ⁇ exon2Ncol ⁇ '-CTA TCC CAT GGG CAC AGT TGT TTC AGG TCC -3' LpMADS6 promoter:
  • KP09 ⁇ ⁇ '- ATT GAA TTC GAG CGT TGA TTT CGT CCA -3'
  • KP092 ⁇ '-TAA GAG CTC ACT CAG ATA GCA CTA CCA-3' LpMADS6 promoter and 1 st intron:
  • KP09 ⁇ ⁇ '-ATT GAA TTC GAG CGT TGA TTT CGT CCA-3'
  • KP102 ⁇ '- TAT CCA TGG TGG AGT TGT AGT TGC AG -3'
  • KP064 ⁇ '-ATT GAA TTC TCG CCG GTA AGG GCA TCT CT-3'
  • KP06 ⁇ ⁇ '-ATT GAG CTC TCA GGC TGC AGT TGT G-3' LpMADS7 promoter and 1 st intron: KP106: 5'- ATT GAG CTC TCG CCG GTA AGG GCA TCT -3' KP104: ⁇ '- AAT CCA TGG CCA CGG CAT CTT GCG A -3'
  • the vector CON ID NO: 4 was cleaved with EcoRI and Narl. The protruding ends of the 1814 bp fragment were filled in with Klenow fragment (blunt ended).
  • the 1814bp LpMADS ⁇ promoter fragment was inserted into the Smal cut pAHC27 (Christensen and Quail; 1996), thereby replacing the Z. mays Ubiquitin 1 promoter with the ⁇ ' untranscribed region of L. perenne MADS ⁇ .
  • the vector pM5P-M5l ⁇ -GUS (CON ID NO: 18) was derived from the vector CON ID NO: 4 by cleavage with Smal and Aflll, followed by a fill-in reaction with Klenow fragment and subsequent re-ligation. Deleted nucleotides from SEQ ID NO: 2 are 2282 to 4322 (including).
  • Cultivation of Lolium perenne plants Lolium perenne plants were grown as described previously (Jensen et al., 2001 ). Four days prior to vernalization leaves were cut approximately 5 cm above the soil. For recovery of young leaf material seeds were germinated in 25 x 25 cm square petridishes on two layers of tap water soaked 3MM paper without specific light regime.
  • Leaves of 7 to 14 day old Lolium perenne seedlings were cut into pieces of 2 - 3cm lenght and put onto tap water soaked 3MM paper prior to further treatment.
  • Leaves from mature Lolium perenne plants in long day conditions after >12 weeks of vernalization were cut into pieces of 2 - 3 cm le ⁇ ght and put onto tap water soaked 3MM paper before further treatment.
  • the abaxial epidermis was removed by cutting with a razor blade from the adaxial side, leaving the abaxial epidermis intact. The epidermis was then peeled off.
  • leaves were put onto tap water soaked 3MM paper with the mesophyll side towards the paper.
  • the parts of the stems of secondary induced Lolium perenne that contained the floral apices / flowers, were cut longitudinally with a razor blade.
  • the sections Prior to further treatment, the sections were placed on tap water soaked 3MM paper, the plane of the sections facing downwards. 5 Prior to particle bombardment all tissues were transfered onto 10 % (w/v) sorbitole solution for at least 90 min. For bombardment, tissues were placed on 0,7 % agarose / tap water plates. Tissue was placed upside down
  • Particles were rinsed three times with 1 ml of 100 % ethanol by centrifugation for 5 seconds at
  • Bombardment parameters for the tested tissues were determined as the following:
  • tissue was vacuum-infiltrated for 5 min with X-Gluc buffer. Staining was performed 0 for 2 - 16 h at 37°C. Subsequently, the X-Gluc buffer was exchanged by 96 % ethanol to remove the chlorophyll from green tissues. Finally, tissues were incubated in an aqueous solution of chloralhydrate to achieve transparent tissues.
  • X-Gluc buffer 388 ⁇ M X-Gluc; 100 m NaP0 4 , pH 7,0; 0,1 % Triton X-100; ⁇ OO ⁇ M K 3 Fe(CN) 6 ; ⁇ OO ⁇ M K 4 Fe(CN) 6 ; 10 mM EDTA, pH 7,0; 20 % ethanol.
  • Chloralhydrate solution 67 % (w/v) chloralhydrate; 8,3 % (v/v) glycerol in H 2 0
  • Plasmids of interest were introduced into Lolium perenne together with pAHC20 0 (Christensen and Quail, 1996) harbouring the Bar gene, which confers resistance to the herbicide BASTA®.
  • pAHC20 0 Christensen and Quail, 1996) harbouring the Bar gene, which confers resistance to the herbicide BASTA®.
  • BASTA® herbicide BASTA®
  • For particle bombardment highly embryogenic callus induced from meristems or mature embryos was used.
  • Two different ryegrass cultivars (ACTION and TELSTAR) and one propagated clone (F6) were used as source for the callus production.
  • Isolated embryos and meristems were cultured on a MS-based (Murashige and Skoog, 1962) ⁇ callus induction medium (CM) containing 3 % sucrose, 4 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 100 mg/l casein hydrolysate and 0.3 % (w/v) gelrite (Kelco) for 12-26 weeks in the dark at 23°C. Calli were maintained by subculturing every third week on fresh CM-medium.
  • CM callus induction medium
  • an osmotic pre-treatment for 4 hours were given by transferring small calli (2- 4mm) to a solid MS-based medium supplemented with 3 % sucrose, 3mg/l 2,4-D, 0.25 M sorbitol, 0.2 ⁇ M mannitol and 0,3 % w/v Gelrite.
  • Bombardment was performed with a particle inflow gun (Finer et al., 1992) according to the optimised protocol described by Spangenberg et al.
  • tissue pieces Prior to bombardment, tissue pieces (3-4 mm) were transferred for osmotic pretreatment in liquid medium containing 30g/l sucrose, 3mg/l 0 2,4-D, 0.2 ⁇ M sorbitol and 0.2 ⁇ M mannitol for 30 min, and then transferred to the same medium solidified with 0.3% gelrite and incubated overnight in the dark.
  • Gold particles (1.0 ⁇ m), coated with 12 ⁇ g of a mixture of pLPTFLI and pAHC20 at a molar ratio of 1 :1 were used for particle bombardment with a Bio-Rad PDS-1000 He Biolistic device (Biorad, Hercules, California) at 1300 Psi.
  • calli were placed on MS ⁇ medium ⁇ supplemented with 2 mg/l bialaphos (Shinyo Sangyo Ltd., Japan) and grown at 2 ⁇ ⁇ 1 °C under 16 hrs light. After four to five successive rounds of selection at three weeks interval, putative transgenic plants were regenerated from the calli by supplementing the selection medium with 0.2 mg/l kinetin. Each callus tissue gave between one to four explants, which were transferred to soil and grown to maturity under greenhouse conditions. Results
  • FSD Family Specific Domain Differential Display
  • the aim of the screening was to isolate MADS box genes with an apparent differential spatial or temporal expression pattern.
  • the majority of the cDNA fragments amplified with the FSD primer displayed constitutive expression during floral transition.
  • a number of the cDNA fragments showed up- or down-regulation during primary and/or secondary induction ( Figure 1 ).
  • Figure 1 Those fragments showing a suitable expression pattern from Figure 1 (specific up- regulation in apices during primary and/or secondary induction) were cloned and sequenced for identification.
  • full-length cDNA clones were isolated from a ryegrass cDNA library produced from reproductive apices during the floral transition/development.
  • LpMADS1-3 were expressed at all stages examined, but at very low level during the vegetative stage.
  • LpMADS4- ⁇ were expressed within 2-3 weeks of secondary induction while LpMADS ⁇ was expressed within the first 3 days.
  • LpMADS7 was expressed within 3-7 days, LpMADS9 was expressed within 1-2 weeks.
  • LpMADS1-3 were expressed at all stages examined but at very low level during the vegetative stage, LpMADS ⁇ was expressed within 3- ⁇ weeks of secondary induction, ⁇ while the others (LpMADS4-7) were not expressed in leaves at any stage.
  • LpMADS1-7 and 9 were expressed at different levels, except LpMADS9, which was not expressed in stems. None of the LpMADS genes were expressed in seedlings.
  • LpMADS1-3 showed expression in the roots, but LpMADS2-3 were expressed at low 0 levels only.
  • LpMADS2, 4 and 6 were expressed in green calli only, while LpMADS ⁇ was also expressed in white callus.
  • LpMADS4, ⁇ , 6 and 7 were selected because of their specific pattern of expression during flower induction/development. Based on real-time RT-PCR experiments, it was shown ⁇ that they are not expressed in leaves or roots but are expressed in the shoot apices after secondary induction and in reproductive organs (flowers, stems and knees) (see Figure 2).
  • the 4 MADS box genes with the wanted expression characteristics fall into one group in the phylogenetic analysis (see figure 3).
  • LpMADS 4-7 promoters and LpMADS4-7 promoters and introns were also cloned into L ⁇ O and ⁇ L ⁇ O without Actin intron, respectively.
  • a schematic drawing of the vectors I37 and L ⁇ O is presented in figure 4.
  • CON ID NO: 1 , CON ID NO: 3, CON ID NO: ⁇ and CON ID NO: 7 illustrate the expression cassettes of GUS driven by promoters from LpMADS4, LpMADS ⁇ , LpMADS6 and
  • CON ID NO: 2 CON ID NO: 4
  • CON ID NO: 6 CON ID NO: 8 0
  • CON ID NO: 8 0 illustrates the expression cassettes of GUS driven by promoters and introns from LpMADS4,
  • CON ID NO: 9 illustrates the expression cassettes of Barnase driven by promoters LpMADS4, LpMADS5, LpMADS6 and LpMADS7, respectively.
  • CON ID NO: 10 illustrates the expression cassettes of Barnase driven by promoters and introns from LpMADS4, LpMADS ⁇ , LpMADS ⁇ and LpMADS7, respectively, The different constructs are illustrated in figure 9 to 16.
  • CON ID NOS: 1 , 2, 3, 4, 7 and 8 were transformed via biolistical bombardment into Lolium (ryegrass) and Festuca (red fescue). Transient GUS staining was performed in order to test for promoter activity in the callus phase.
  • Barstar have been generated, in order to neutralize the toxic effect of Barnase in the callus phase.
  • the Barstar gene can then be crossed out in a later phase of the product development.
  • CON ID NOS: 4 and 8 containing LpMADS ⁇ and 7 promoter/intron fusion to GUS showed no GUS staining in transiently transformed calli.
  • LpMADS4, LpMADS ⁇ and LpMADS7 constructs were confirmed by transient expression studies in different plant tissues using the udiA GUS-reporter ⁇ gene.
  • LpMADS ⁇ CON ID NO: 4
  • GUS-positive spots highest number and strongest staining.
  • the expression was restricted to floral primordial tissue, demonstrating the expected tissue and developmental specific expression (see figure 18).
  • the transient test included the testing of the MADS ⁇ promoter region (SEQ ID NO: 2) controlling the GUS reporter gene uidA from E. coli, and an investigation of the influence of the first intron on the transcriptional expression of LpMADS ⁇ .
  • CON ID NO: 4 MADS ⁇ with its native first intron
  • CON ID NO: 17 which contained only the ⁇ ' non-transcribed genomic region of LpMADS ⁇ fused to uidA from E. coli.
  • the constructs are illustrated in figure 17. Young non- induced leaves, secondary induced leaves and floral primordia were bombarded with the different constructs, respectively, and the expression pattern was recorded by counting the resulting spots after histochemical staining with X-Gluc (see figure 19).
  • the MADS ⁇ promoter alone without its native intron shows floral primordial tissue specific expression, as also illustrated by real time PCR analysis (see above).
  • transgenic L. perenne lines expressing the UdiA GUS-reporter gene under the control of the LpMADS ⁇ and LpMADS7 promoters were generated with CON ID NO 4 and CON ID NO 8, respectively.
  • GUS stainings of aerial tissues from vegetative plants, aerial tissues from plants after 12 weeks of vernalization, aerial tissues from secondary induced plants (2-3 weeks, 4- ⁇ weeks and 8-9 weeks after shift to secondary induction), stem and knee sections and developing flowers were performed.
  • ⁇ GUS activity and blue staining could be detected after 4 to ⁇ weeks secondary induction in the developing inflorescence and culm nodes of plants transformed with CON ID NO 4.
  • the LpMADS ⁇ promoter conferred GUS activity in later stages of the floral development (see figure 21 ).
  • Panel A of figure 21 a part of the inflorescence of a L. perenne line transformed with CON ID NO 4 is shown 7 weeks after secondary induction. Spikelets 0 consisting of several flowers showed blue staining in each of the flowers.
  • Panel B and C of figure 21 show opened flowers with the detached ovary and stigma, where the majority of the GUS staining was observed in the ovary.
  • Plants transformed with CON ID NO 8 showed GUS staining after 2 to 4 weeks of secondary induction, and, in contrast to CON ID NO 4, the Gus activity decreased as the 6 inflorescence differentiated to the fully developed floral structures. This indicates, that the LpMADS7 promoter was active in a temporal and developmental window for the first ⁇ th to 6th weeks of secondary induction. In some cases weak blue staining in culm nodes could be detected.
  • Shown in figure 22 is the developing inflorescence, 3 weeks after secondary induction, of a transgenic L. perenne line expressing the UdiA GUS reporter gene under the control of the LpMADS 7 promoter (CON ID NO 8). In panel A the overall appearance of the developing inflorescence is shown.
  • Panel C shows an enlargement of three developing spikelets. Also Gus staining could be detected in a distinct region below the developing inflorescence in the junction zone to the stem (peduncle) as shown in panel B.
  • the LpMADS7 promoter was temporarily active in the time frame 2 to 4 weeks after shift to long day at 20°C 0 and with activity decreasing in the later stages of floral development.
  • LpMADS ⁇ and LpMADS7 promoters in combination with their own first exon and intron showed no GUS expression in vegetative plant structures or in tissues from plants under vernalization, and the GUS expression observed was in agreement with the expression data presented in the real-time RT-PCR experiment shown in figure 2.
  • Both isolated ⁇ LpMADS ⁇ and LpMADS7 promoters fused to the GUS gene and retransformed into L. perenne showed GUS expression in the same time and developmental frame as the endogenous genomic copies of the LpMADS ⁇ and LpMADS7 genes as measured by real-time RT-PCR. This demonstrates the potential of the LpMADS ⁇ and LpMADS7 promoters as tools to direct transgene expression to specific reproductive structures after shift to reproductive growth in 0 Lolium perenne.
  • Festuca rubra plants were transformed with CON ID NO 16 (LpMADS7promoter- intron::Barnase) and a set of 38 transgenic lines were regenerated. 12 lines were identified to ⁇ have the CON ID NO 16 integrated in the genome. These plants were vernalized together with control plants and flowering was induced by 12 weeks of vernalization and a following secondary induction with long day light at 20°C. After several weeks of secondary floral induction the flowering phenotype was scored, and 4 transgenic lines showed no development of any floral structures.
  • CON ID NO 16 LpMADS7promoter- intron::Barnase
  • Figure 23 shows three of those lines in comparison with one control line induced to flower, thus demonstrating the potential of the LpMADS7 promoter to specifically direct expression of a cytotoxic gene to floral determined tissues of grasses for the purpose of ablation of reproductive tissues, including stems, inflorescences and flowers.
  • the regeneration of plants from callus transformed with the LpMADS7promoter-intron::Barnase construct demonstrates the lack of expression (tightness) by the LpMADS7 promoter-intron in in vitro cultured callus tissue.
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Abstract

L'invention concerne une pluralité de promoteurs à boîte MADS spécifiques aux cellules déterminées florales, issus de plantes monocotylédones, ainsi que des cassettes d'expression contenant de tels promoteurs, destinées à être employées dans l'expression de gènes souhaités de cellules déterminées florales. L'invention concerne par ailleurs des applications desdites cassettes d'expression et desdits promoteurs, notamment l'expression spécifique de gènes létaux ou de constructions antisens destinés à inhiber la floraison. Lesdits gènes ou constructions peuvent servir à augmenter la capacité fourragère d'herbes, ou à empêcher l'ensemencement de plantes. L'invention concerne également des procédés de modification de plantes de manière que celles-ci contiennent lesdites cassettes, ainsi que des plantes et des lignées cellulaires transformées.
PCT/EP2003/011038 2002-09-27 2003-09-23 Promoteurs specifiques aux tissus issus de plantes Ceased WO2004035797A2 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006005520A2 (fr) 2004-07-08 2006-01-19 Dlf-Trifolium A/S Moyens et procedes de commande de la floraison chez des plantes
CN103299895A (zh) * 2013-02-27 2013-09-18 中国农业科学院烟草研究所 一种胞质雄性不育系的快速创制方法
WO2015168124A1 (fr) * 2014-04-28 2015-11-05 The Trustees Of The University Of Pennsylvania Compositions et procédés permettant de réguler la croissance et le développement de plantes
CN108752446A (zh) * 2018-06-21 2018-11-06 江苏省太湖常绿果树技术推广中心 一种杨梅MrLFY基因及其应用
CN112831504A (zh) * 2021-03-16 2021-05-25 昆明理工大学 三七WRKY转录因子基因PnWRKY9及其应用
CN113322257A (zh) * 2021-05-31 2021-08-31 昆明理工大学 一种三七诱导型启动子ppo1及其应用
CN113373145A (zh) * 2021-05-31 2021-09-10 昆明理工大学 一种三七诱导型启动子ppl1及其应用
US11124801B2 (en) 2018-04-18 2021-09-21 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
CN113652426A (zh) * 2021-08-20 2021-11-16 昆明理工大学 一种三七诱导型启动子r1及其应用
US12371702B2 (en) 2018-04-18 2025-07-29 Pioneer Hi-Bred International, Inc. Improving agronomic characteristics in maize by modification of endogenous mads box transcription factors

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CN107815454B (zh) * 2017-11-28 2021-02-19 云南省烟草农业科学研究院 一种烟草开花期调控基因NtMADS1及其克隆方法与应用
CN108094058A (zh) * 2018-02-26 2018-06-01 深圳文科园林股份有限公司 一种采用人工光源栽培高羊茅的技术

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Publication number Priority date Publication date Assignee Title
EP0672155A1 (fr) * 1992-06-30 1995-09-20 Asgrow Seed Company Procede d'obtention d'une plante a morphologie florale modifiee, et procede de protection des plantes contre les insectes nuisibles
CA2324453A1 (fr) * 1998-03-19 1999-09-23 Roland Gleissner Association de genes pour la regulation de l'induction de la floraison chez les plantes cultivees et les plantes d'ornement
WO2000037488A2 (fr) * 1998-12-21 2000-06-29 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Nouveaux genes a boite mads et utilisation de ces genes
WO2000055172A1 (fr) * 1999-03-17 2000-09-21 Carter Holt Harvey Limited Plantes a capacite de reproduction modifiee
AUPR087300A0 (en) * 2000-10-19 2000-11-16 Agresearch Limited Manipulation of flowering and plant architecture

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006005520A2 (fr) 2004-07-08 2006-01-19 Dlf-Trifolium A/S Moyens et procedes de commande de la floraison chez des plantes
CN103299895A (zh) * 2013-02-27 2013-09-18 中国农业科学院烟草研究所 一种胞质雄性不育系的快速创制方法
CN103299895B (zh) * 2013-02-27 2016-01-13 中国农业科学院烟草研究所 一种烟草胞质雄性不育系的快速创制方法
WO2015168124A1 (fr) * 2014-04-28 2015-11-05 The Trustees Of The University Of Pennsylvania Compositions et procédés permettant de réguler la croissance et le développement de plantes
US11584937B2 (en) 2014-04-28 2023-02-21 The Trustees Of The University Of Pennsylvania Compositions and methods for controlling plant growth and development
US11421242B2 (en) 2018-04-18 2022-08-23 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US12371702B2 (en) 2018-04-18 2025-07-29 Pioneer Hi-Bred International, Inc. Improving agronomic characteristics in maize by modification of endogenous mads box transcription factors
US12234470B2 (en) 2018-04-18 2025-02-25 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
US11124801B2 (en) 2018-04-18 2021-09-21 Pioneer Hi-Bred International, Inc. Genes, constructs and maize event DP-202216-6
CN108752446A (zh) * 2018-06-21 2018-11-06 江苏省太湖常绿果树技术推广中心 一种杨梅MrLFY基因及其应用
CN112831504B (zh) * 2021-03-16 2023-03-24 昆明理工大学 三七WRKY转录因子基因PnWRKY9及其应用
CN112831504A (zh) * 2021-03-16 2021-05-25 昆明理工大学 三七WRKY转录因子基因PnWRKY9及其应用
CN113373145A (zh) * 2021-05-31 2021-09-10 昆明理工大学 一种三七诱导型启动子ppl1及其应用
CN113373145B (zh) * 2021-05-31 2023-06-16 昆明理工大学 一种三七诱导型启动子ppl1及其应用
CN113322257B (zh) * 2021-05-31 2023-06-16 昆明理工大学 一种三七诱导型启动子ppo1及其应用
CN113322257A (zh) * 2021-05-31 2021-08-31 昆明理工大学 一种三七诱导型启动子ppo1及其应用
CN113652426A (zh) * 2021-08-20 2021-11-16 昆明理工大学 一种三七诱导型启动子r1及其应用
CN113652426B (zh) * 2021-08-20 2023-06-20 昆明理工大学 一种三七诱导型启动子r1及其应用

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