WO2004035797A2

WO2004035797A2 - Tissue-specific promoters from plants

Info

Publication number: WO2004035797A2
Application number: PCT/EP2003/011038
Authority: WO
Inventors: Klaus K. Nielsen; Claus H. Andersen; Ingo Lenk; Klaus Petersen; Thomas Didion
Original assignee: Riso National Laboratory; DLF Trifolium AS
Current assignee: Riso National Laboratory; DLF Trifolium AS
Priority date: 2002-09-27
Filing date: 2003-09-23
Publication date: 2004-04-29
Anticipated expiration: 2005-03-27
Also published as: NZ539360A; WO2004035797A3; GB0222467D0; AU2003293602A1

Abstract

A number of floral determined cell specific MADS box promoters from rye-grass (Lolium perenne) are described, together with expression cassettes including such promoters for use in expression of desired genes in floral determined cells. Applications of such expression cassettes and promoters are described, including the specific expression of lethal genes or antisense constructs to inhibit flowering. This may be used to increase the forage value of grasses, or to prevent seeding of plants. Methods of modifying plants to contain such cassettes are also described, as well as transformed plants and cell lines.

Description

TISSUE-SPECIFIC PROMOTERS FROM PLANTS

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. 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. 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. However, evidence for conserved function of specific MADS box genes between trees and grasses has not yet been shown. On the contrary, 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.

In its broadest terms, 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 inventors isolated a total of sixteen MADS box genes from Lolium perenne and investigated their expression patterns. Of the promoters investigated, only four displayed a suitable floral determined tissue-specific expression pattern. Thus, in a first aspect, 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.

Thus, the skilled man would understand that 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.

It has been observed that expression controlled by the promoters occurs at reduced levels in some of other tissues of the plant. However, this may not be detrimental if the product is not toxic or where the site of action of the expressed product is only floral determined tissues and not any other tissues. For example, 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. On the other hand, 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. For example, 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, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitosa, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, Trisetum flavescens.

The term "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. The term "inflorescence" refers to the total number of reproductive tissues or structures that the shoot apical cells differentiate into.

The terms "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. Thus, "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.

The terms "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.

In particular, 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. However, 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.

The term "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. Examples of 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. 2000; Simpson and Dean 2002), Meristem Identity genes, such as TFL, LFY, and the AP1 -family (MADS box class of genes) (Simpson and Dean 2002), genes determining plant architecture and/or development, 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 plant hormone biosynthesis, sensing or signalling (for example, Gibberellins, Cytokinins and Auxins) (McCourt 1999), genes involved in light perception and signalling such as Cryptochromes, Phytochromes, and CONSTANS (CO) (Mouradov et. A , 2002), and genes acting as regulators of the cell cycle (Vandepoele et al., 2002).

As used herein, 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. For example, a polynucleotide sequence that produces a lethal product may be selected from those that encode ribonucleases such as Barnase (Hartley, 1988), from B. amyloliquefaciens, RNase T1 (Mariani et al., 1990; Mariani et al., 1992; Reynaerts et al., 1993), from A. aryzae, bovine RNase A (Carsana et al., 1988), RNase I (Zhu LQ et al., 1990), and RNase H (Wu H. et al., 1998) from E.coli and set of plant RNases (family of S-proteins) (Norioka et al., 1996). Alternatively, 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 β-1-4-glucanases or β-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). "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. Examples of 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).

Furthermore, preventing the development of flowering parts of a plant will prevent seed production. Therefore, 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.

Alternatively, 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 yield (Okita et al., 2001 ), an environment or stress tolerance gene (Kasuga et al., 1999), or the like.

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. In particular, 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.

For example, 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. Furthermore, although 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. Therefore, the entire intron has been observed to cause an increase in expression level, which may be even further increased by the use of a truncated version. This may also be applicable to the promoters and their associated introns of the present invention. In this aspect, 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.

The term "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.

The terms "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. The term "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.

Using the information provided by the present invention, 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. By "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. Normally, 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. The 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). In the case that the similar and inventive sequences are mixed together and denatured simultaneously, 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. Thus for example either 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. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, 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. al., 2002) 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).

Furthermore, 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. al., 2002) LFY, the AP1- 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).

In addition, 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. Without wishing to be bound by theory, in forage grasses, 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. It should be noted that 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. As such, 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. Thus, in a further aspect, 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. Alternatively, 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.

Thus, the skilled man would understand that 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. In a grass plant, 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.

In a further aspect there is provided 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.

In a yet further aspect there is provided a polynucleotide with a sequence comprising SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, fragments or derivatives thereof. These 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. (eds), Transformation of plants and soil micro organisms, Cambridge, UK: University Press (1995); Bechthold et al., 1993; Duan et al., 1996; Shimamoto et al., 1994, each of which is incorporated herein by reference). 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. Therefore, 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. However, 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.

Thus, 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. However, transformation of the callus using promoters MADS4 (SEQ ID NO:1 ) and

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. Such a lethal product and counteracting product may, for example, be the Barnase/Barstar genes discussed above.

Thus, 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,

- inducing said second promoter to express the product of said second polynucleotide fragment, and

- selecting and vegetatively propagating any transformants. The plant may, for example, be any of those previously described hereinabove.

The first promoter and first polynucleotide fragment have been described above as being located on a first expression cassette with the second promoter and the second polynucleotide fragment located on a second expression cassette. Generally, each expression cassette may be either located on separate polynucleotide molecules, such as plasmids, or may be located on the same polynucleotide molecule.

An example of an inducible promoter suitable for the above method may be an ethanol- inducible system (Roslan et al., 2001 ; Salter et al., 1998).

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. For example, to overcome leakage, 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.

Thus, in a yet further aspect, 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. Alternatively, the inhibitor may be under the control of a weak constitutively active promoter. In this aspect, 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. However in the floral determined 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.

Thus, in still a further aspect, 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. For example, 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.

Upon administration of a suitable signal for expressing the second polynucleotide fragment, the effects of the first polynucleotide fragment under the control of the promoter of the present invention will be counteracted and the floral determined cells allowed to develop into inflorescence. Embodiments of the present invention will now be described by way of example, with reference to the accompanying drawings in which:

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:

1) vegetative

2) 6 weeks primary induction

3) 12 weeks primary induction

4) 12 weeks primary induction and 3 days secondary induction 5) 12 weeks primary induction and 1 week secondary induction

6) 12 weeks primary induction and 2 week secondary induction

7) 12 weeks primary induction and 3 week secondary induction

8) 12 weeks primary induction and 5 week secondary induction

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.

1 ) Apices vegetative

2) 6 weeks primary induction

3) 12 weeks primary induction

6) 12 weeks primary induction and 2 week secondary induction

7) 12 weeks primary induction and 3 week secondary induction

8) 12 weeks primary induction and 5 week secondary induction leaves (leaf blades harvested at three different time point during the floral transition):

9) Vegetative

10) 12 weeks primary induction

11 ) 12 weeks primary induction and 5 week secondary induction Other (tissues were all harvested from plants with fully developed flowers): 12) Flowers (mature flowers including all flower tissues but without seeds)

13) Stems (stems tissue without intercalary meristems (knees) and leaves)

14) Knees (intercalary meristems from stems)

15) Seedlings (1-week old shoots)

16) Roots (washed roots without any aerial tissues) 17) Green calli

18) White calli

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). 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

(CON ID NO:17) and the LpMADS5promoter-Δintron::GUS fusion (CON ID NO: 18)

Figure 18 illustrates transient expression studies on the influence of the first intron of LpMADSδ 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).

The 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. EXAMPLES Materials and Methods

Plant material Shoot 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.

RNA extraction and mRNA purification

RNA was extracted using the FastRNA Green Kit supplied by BIO101 , Inc. (Vista, CA, USA) according to the manufacturer's recommendation. Total RNA samples were treated with RNase-free DNasel to remove residual DNA and mRNA was purified from total RNA using Dynabeads Oligo (dT)25 from Dynal A.S (N-0212 Oslo, Norway).

Differential display analysis

Family Specific Domain differential display (FSD) analysis was carried out using the DisplayPROFILE-FSD Kit supplied by Display Systems Biotech Inc. (Vista, CA, USA). Purified mRNA derived from 5μg of DNase-free RNA was used for the synthesis of the cDNA templates as described by the manufacturer (Display Systems Biotech Inc.). Double-stranded cDNA was prepared and digested with Taql restriction enzyme and a specific DNA adapter was ligated to the ends of the cDNA fragments. Subsequently, PCR was performed with 0.5μl 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).

Gels were dried onto the glass plate and exposed to X-ray films (Kodak XK-1 ) overnight at -80°C. X-ray films and gels were aligned, and bands of interest were excised, eluted in 50μl of ddH₂0 at 95°C for 5 min and re-amplified by PCR according to the manufacturer's protocol (Display Systems Biotech). The PCR products were analysed on agarose gels. 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. Differentially expressed clones, which showed homology to MADS box genes by comparison with nucleotide sequences in the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov) database with the BLASTN search program were used as probes to screen a shoot apex cDNA library.

Screening and sequencing of cDNA clones

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 ³²P-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).

Quantitation of mRNA levels using Real-time PCR

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 5μg 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:

LpMADSI : 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'

LpMADSβ: KP053: 5'-CTC AAG CGT AAG GAA CAA-3' KP054: 5'-CAC ACT TAG ATA GTT CAC-3"

LpMADS7: 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.

Screening and sequencing of Genomic DNA clones A 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 10⁶ independent clones of an average size of 14 kb and more than 10⁹ pfu/ml was screened with ³²P-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).

Construction of expressing cassettes

Promoters were PCR amplified with primers that had been restricted in order to ligate them into either I37 or L50 (see Figure 3). For constructs including both the MADS promoter and first intron, 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. Primers (DA56 and DA61) 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). The same strategy was used for cloning LpMADSδ, 6 and 7 promoter or promoter-intron into 137 and L50. However, LpMADSδ promoter was restricted with Clal and Sacl and LpMADSδ promoter-intron with Clal and Ncol. LpMADS6 promoter was restricted with EcoRI and Sacl and LpMADS6 promoter-intron with EcoRI and Ncol. Finally, 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'

LpMADS4 promoter and 1^st intron:

DA56: 5'- GAG CTC GGT AAA TCC CAT GAA TCG GTG -3'

DA61 : 5'- AAA CCA TGG CAT TGT AGC AGA GTG TTG G -3' LpMADSδ promoter: MADSpromClalA: 5'-GGA ATC ATC GAT TGA AGG TGA TGT GGA GAC-3'

MADS5UTR5' : 5'-GTT CCT CCA TGG ATG C-3' LpMADSδ promoter and 1^st intron:

MADSδpromClalA: δ'-GGA ATC ATC GAT TGC AGG TGA TGT GGA GAC-3'

MADSδexon2Ncol: δ'-CTA TCC CAT GGG CAC AGT TGT TTC AGG TCC -3' LpMADS6 promoter:

KP09δ: δ'- ATT GAA TTC GAG CGT TGA TTT CGT CCA -3'

KP092: δ'-TAA GAG CTC ACT CAG ATA GCA CTA CCA-3' LpMADS6 promoter and 1^st intron:

KP09δ: δ'-ATT GAA TTC GAG CGT TGA TTT CGT CCA-3' KP102: δ'- TAT CCA TGG TGG AGT TGT AGT TGC AG -3'

LpMADS7 promoter:

KP064: δ'-ATT GAA TTC TCG CCG GTA AGG GCA TCT CT-3'

KP06δ: δ'-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: δ'- AAT CCA TGG CCA CGG CAT CTT GCG A -3'

Transient expression studies: Constructs

For cloning of the vector pMδP-GUS(CON ID NO: 17), 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 LpMADSδ promoter fragment was inserted into the Smal cut pAHC27 (Christensen and Quail; 1996), thereby replacing the Z. mays Ubiquitin 1 promoter with the δ' untranscribed region of L. perenne MADSδ.

The vector pM5P-M5lΔ-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.

Tissue preparation for particle bombardment

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 leπght and put onto tap water soaked 3MM paper before further treatment. Optionally, 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. Prior to further treatment, 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. 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.

Particle bombardment 0 Particle bombardment was carried out with a Helios™ Gene Gun system (Bio-Rad

Laboratories, Inc., USA) and the associated equipment.

Particle coating

2δ mg of 1 μm gold particles were coated with the appropriate plasmid DNA by δ subsequent addition of δO μl of δO mM spermidin, δO μl of 1 μg/μl plasmid DNA, δO μl of

1 M CaCI₂ to a 1 ,5 ml microreaction tube. The tube was vortexed during and sonicated between each addition step. The particles were settled by a 10 min incubation at room temperature.

Particles were rinsed three times with 1 ml of 100 % ethanol by centrifugation for 5 seconds at

13.000 rpm in a SIGMA 202MC centrifuge. 0 Particles were resuspended in 3 ml of 100 % ethanol, 10 μg/ml PVP 360. The suspension was soaked into a tefzel tubing, which was inserted into the tubing prep station. The tubing had been predried in the prep station for at least 1δ min by nitrogen stream at 0.4 Ipm.

Particles were settled for 4 min, the liquid was soaked out and the tubing was immediately turned by 180°. After 1 min the tubing support cylinder was switched to continuous rotation for 5 another 1 min before the inner tubing was dried by nitrogen stream at 0.4 Ipm and rotation for at least 10 min. The coated tubing was cut into pieces of approximately 1 cm which were stored under dry conditions until bombardment was carried out.

Particle bombardment parameters Bombardment parameters for the tested tissues were determined as the following:

(1 ) young leaves: 8 - 12 bar helium

(2) secondary induced leaves: 12 bar helium

(3) floral tissue: 8,5 bar helium

5 After bombardment the tissue was left for approx. 24 h on the agarose plates before X-

Gluc staining was carried out.

X-Gluc staining

The 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 μM X-Gluc; 100 m NaP0₄, pH 7,0; 0,1 % Triton X-100; δOO μM K₃Fe(CN)₆; δOO μM K₄Fe(CN)₆; 10 mM EDTA, pH 7,0; 20 % ethanol. δ Chloralhydrate solution: 67 % (w/v) chloralhydrate; 8,3 % (v/v) glycerol in H₂0

Stable expression studies - plant transformation Transformation of Lolium perenne

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®. 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) δ 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. Prior to bombardment, 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, 0.2δ 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. (199δ) with a few modifications: bombardment pressure was 8 bar and 300 μg gold particles 0.6 μm (Biorad) were coated with 0.6 μg plasmid DNA (pLPTFL and pAHC20 at a molar ratio of δ 2:1 ) according to Vain et al. (1993). The following day, calli were transferred to CM-medium supplemented with 2 mg/l bialaphos (Meiji Seika Kaisha, LTD, Tokyo) and grown at 23°C under 16 hrs light. Selection at three weeks interval was performed until vigorously growing callus was obtained. Putative transgenic plants were regenerated by transferring calli to hormone free medium RM (MS-medium containing 3% sucrose and 2 mg/l bialaphos). Rooted plantlets were 0 transferred to soil and grown to maturity under greenhouse conditions.

Transformation of Festuca rubraPlasmids of interest were introduced into red fescue together with pAHC20 (Christensen and Quail, 1996) harboring the Bar gene, which confers resistance to the herbicide BAST A®. Friable, embryogenic calli, ready for particle bombardment δ were prepared by growing excised embryos on a MS (Murashige and Skoog, 1962)-based callus-induction medium (MSδ) for 10-12 weeks at 2δ°C in the dark. The MSδ medium was supplemented with δ mg/l 2,4-dichlorphenoxyacetic acid (2,4-D), δOO mg/l casein hydrolysate and 3% (w/v) sucrose and solidified with 0.3% gelrite. 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, 0.2δ M sorbitol and 0.2δ 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 μm), coated with 12 μg 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. Following bombardment, calli were placed on MSδ medium δ supplemented with 2 mg/l bialaphos (Shinyo Sangyo Ltd., Japan) and grown at 2δ ± 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

Family Specific Domain Differential Display (FSD) was performed on samples made from ryegrass apices harvested at different time points during the floral transition (primary and δ secondary floral induction). After several rounds of screening using FSD, 9 MADS box cDNA fragments were isolated from Lolium perenne. Additional 7 MADS box cDNA clones were isolated through cDNA Library screenings using a conserved MADS domain as probe and Yeast Two Hybrid studies.

0 The aim of the screening was to isolate MADS box genes with an apparent differential spatial or temporal expression pattern. As illustrated in the FSD gel of Figure 1 , the majority of the cDNA fragments amplified with the FSD primer displayed constitutive expression during floral transition. However, a number of the cDNA fragments showed up- or down-regulation during primary and/or secondary induction (Figure 1 ). δ 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. Upon identification and verification, full-length cDNA clones were isolated from a ryegrass cDNA library produced from reproductive apices during the floral transition/development. 0 A total of 8 different full-length MADS box cDNA clones (MADS1 to 7 and 9) were isolated and sequenced. Upon sequencing their expression pattern was investigated/verified by use of real time quantitative PCR (see Figure 2). The expression pattern of additional LpMADS box genes (LpMADS10-16), which were isolated by cDNA library screenings and Yeast Two Hybrid studies are also shown in Figure 2 for comparison. In the following results, no detectable δ gene expression has been interpreted as no expression

These experiments revealed that, in shoot apices, LpMADS1-3 were expressed at all stages examined, but at very low level during the vegetative stage. LpMADS4-δ were expressed within 2-3 weeks of secondary induction while LpMADSδ was expressed within the first 3 days. LpMADS7 was expressed within 3-7 days, LpMADS9 was expressed within 1-2 weeks.

In leaves, LpMADS1-3 were expressed at all stages examined but at very low level during the vegetative stage, LpMADSΘ was expressed within 3-δ weeks of secondary induction, δ while the others (LpMADS4-7) were not expressed in leaves at any stage.

In the flowers, stems and knees, 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.

In the callus, LpMADS2, 4 and 6 were expressed in green calli only, while LpMADSβ was also expressed in white callus.

LpMADS4, δ, 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 δ 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).

In silico analysis of the different cloned cDNA sequences support the expression data gained by real time RT-PCR analysis.

The 4 MADS box genes with the wanted expression characteristics (LpMADS4, δ, 6 0 and 7) fall into one group in the phylogenetic analysis (see figure 3).

The common expression pattern of those 4 genes is further supported by an in silico analysis of the 4 promoter and first intron regions for cis-acting elements. As illustrated in table

1 all the four promoter sequences contain LEAFY binding elements and GA-MYB related binding elements. Both elements are specific for genes regulated upon flower development δ (Deyholos and Sieburth, 2000; Busch et al., 1999; Gocal et al., 2001 ).

Table 1. Motifs common to all four genomic sequences of LpMADS4, LpMADSδ, LpMADSΘ and LpMADS7

Cloning of LpMADS Promoters into 137 and L50

Recent publications on MADS box genes have uncovered that regulatory elements for δ proper transcriptional regulation can be found not only in the promoter region upstream the gene encoding sequence, but also in the intron sequence of the genes. Especially the largest intron (most often the first intron in the sequence) contained regulatory elements necessary for the specific expression of the gene (Deyholos and Sieburth, 2000, Busch et al., 1999). Therefore, in order to analyse the potential role of the first intron of the LpMADS4-7 genes in the 0 specific expression pattern of their respective promoters, we compared the expression patterns of promoter-GUS constructs with or without the first intron sequence.

Promoters from LpMADS4-7, and promoters and introns from LpMADS4-7 were cloned into I37 and I37 without Actin intron, respectively. For expression of the Barnase gene, the

LpMADS 4-7 promoters, and LpMADS4-7 promoters and introns were also cloned into LδO and δ LδO without Actin intron, respectively. A schematic drawing of the vectors I37 and LδO is presented in figure 4.

CON ID NO: 1 , CON ID NO: 3, CON ID NO: δ and CON ID NO: 7 illustrate the expression cassettes of GUS driven by promoters from LpMADS4, LpMADSδ, LpMADS6 and

LpMADS7, respectively. CON ID NO: 2, CON ID NO: 4, CON ID NO: 6 and CON ID NO: 8 0 illustrates the expression cassettes of GUS driven by promoters and introns from LpMADS4,

LpMADSδ, LpMADSβ and LpMADS7, respectively. CON ID NO: 9, CON ID NO: 11 , CON ID NO: 13 and CON ID NO: 1 δ illustrate the expression cassettes of Barnase driven by promoters LpMADS4, LpMADS5, LpMADS6 and LpMADS7, respectively. CON ID NO: 10, CON ID NO: 12, CON ID NO: 14 and CON ID NO: 16 illustrate the expression cassettes of Barnase driven by promoters and introns from LpMADS4, LpMADSδ, LpMADSβ and LpMADS7, respectively, The different constructs are illustrated in figure 9 to 16.

Transformation of Lolium (ryegrass) and Festuca (red fescue) with CON ID NO: 1-8

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.

Both Lolium and Festuca transiently transformed with CON ID NOS: 1 , 2, 3 and 7 showed visible GUS staining in callus. This indicates that the promoters LpMADS4 with the actin intron or with its own intron, LpMADS 5 with actin intron and LpMADS 7 with actin intron are not tight in callus phase, and that unwanted expression of Barnase in the callus phase may thus occur. In order to enable the transformation of the promoters LpMADS4, δ and 7, and

LpMADS4 promoter/intron Barnase fusion, Lolium and Festuca lines constitutively expressing

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.

It turned out that the actin intron from rice conferred constitutive expression to any promoter fused to during our investigations. This general feature of the rice action intron (i.e. conferring enhanced and constitutive expression of tissue specific promoters) has previously been described (W09109948).

In contrast CON ID NOS: 4 and 8 containing LpMADSδ and 7 promoter/intron fusion to GUS showed no GUS staining in transiently transformed calli. These results suggest that LpMADSδ and 7 promoter/intron were tight in the callus phase and that an unwanted expression of Barnase in the callus phase is hot to be expected.

TRANSIENT EXPRESSION STUDIES Comparison of the expression pattern of the 5' regulatory regions of LpMADS4, 5 and 7 in transient assays

The expression patterns of the LpMADS4, LpMADSδ and LpMADS7 constructs were confirmed by transient expression studies in different plant tissues using the udiA GUS-reporter δ gene.

Leaves from two week old seedlings, leaves from secondary induced plants and floral primordial tissue from 2- 3 weeks secondary induced Lolium perenne plants were subjected to particle bombardment with constructs CON ID NO: 2, CON ID NO: 4 and CON ID NO: 8, respectively. Figure 18 illustrates the results from these experiments. 0 The transient expression studies in young leaves, secondary induced leaves and floral primordial tissue showed the floral determined cell specific expression of LpMADS4, δ and 7.

The results confirmed the data obtained by the real time PCR expression analysis, with relative expression levels reflecting the expression levels quantitatively determined by real time-PCR

(see above). δ Among the three MADS constructs investigated, LpMADSδ (CON ID NO: 4) gave the most GUS-positive spots (highest number and strongest staining). For all three constructs the expression was restricted to floral primordial tissue, demonstrating the expected tissue and developmental specific expression (see figure 18).

Single spots observed on leaf tissue in very rare cases were most likely the result of 0 contamination, construct rearrangement or rare integration events into an transcriptionally active site of the genome. Nevertheless, all spots were included in the number of counted spots illustrated in figure 18.

Influence of the first intron on the expression of LpMADSδ in transient assays δ A more detailed analysis was carried out with LpMADSδ. The transient test included the testing of the MADS δ 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 LpMADSδ. In order to investigate the role of the first intron on expression level and tissue specificity, three different constructs were tested: 1) CON ID NO: 4 (MADSδ with its native first intron), 2) CON ID NO: 18, where a major portion of the first intron was deleted leaving δδ6 nt, and 3) CON ID NO: 17, which contained only the δ' non-transcribed genomic region of LpMADSδ 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 MADSδ promoter alone without its native intron shows floral primordial tissue specific expression, as also illustrated by real time PCR analysis (see above).

No GUS positive spots were observed in vegetative tissue such as young leaves, secondary induced leaves or stem tissue below the upper most knee when delivering the construct without an intron, CON ID NO: 17, transiently into these tissues by particle bombardment. GUS staining was still observed exclusively in floral primodial tissue, although the level of expression was lower compared to the construct CON ID NO: 4 (containing the native first intron), resulting in both fewer and more faint spots.

By comparison of the GUS expression conferred by CON ID NO: 4 and CON ID NO:

18, the latter construct (with the partly deleted intron) gave the same tissue specificity but a higher number of spots with higher staining intensity, indicating that portion of the intron with the capability of restricting the expression level to a certain extend was deleted (see figure 21 ). This result could not be expected.

One possible interpretation of this result is that the deletion of the central intronic part of LpMADSδ (CON ID NO: 18) removed putative binding sites for transcription factors resulting in an increased, but still tissue specific GUS-expression. Thus, the result of this study suggests that the first intron is not essential for the tissue specific expression of the MADSδ promoter, but that it contains regulatory elements of importance for specifying the level of gene expression in the floral determined tissue.

STABLE EXPRESSION STUDIES - 36 -

Promoter expression studies in stably transformed Lolium plants

In agreement with the transient expression results, stable transformation studies with the constructs containing the different promoters in combination with the actin intron (Con ID1 , 3 δ and 7) showed non-specific constitutive expression of the UdiA G US-reporter gene in δ transgenic Lolium plants. None of the transgenic lines transformed with these plasmids were pursued any further.

Several independent transgenic L. perenne lines expressing the UdiA GUS-reporter gene under the control of the LpMADSδ and LpMADS7 promoters were generated with CON ID NO 4 and CON ID NO 8, respectively. The transgenic L. perenne plants transformed with CON 0 ID NO 4 and CON ID NO 8 were analysed in detail through a whole life cycle.

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-δ weeks and 8-9 weeks after shift to secondary induction), stem and knee sections and developing flowers were performed. δ GUS activity and blue staining could be detected after 4 to δ weeks secondary induction in the developing inflorescence and culm nodes of plants transformed with CON ID NO 4. In addition, the LpMADSδ promoter conferred GUS activity in later stages of the floral development (see figure 21 ). In 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 δth 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. Blue staining can be seen in the developing flowers on the developing δ spikelets. 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.

In accordance with the real-time RT-PCR results shown in figure 2, 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.

In conclusion, the LpMADSδ 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 δ LpMADSδ 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 LpMADSδ and LpMADS7 genes as measured by real-time RT-PCR. This demonstrates the potential of the LpMADSδ and LpMADS7 promoters as tools to direct transgene expression to specific reproductive structures after shift to reproductive growth in 0 Lolium perenne.

Mads7 promoter Barnase expression in Festuca:

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 δ 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. 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. In addition, 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.

References

Allen RD, Bernier F, Lessard PA, Beachy RN. Nuclear factors interact with a soybean beta-conglycinin enhancer. Plant Cell 1 :623-631 (1989).

Berzal-Herranz et al., In vitro selection of active hairpin ribozymes by sequential RNA- δ catalyzed cleavage and ligation reactions. Genes and Devel., 6:129-134, 1992.

Busch MA, Bomblies K, Weigel D. Activation of a floral homeotic gene in Arabidopsis. Science. 1999 Jul 23;28δ(δ427):δ8δ-7.

Cercos M, Gomez-Cadenas A, Ho THD. Hormonal regulation of a cysteine proteinase gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the co- 0 ordinated gene expression regulated by gibberellins and abscisic acid. Plant J 19: 107-118 (1999).

Chowrira et al, "In vitro and in vivo comparison of hammerhead, hairpin, and hepatitis delta virus self-processing ribozyme cassettes," J. Biol Chem., 269:26866-26864, 1994.

Christensen and Quail 1996; Ubiquitin promoter-based vectors for high-level expression δ of selectable and/or screenable marker genes in monocotyledonous plants; Transgenic Research 6, 213-218.

Collier R.J., Diphtheria toxin: mode of action and structure. Bacteriol. Rev. 39:64-86 (1976).

Colot V, Robert LS, Kavanagh TA, Bevan MW, Thompson RD. Localization of 0 sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco. EMBO J 6:3569-3664 (1987)

Drews GN, Bowman JL, Meyerowitz EM.; 1991 ; Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell 14;65(6):991-1002.

Elliott KA , Shirsat AH. Promoter regions of the extA extensin gene from Brassica napus δ control activation in response to wounding and tensile stress Plant Mol Biol 37:676-687 (1998).

Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, Somssich IE. Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J 18: 4689-4699. Flanagan CA, Hu Y, Ma H. Specific expression of the AGL1 MADS-box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development. Plant J. 1996 Aug;10(2):343-63.

Forster and Symons, "Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites," Cell, 49:211 -220, 1987.

Frutos, R, Rang, C, Royer, F. Managing insect resistance to plants producing Bacillus thuringiensis toxins. CRIT REV BIOTECHNOL 19 (3): 227-276 1999.

Gerlach et al., "Construction of a plant disease resistance gene from the satellite RNA of tobacco ringspot virus," Nature (London), 328:802-806, 1987. Grotewold E., Drummond B. J., Bowen B., Peterson T. The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76:643-663 (1994).

Gubler F, Kalla R, Roberts JK, Jacobsen JV. Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pl alpha-amylase gene promoter. Plant Cell 7:1879-1891 (1996).

Ha SB, An G. Identification of upstream regulatory elements involved in the developmental expression of the Arabidopsis thaliana cabl gene. Proc Natl Acad Sci USA 85: 8017-8021 (1988).

Hattori T, Terada T, Hamasuna S. Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1 : analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1. Plant J 7: 913-925 (1996).

Hobo T, Asada M, Kowyama Y, Hattori T. ACGT-containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent. Plant J 19: 679-689 (1999). Hobo T, Kowyama Y, Hattori T. A bZIP factor, TRAB1 , interacts with VP1 and mediates abscisic acid-induced transcription. Proc Natl Acad Sci USA 96: 15348-15353 (1999).

Hong JC, Cheong YH, Nagao RT, Bahk JD, Key JL, Cho MJ. Isolation of two soybean G-box binding factors which interact with a G-box sequences of an auxin-responsive gene. Plant J 8: 199-211 (1995). Huang H, Tudor M, Su T, Zhang Y, Hu Y, Ma H.; DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation. Plant Cell 1996 Jan;8(1 ):81 -94.

Huang H., Mizukami Y., Hu Y., Ma H.; Isolation and characterization of the binding sequences for the product of the Arabidopsis floral homeotic gene AGAMOUS. Nucleic Acids Res. 21 :4769-4776 (1993).

Huang N, Sutliff TD, Litts JC, Rodriguez RL. Classification and characterization of the rice alpha-amylase multigene family. Plant Mol Biol 14:666-668 (1990).

Hwang YS, Karrer EE, Thomas BR, Chen L, Rodriguez RL. Three cis-elements required for rice alpha-amylase Amy3D expression during sugar starvation. Plant Mol Biol 36:331-341 (1998).

Itzhaki H, Maxson JM, Woodson WR. An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GSTI) gene. Proc Natl Acad Sci USA 91 :8925-8929 (1994). Jagannath, A, Bandyopadhyay, P, Arumugam, N, et al. The use of a Spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high- frequency generation of transgenic male sterile lines in Brassica juncea L. MOL BREEDING 8 (1): 11-23 2001.

Jensen CS, Salchert K, Nielsen KK.; A TERMINAL FLOWER1-like gene from perennial ryegrass involved in floral transition and axillary meristem identity. Plant Physiol, March 2001 , Vol. 125, pp. 1617-1628.

Johansen B, Pedersen LB, Skipper M, Frederiksen S. MADS-box gene evolution- structure and transcription patterns. Mol Phylogenet Evol 2002 Jun;23(3):458-80.

Jongedijk, E, Tigelaar, H, Vanroekel, JSC, et al. Synergistic activity of chitinases and beta-1 , 3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 86 (1- 3): 173-180 1995.

Kagaya Y, Ohmiya K, Hattori T; RAV1 , a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27: 470-478 (1999). Kasuga, M, Liu, Q, Miura, S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. NAT BIOTECHNOL 17 (3): 287-291 1999.

Kieber J.J, Signer E.R., Cloning And Characterization Of An Inorganic Pyrophosphatase Gene From Arabidopsis thaliana, Plant Molecular Biology:16 (2): 346-348 (1991 ).

Kim and Cech, "Three dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena," Proc. Nat'l Acad. Sci. USA, 84:8788-8792, 1987.

King RW, Moritz T, Evans LT, Junttila O, Herlt AJ. Long-day induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex. Plant Physiol 2001 Oct;127(2):624-32.

Koprek, T, McElroy, D, Louwerse, J, et al. Negative selection systems for transgenic barley (Hordeum vulgare L.): comparison of bacterial codA- and cytochrome P460 gene- mediated selection. PLANT J 19 (6): 719-726 1999. Kreis M, Williamson MS, Forde J, Schmitz D, Clark J, Buxton B, Pywell J, Marris C,

Henderson J, Harris N, Shewry PR, Forde BG, Miflin BJ. Differential gene expression in the developing barley endosperm. Philos Trans R Soc Lond B314:35δ-365 (1986).

Lelievre J-M, Oliveira LO, Nielsen NC. δ'-CATGCAT-3' elements modulate the expression of glycinin genes. Plant Physiol 98:387-391 (1992). Lessard PA, Allen RD, Bernier F, Crispino JD, Fujiwara T, Beachy RN. Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes. Plant Mol Biol 16:397-413 (1991 ).

Levy et al. (in preparation).

Logemann E, Parniske M, Hahlbrock K. Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Natl Acad Sci USA 92:5905-6909 (1995) .

Lohmann JU, Weigel D Building beauty: The genetic control of floral patterning. Developmental Cell. 2 (2): 135-142 FEB 2002. Lovegrove A, Hooley R Gibberel n and abscisic acid signalling in aleurone Trends Plant Sci 2000 Mar, 5(3) 102-10

Marcotte WR Jr, Russell SH, Quatrano RS Abscisic acid-responsive sequences from the Em gene of wheat Plant Cell 1 969-976 (1989) Martin-Tπllo M, Martinez-Zapater JM Growing up fast manipulating the generation time of trees Current Opinion In Biotechnology 13 (2) 151 -155 Apr 2002

McCourt P Genetic analysis of hormone signalling Annual Review of Plant Physiology and plant Molecular Biology 50 219-243 1999

Montgomery J, Goldman S, Deikman J, Margossian L, Fischer RL Identification of an ethylene-responsive region in the promoter of a fruit ripening gene Proc Natl Acad Sci USA 90 5939-6943 (1993)

Moπshima A Identification of preferred binding sites of a hght-inducible DNA-binding factor (MNF1 ) within 5'-upstream sequence of C4-type phosphoenolpyruvate carboxylase gene in maize Plant Mol Biol 38 633-646 (1998) Moπta A, Umemura T, Kuroyanagi M, Futsuhara Y, Perata P, Yamaguchi J Functional dissection of a sugar-repressed α-amylase gene (RamylA) promoter in rice embryos FEBS Lett 423 81-85 (1998)

Mouradov A, Cremer F, Coupland G Control of flowering time Interacting pathways as a basis for diversity Plant Cell14 111-130 Suppl S 2002 Nambara E, Hayama R, Tsuchiya Y, Nishimura M, Kawaide H, Kamiya Y, Naito S The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination Dev Biol 2000 Apr 15, 220(2) 412-23

Ng M, Yanofsky MF Three ways to learn the ABCs Current opinion in plant Biology 3 (1 ) 47-62 FEB 2000 Ohgishi M, Oka A, Morelli G, Ruberti I, Aoyama T Negative autoregulation of the

Arabidopsis homeobox gene ATHB-2 Plant J 25 389-398 (2001 )

Okita, TW, Sun, JD, Sakulnngharoj, C, et al Increasing rice productivity and yield by manipulation of starch synthesis NOVART FDN SYMP 236 135-152 2001 Palukaitis et al., Characterization of a viroid associated with avacado sunblotch disease. Virology, 99:146-161 , 1979.

Probst JC: Antisense oligodeoxynucleotide and ribozyme design METHODS-A COMPANION TO METHODS IN ENZYMOLOGY 22 (3): 271-281 NOV 2000. Prody et al., "Autolytic processing of dimeric plant virus satellite RNA." Science,

231 :1677-1580, 1986.

Proveniers M, Smeekens S. The Arabidopsis homeobox gene ATH1 and floral transition. Developmental Biology. 186 (2): A49-A49 JUN 15 1997.

Rathore, KS, Chowdhury, VK, Hodges, TK Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts. PLANT MOL BIOL 21 (5): 871-884 1993.

Reidt W, Wohlfarth T, Ellerstrom M, Czihal A, Tewes A, Ezcurra I, Rask L, Baumlein H. Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product. Plant J 21 : 401-408 (2000). Roslan HA, Salter MG, Wood CD, White MRH, Croft KP, Robson F, Coupland G,

Doonan J, Laufs P, Tomsett AB, Caddick MX Characterization of the ethanol-inducible ale gene-expression system in Arabidopsis thaliana Plant Journal 28 (2): 225-235 OCT 2001

Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15:6690-5700 (1996).

Sablowski RWM, Moyano E, Culianez-Macia FA, Schuch W, Martin C, Bevan M. A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. EMBO J 13:128-137 (1994).

Salter MG, Paine JA, Riddell KV, Jepson I, Greenland AJ, Caddick MX, Tomsett AB Characterisation of the ethanol-inducible ale gene expression system for transgenic plants Plant Journal 16 (1 ): 127-132 Oct 1998.

Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G. Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. SCIENCE 288 (5471 ): 1613-1616 JUN 2 2000. Sato F, Kitajima S, Koyama T, Yamada Y. Ethylene-induced gene expression of osmotin-like protein, a neutral isoform of tobacco PR-5, is mediated by the AGCCGCC cis- sequence. Plant Cell Physiol 37: 249-255 (1996).

Sharma, A, Sharma, R, Imamura, M, et al. Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice. FEBS LETT 484 (1 ): 7-11 OCT 27 2000.

Shen Q, Ho TH. Functional dissection of an abscisic acid(ABA)-inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cis-acting element. Plant Cell 7:295-307 (1996). Shirsat A, Wilford N, Croy R, Boulter D. Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco. Mol Gen Genet 215:326-331 (1989)

Simpson G. G., and Dean C. Arabidopsis, the rosetta stone of flowering time? Science

296: 285-289. 2002.

Solano R., Nieto C, Avila J., Canas L., Diaz I., Paz-Ares J. Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida. EMBO

J. 14:1773-1784 (1996).

Steindler C, Matteucci A, Sessa G, Weimar T, Ohgishi M, Aoyama T, Morelli G, Ruberti

I. Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression. Development 1999 Oct;126 (19):4235-4δ. Stewart, CN. The utility of green fluorescent protein in transgenic plants. PLANT CELL

REP 20 (5): 376-382 2001.

Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G.

CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis.

NATURE 410 (6832): 1116-1120 APR 26 2001. Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G,

SymonsRH. Avocado sunblotch viroid: primary sequence and proposed secondary structure.

Nucleic Acids Res. 1981 Dec 11 ;9(23):6527-37.

Symons, "Avacado sunblotch viroid: primary sequence and proposed secondary structure." Nucl. Acids Res., 9:6527-6537, 1981. Tamagnone L, Merida A, Parr A, Mackay S, Culianez-Macia FA, Roberts K, Martin C The AmMYB308 and AmMYB330 transcription factors from antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell 10: 135-154 (1998).

Thomas Albert, Julie Wells, Jens-Oliver Funk, Andrea Pullner, Eva-Elisabeth Raschke, Gertraud Stelzer, Michael Meisterernst, Peggy J. Farnham, and Dirk Eick; 2001 ; The Chromatin Structure of the Dual c-myc Promoter P1/P2 Is Regulated by Separate Elements. J. Biol. Chem. 276: 20482-20490.

Thomas MS, Flavell RB. Identification of an enhancer element for the endosperm- specific expression of high molecular weight glutenin. Plant Cell 2:1171-1180 (1990). Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R; Agrobacterium nvmefac/ens-mediated barley transformation. The Plant Journal, 1997, 11(6), 1369-1376.

Toyofuku K, Umemura T, Yamaguchi J. Promoter elements required for sugar- repression of the RAmy3D gene for alpha-amylase in rice. FEBS Lett 428:276-280 (1998).

Uimari and Strommer; 1997; Myb26: a MYB-like protein of pea flowers with affinity for promoters of phenylpropanoid genes. Plant J.;12(6):1273-84.

Vandepoele K, Raes J, De Veylder L, Rouze P, Rombauts S, Inze D. Genome-wide analysis of core cell cycle genes in Arabidopsis. Plant Cell 14 (4): 903-916 APR 2002.

Venglat, SP, Dumonceaux, T, Rozwadowski, K, et al. The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis. P NATL ACAD SCI USA 99 (7): 4730-4735 2002.

Wang Z-Y, Kenigsbuch D, Sun L, Harel E, Ong MS, Tobin EM. A myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene. Plant cell 9:491-507 (1997).

Wang, H, Zhou, YM, Gilmer, S, et al. Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology. PLANT J 24 (5): 613-623 2000.

WO9807875-A1 Guilley H, Jonar G, Richards K, et al. Inducing viral resistance in plant

Worrall et al., Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Plant Cell 4:759-771 (1992). Wu C, Washida H, Onodera Y, Harada K, Takaiwa F. Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression. Plant J 23: 415-421 (2000).

Yanagisawa S., Schmidt R. J. Diversity and similarity among recognition sequences of Dof transcription factors. Plant J. 17:209-214 (1999).

Ziemienowicz, A. Plant selectable markers and reporter genes. ACTA PHYSIOL PLANT 23 (3): 363-374 2001.

Claims

1. A method of controlling expression of a gene product in floral determined cells, said method comprising: transforming a plant cell with an expression cassette, 5 said expression cassette comprising a promoter sequence and at least one associated polynucleotide fragment, wherein said promoter sequence is capable of controlling preferential expression of a product from said at least one associated polynucleotide fragment in floral determined cells, and expressing the product from at least one associated polynucleotide fragment in floral 0 determined cells.

2. A method according to claim 1 , wherein said plant is a monocot.

3. A method according to any preceding claim, wherein said plant is a monocot δ selected from the grass family of Poaceae.

4. A method according to any preceding claim, wherein said plant is selected from the group consisting of Phleum species, Dactylis species, Lolium species, Festulolium species, Festuca species, Poa species, Bromus species, Agrostis species, Arrhenatherum species, 0 Phalaris species, and Trisetum species,

δ. A method according to any preceding claim, wherein said plant is selected from the group consisting of Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, δ Festulolium loliaceum, Festulolium holmbergii, Festulolium pabulare, Festuca pratensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitosa, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, and Trisetum flavescens.

6. A method according to any preceding claim, wherein said promoter sequence δ of the expression cassette is active in the shoot apex at a early time point once the floral transition has been initiated.

7. A method according to any preceding claim, wherein said product to be expressed by the promoter of the expression cassette substantially or completely prevents 0 flowering, delays or accelerates flowering, or renders the plant sterile.

8. A method according to any preceding claim, wherein said product to be expressed by the promoter of the expression cassette is selected from the group consisting of a polypeptide lethal to the plant, a polynucleotide fragment or a nucleotide sequence which is at 5 least in part complementary to at least a portion of a gene essential for the development of inflorescence tissue and stems.

9. A method according to claim 8, wherein said product is encoded by a gene or combination of genes which encode a polypeptide, or an antisense RNA or ribozymes which 0 significantly disrupt a target cell leading thereby to the death of the target cell.

10. A method according to claim 9, wherein said product is selected from the group consisting of ribonucleases, nucleases, diphtheria toxin A chain (DTA), enzymes and inhibitors of plant cell wall synthesis. 5

11. A method according to claim 9, wherein said product arrests cell metabolism and/or cell growth thereby leading to cell death.

12. A method according to claim 8, wherein said sense or anti-sense polynucleotide fragment is selected from the group consisting of Flowering Time genes, Meristem Identity genes, genes determining plant architecture, genes determining plant development, Homeo box genes, Floral Morphology genes, genes involved in plant hormone biosynthesis, genes involved in plant hormone sensing, genes involved in plant hormone signalling, genes involved in light perception, genes involved in light signalling, genes acting as regulators of the cell cycle, genes involved in cell wall synthesis/metabolism/degradation, and genes involved in photosynthesis.

13. A method according to claim 10, wherein said ribonuclease is selected from the group consisting of Barnase from B.amyloliquefaciens, RNase T1 from A.aryzae, bovine RNase

A, RNase I from E.coli, RNase H from E.coli and plant RNases.

14. A method according to claim 10, wherein said nuclease is a restriction endonuclease.

15. A method according to claim 10, wherein said enzyme is selected from the group consisting of glucanases, ubiquitins and acid pyrophosphatases.

16. A method according to claim 7, wherein said product to be expressed by the promoter of the expression cassette to delay flowering is selected from the group consisting of

FLOWERING LOCUS T, SOC1 (AGL20), CONSTANS (CO), LFY, the AP1 -family (MADS box class of genes), genes involved in plant hormone biosynthesis, sensing or signalling, antisense sequences to known flowering repressors, homeobox genes, floral morphology genes, genes involved in light perception and signalling and genes acting as regulators of the cell cycle.

17. A method according to claim 1 , wherein said product to be expressed by the promoter of the expression cassette is selected from the group consisting of an insect resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance protein, a herbicide resistance gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a negative selectable marker gene, a reporter gene, a gene which expresses a protein affecting plant agronomic characteristics including plant morphology, plant architecture, and seed yield, an environment tolerance gene, and a stress tolerance gene.

18. A method according to claim 1 , wherein said promoter sequence is a MADS box gene promoter which preferentially expresses the product of said at least one associated polynucleotide fragment in the floral determined tissue of the plant.

19. A method according to any preceding claim, wherein said promoter sequence is a subset of the MADS box gene promoters.

20. A method according to any one of claims 1 to 18, wherein said promoter sequence is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, fragments, derivatives or homologues thereof.

21. A method according to claim 20, wherein said promoter sequence is selected from the group consisting of bases 1-2381 of SEQ ID NO. 1 , bases 1-1751 of SEQ ID NO. 2, bases 1-2390 of SEQ ID NO. 3 and bases 1 -3443 of SEQ ID NO. 4, fragments, derivatives or homologues thereof.

22. A method according to any one of claims 19 to 21 , wherein said promoter sequence includes a first intron.

23. A method according to claim 22, wherein said intron is selected from the group consisting of bases 2672-4617 of SEQ ID NO. 1 , bases 1996-4595 of SEQ ID NO. 2, bases 2672-5902 in SEQ ID NO. 3 and bases 3841-5681 of SEQ ID NO. 4.

24. A method according to claim 22 or 23, wherein said intron and said promoter are selected from the same SEQ ID NO.

25. A method according to claim 22 or 23, wherein said intron and said promoter δ are selected from different SEQ ID NOS.

26. 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, 0 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 δ 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 substantially or completely prevent or delay reproductive tissue growth

0 27. A method according to claim 26, wherein said product prevents development of inflorescence through being lethal to the floral determined cells.

28. An expression cassette comprising a promoter sequence and a polynucleotide fragment, wherein said promoter expresses a product from said polynucleotide fragment in floral 5 determined cells of a plant.

29. An expression cassette according to claim 28, wherein said polynucleotide fragment expresses a product which prevents and/or delays said floral determined cells from developing into inflorescence.

30. A transformed plant cell containing an expression cassette as described in claim 28 or claim 29.

31. A plant tissue or a plant comprising transformed plant cells as described in claim 30, and plants, seeds or pollen derived therefrom.

32. A polynucleotide sequence, fragment, derivative or homologue thereof, selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

33. A polynucleotide sequence, fragment, derivative or homologue thereof, selected from the group consisting of bases 1 -2381 of SEQ ID NO. 1 , bases 1-1761 of SEQ ID NO. 2, bases 1-2390 of SEQ ID NO. 3 and bases 1-3443 of SEQ ID NO. 4.

34. 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 or SEQ ID NO:4, fragments or derivatives thereof, and a polynucleotide fragment whose expression is under the control of said promoter sequence, and selecting any transformants and vegetatively propagating them.

36. 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,

- selecting and vegetatively propagating any transformants.

36. A method according to claim 35, wherein said first expression cassette is located on a first polynucleotide molecule and said second expression cassette is located on a second polynucleotide molecule.

37. A method according to claim 35, wherein said first expression cassette and said second expression cassette are located on the same polynucleotide molecule.

38. A method according to claim 35, wherein said inducible promoter is an ethanol- inducible system.

39. A transformed plant cell comprising a first expression cassette as described in claim 28, and a second expression cassette, wherein said second expression cassette comprises a second promoter and a second polynucleotide fragment, wherein said second promoter expresses said second product from said second polynucleotide fragment in non- target tissue, and wherein said second product inhibits the first product of the first polynucleotide fragment of the first expression cassette described in claim 28.

40. A transformed plant cell comprising a first expression cassette as described in claim 28, and a second expression cassette, wherein said second expression cassette comprises a second promoter and second polynucleotide fragment, wherein said second promoter constitutively expresses said second polynucleotide fragment at low levels in all tissues of the plant, and wherein said second product inhibits the first product of the first polynucleotide fragment of the first expression cassette described in claim 28.

41. A method according to any of claims 1 to 27 or 34 to 38 wherein said promoters are included in a system for biological containment of a transformed plant.

42. A method according to claim 41 , wherein said system is used in conjunction with a second, inducible system.